Structural Variants Create New Topological-Associated Domains and Ectopic Retinal Enhancer-Gene Contact in Dominant Retinitis Pigmentosa.

The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer-GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.

Chromosome 17 Missing Proteins: Recent Progress and Future Directions as Part of the neXt-MP50 Challenge.

The Chromosome-centric Human Proteome Project (C-HPP), announced in September 2016, is an initiative to accelerate progress on the detection and characterization of neXtProt PE2,3,4 "missing proteins" (MPs) with a mandate to each chromosome team to find about 50 MPs over 2 years. Here we report major progress toward the neXt-MP50 challenge with 43 newly validated Chr 17 PE1 proteins, of which 25 were based on mass spectrometry, 12 on protein-protein interactions, 3 on a combination of MS and PPI, and 3 with other types of data. Notable among these new PE1 proteins were five keratin-associated proteins, a single olfactory receptor, and five additional membrane-embedded proteins. We evaluate the prospects of finding the remaining 105 MPs coded for on Chr 17, focusing on mass spectrometry and protein-protein interaction approaches. We present a list of 35 prioritized MPs with specific approaches that may be used in further MS and PPI experimental studies. Additionally, we demonstrate how in silico studies can be used to capture individual peptides from major data repositories, documenting one MP that appears to be a strong candidate for PE1. We are close to our goal of finding 50 MPs for Chr 17.

Fine mapping of meiotic NAHR-associated crossovers causing large NF1 deletions.

Large deletions encompassing the NF1 gene and its flanking regions belong to the group of genomic disorders caused by copy number changes that are mediated by the local genomic architecture. Although nonallelic homologous recombination (NAHR) is known to be a major mutational mechanism underlying such genomic copy number changes, the sequence determinants of NAHR location and frequency are still poorly understood since few high-resolution mapping studies of NAHR hotspots have been performed to date. Here, we have characterized two NAHR hotspots, PRS1 and PRS2, separated by 20 kb and located within the low-copy repeats NF1-REPa and NF1-REPc, which flank the human NF1 gene region. High-resolution mapping of the crossover sites identified in 78 type 1 NF1 deletions mediated by NAHR indicated that PRS2 is a much stronger NAHR hotspot than PRS1 since 80% of these deletions exhibited crossovers within PRS2, whereas 20% had crossovers within PRS1. The identification of the most common strand exchange regions of these 78 deletions served to demarcate the cores of the PRS1 and PRS2 hotspots encompassing 1026 and 1976 bp, respectively. Several sequence features were identified that may influence hotspot intensity and direct the positional preference of NAHR to the hotspot cores. These features include regions of perfect sequence identity encompassing 700 bp at the hotspot core, the presence of PRDM9 binding sites perfectly matching the consensus motif for the most common PRDM9 variant, specific pre-existing patterns of histone modification and open chromatin conformations that are likely to facilitate PRDM9 binding.

Identification and characterization of Aurora kinase B and C variants associated with maternal aneuploidy.

STUDY QUESTION: Are single nucleotide variants (SNVs) in Aurora kinases B and C (AURKB, AURKC) associated with risk of aneuploid conception? SUMMARY ANSWER: Two SNVs were found in patients with extreme aneuploid concepti rates with respect to their age; one variant, AURKC p.I79V, is benign, while another, AURKB p.L39P, is a potential gain-of-function mutant with increased efficiency in promoting chromosome alignment. WHAT IS KNOWN ALREADY: Maternal age does not always predict aneuploidy risk, and rare gene variants can be drivers of disease. The AURKB and AURKC regulate chromosome segregation, and are associated with reproductive impairments in mouse and human. STUDY DESIGN, SIZE, DURATION: An extreme phenotype sample selection scheme was performed for variant discovery. Ninety-six DNA samples were from young patients with higher than average embryonic aneuploidy rates and an additional 96 DNA samples were from older patients with lower than average aneuploidy rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using the192 DNA samples, the coding regions of AURKB and AURKC were sequenced using next generation sequencing. To assess biological significance, we expressed complementary RNA encoding the human variants in mouse oocytes. Assays such as determining subcellular localization and assessing catalytic activity were performed to determine alterations in protein function during meiosis. MAIN RESULTS AND THE ROLE OF CHANCE: Ten SNVs were identified using three independent variant-calling methods. Two of the SNVs (AURKB p.L39P and AURKC p.I79V) were non-synonymous and identified by at least two variant-identification methods. The variant encoding AURKC p.I79V, identified in a young woman with a higher than average rate of aneuploid embryos, showed wild-type localization pattern and catalytic activity. On the other hand, the variant encoding AURKB p.L39P, identified in an older woman with lower than average rates of aneuploid embryos, increased the protein's ability to regulate alignment of chromosomes at the metaphase plate. These experiments were repeated three independent times using 2-3 mice for each trial. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Biological significance of the human variants was assessed in an in vitro mouse oocyte model where the variants are over-expressed. Therefore, the human protein may not function identically to the mouse homolog, or the same in mouse oocytes as in human oocytes. Furthermore, supraphysiological expression levels may not accurately reflect endogenous activity. Moreover, the evaluated variants were identified in one patient each, and no trial linking the SNV to pregnancy outcomes was conducted. Finally, the patient aneuploidy rates were established by performing comprehensive chromosome screening in blastocysts, and because of the link between female gamete aneuploidy giving rise to aneuploid embryos, we evaluate the role of the variants in Meiosis I. However, it is possible that the chromosome segregation mistake arose during Meiosis II or in mitosis in the preimplantation embryo. Their implications in human female meiosis and aneuploidy risk remain to be determined. WIDER IMPLICATIONS OF THE FINDINGS: The data provide evidence that gene variants exist in reproductively younger or advanced aged women that are predictive of the risk of producing aneuploid concepti in humans. Furthermore, a single amino acid in the N-terminus of AURKB is a gain-of-function mutant that could be protective of euploidy. STUDY FUNDING/COMPETING INTERESTS: This work was supported by a Research Grant from the American Society of Reproductive Medicine and support from the Charles and Johanna Busch Memorial Fund at Rutgers, the State University of NJ to K.S. and the Foundation for Embryonic Competence, Inc to N.T. The authors declare no conflicts of interest.

17q23.2q23.3 de novo duplication in association with speech and language disorder, learning difficulties, incoordination, motor skill impairment, and behavioral disturbances: a case report.

BACKGROUND: Chromosomal rearrangements involving 17q23 have been described rarely. Deletions at 17q23.1q23.2 have been reported in individuals with developmental delay and growth retardation, whereas duplications at 17q23.1q23.2 appear to segregate with clubfoot. Dosage alterations in the TBX2 and TBX4 genes, located in 17q23.2, have been proposed to be responsible for the phenotypes observed in individuals with 17q23.1q23.2 deletions and duplications. In this report, we present the clinical phenotype of a child with a previously unreported de novo duplication at 17q23.2q23.3 located distal to the TBX2 and TBX4 region. CASE PRESENTATION: We report a 7.5-year-old boy with speech and language disorder, learning difficulties, incoordination, fine motor skill impairment, infrequent seizures with abnormal EEG, and behavior disturbances (mild self-inflicted injuries, hyperactivity-inattention, and stereotyped hand movements). Chromosomal microarray revealed a 2-Mb duplication of chromosome 17q23.2q23.3. Both parents did not have the duplication indicating that this duplication is de novo in the child. CONCLUSIONS: The duplicated region encompasses 16 genes. It is possible that increased dosage of one or more genes in this region is responsible for the observed phenotype. The TANC2 gene is one of the genes in the duplicated region.It encodes a member of the TANC (tetratricopeptide repeat, ankyrin repeat and coiled-coil containing) family which includes TANC1 and TANC2. These proteins are highly expressed in brain and play major roles in synapsis regulation. Hence, it is suggestive that TANC2 is the likely candidate gene responsible for the observed phenotype as an increased TANC2 dosage can potentially alter synapsis, resulting in neuronal dysfunction and the neurobehavioral phenotype observed in this child with 17q23.2q23.3 duplication.

Detection of inherited chromosomally integrated HHV-6 (ciHHV-6) in a marker chromosome.

OBJECTIVES: Inherited chromosomally integrated human herpesvirus-6 (ciHHV-6) is characterised by the complete HHV-6 genome integration into the host germ line genome and is vertically transmitted with a Mendelian inheritance. By now, the only relationship between ciHHV-6 and diseases seems to be with angina pectoris. METHODS: We report a case of an 82-year-old man diagnosed with diffuse large B-cell lymphoma (DLBCL) on October 2014. To substantiate the suspicion of ciHHV-6, we analysed peripheral blood mononuclear cells, bone marrow biopsy and pleural effusion-derived mesothelial cells with PCR, RT-PCR and FISH. RESULTS: Virological routine screening by PCR showed the absence of HHV-8 and EBV infections, while the presence of HHV-6 DNA (ie, U22, U42 and U94 HHV-6 genes), with a viral load of about 1.0 genome per cell, strongly suggests ciHHV-6. The RT-PCR showed the positivity only for the immediate-early U94, at low levels of transcription (100±15 transcripts/1 µg RNA). FISH analysis reported a case of inherited ciHHV-6 in 17p chromosome region and, for the first time, in a marker chromosome. CONCLUSIONS: This is the first case of inherited ciHHV-6 in a marker chromosome, possibly elucidating the role of this abnormality in the biology of DLBCL.

Meta-analysis of genome-wide association studies identifies two loci associated with circulating osteoprotegerin levels.

Osteoprotegerin (OPG) is involved in bone homeostasis and tumor cell survival. Circulating OPG levels are also important biomarkers of various clinical traits, such as cancers and atherosclerosis. OPG levels were measured in serum or in plasma. In a meta-analysis of genome-wide association studies in up to 10 336 individuals from European and Asian origin, we discovered that variants >100 kb upstream of the TNFRSF11B gene encoding OPG and another new locus on chromosome 17q11.2 were significantly associated with OPG variation. We also identified a suggestive locus on chromosome 14q21.2 associated with the trait. Moreover, we estimated that over half of the heritability of OPG levels could be explained by all variants examined in our study. Our findings provide further insight into the genetic regulation of circulating OPG levels.

Whole-exome imputation of sequence variants identified two novel alleles associated with adult body height in African Americans.

Adult body height is a quantitative trait for which genome-wide association studies (GWAS) have identified numerous loci, primarily in European populations. These loci, comprising common variants, explain <10% of the phenotypic variance in height. We searched for novel associations between height and common (minor allele frequency, MAF ≥5%) or infrequent (0.5% < MAF < 5%) variants across the exome in African Americans. Using a reference panel of 1692 African Americans and 471 Europeans from the National Heart, Lung, and Blood Institute's (NHLBI) Exome Sequencing Project (ESP), we imputed whole-exome sequence data into 13 719 African Americans with existing array-based GWAS data (discovery). Variants achieving a height-association threshold of P < 5E-06 in the imputed dataset were followed up in an independent sample of 1989 African Americans with whole-exome sequence data (replication). We used P < 2.5E-07 (=0.05/196 779 variants) to define statistically significant associations in meta-analyses combining the discovery and replication sets (N = 15 708). We discovered and replicated three independent loci for association: 5p13.3/C5orf22/rs17410035 (MAF = 0.10, ß = 0.64 cm, P = 8.3E-08), 13q14.2/SPRYD7/rs114089985 (MAF = 0.03, ß = 1.46 cm, P = 4.8E-10) and 17q23.3/GH2/rs2006123 (MAF = 0.30; ß = 0.47 cm; P = 4.7E-09). Conditional analyses suggested 5p13.3 (C5orf22/rs17410035) and 13q14.2 (SPRYD7/rs114089985) may harbor novel height alleles independent of previous GWAS-identified variants (r(2) with GWAS loci <0.01); whereas 17q23.3/GH2/rs2006123 was correlated with GWAS-identified variants in European and African populations. Notably, 13q14.2/rs114089985 is infrequent in African Americans (MAF = 3%), extremely rare in European Americans (MAF = 0.03%), and monomorphic in Asian populations, suggesting it may be an African-American-specific height allele. Our findings demonstrate that whole-exome imputation of sequence variants can identify low-frequency variants and discover novel variants in non-European populations.

Functional variations modulating PRKCA expression and alternative splicing predispose to multiple sclerosis.

The protein kinase C alpha (PRKCA) gene, encoding a Th17-cell-selective kinase, was repeatedly associated with multiple sclerosis (MS), but the underlying pathogenic mechanism remains unknown. We replicated the association in Italians (409 cases, 723 controls), identifying a protective signal in the PRKCA promoter (P = 0.033), and a risk haplotype in intron 3 (P = 7.7 × 10(-4); meta-analysis with previously published data: P = 4.01 × 10(-8)). Expression experiments demonstrated that the protective signal is associated with alleles conferring higher PRKCA expression levels, well fitting our observation that MS patients have significantly lower PRKCA mRNA levels in blood. The risk haplotype was shown to be driven by a GGTG ins/del polymorphism influencing the heterogeneous nuclear ribonucleoprotein H-dependent inclusion/skipping of a PRKCA alternative exon 3*. Indeed, exon 3* can be present in two different versions in PRKCA mRNAs (out-of-frame 61 bp or in-frame 66 bp long), and is preferentially included in transcripts generated through a premature polyadenylation event. The GGTG insertion downregulates 3* inclusion and shifts splicing towards the 66 bp isoform. Both events reduce the nonsense-mediated mRNA-decay-induced degradation of exon 3*-containing mRNAs. Since we demonstrated that the protein isoform produced through premature polyadenylation aberrantly localizes to the plasma membrane and/or in cytoplasmic clusters, dysregulated PRKCA 3* inclusion may represent an additional mechanism relevant to MS susceptibility.

Application of whole-exome sequencing for detecting copy number variants in CMT1A/HNPP.

Large insertions and deletions (indels), including copy number variations (CNVs), are commonly seen in many diseases. Standard approaches for indel detection rely on well-established methods such as qPCR or short tandem repeat (STR) markers. Recently, a number of tools for CNV detection based on next-generation sequencing (NGS) data have also been developed; however, use of these methods is limited. Here, we used whole-exome sequencing (WES) in patients previously diagnosed with CMT1A or HNPP using STR markers to evaluate the ability of WES to improve the clinical diagnosis. Patients were evaluated utilizing three CNV detection tools including CONIFER, ExomeCNV and CEQer, and array comparative genomic hybridization (aCGH). We identified a breakpoint region at 17p11.2-p12 in patients with CMT1A and HNPP. CNV detection levels were similar in both 6 Gb (mean read depth = 80×) and 17 Gb (mean read depth = 190×) data. Taken together, these data suggest that 6 Gb WES data are sufficient to reveal the genetic causes of various diseases and can be used to estimate single mutations, indels, and CNVs simultaneously. Furthermore, our data strongly indicate that CNV detection by NGS is a rapid and cost-effective method for clinical diagnosis of genetically heterogeneous disorders such as CMT neuropathy.

The clinical impact and molecular biology of del(17p) in multiple myeloma treated with conventional or thalidomide-based therapy.

Hemizygous deletion of 17p (del(17p)) has been identified as a variable associated with poor prognosis in myeloma, although its impact in the context of thalidomide therapy is not well described. The clinical outcome of 85 myeloma patients with del(17p) treated in a clinical trial incorporating both conventional and thalidomide-based induction therapies was examined. The clinical impact of deletion, low expression, and mutation of TP53 was also determined. Patients with del(17p) did not have inferior response rates compared to patients without del(17p), but, despite this, del(17p) was associated with impaired overall survival (OS) (median OS 26.6 vs. 48.5 months, P < 0.001). Within the del(17p) group, thalidomide induction therapy was associated with improved response rates compared to conventional therapy, but there was no impact on OS. Thalidomide maintenance was associated with impaired OS, although our analysis suggests that this effect may have been due to confounding variables. A minimally deleted region on 17p13.1 involving 17 genes was identified, of which only TP53 and SAT2 were underexpressed. TP53 was mutated in <1% in patients without del(17p) and in 27% of patients with del(17p). The higher TP53 mutation rate in samples with del(17p) suggests a role for TP53 in these clinical outcomes. In conclusion, del(17p) defined a patient group associated with short survival in myeloma, and although thalidomide induction therapy was associated with improved response rates, it did not impact OS, suggesting that alternative therapeutic strategies are required for this group.

HER2, chromosome 17 polysomy and DNA ploidy status in breast cancer; a translational study.

Breast cancer treatment depends on human epidermal growth factor receptor-2 (HER2) status, which is often determined using dual probe fluorescence in situ hybridisation (FISH). Hereby, also loss and gain of the centromere of chromosome 17 (CEP17) can be observed (HER2 is located on chromosome 17). CEP17 gain can lead to difficulty in interpretation of HER2 status, since this might represent true polysomy. With this study we investigated whether isolated polysomy is present and how this effects HER2 status in six breast cancer cell lines and 97 breast cancer cases, using HER2 FISH and immunohistochemistry, DNA ploidy assessment and multiplex ligation dependent probe amplification. We observed no isolated polysomy of chromosome 17 in any cell line. However, FISH analysis did show CEP17 gain in five of six cell lines, which reflected gains of the whole chromosome in metaphase spreads and aneuploidy with gain of multiple chromosomes in all these cases. In patients' samples, gain of CEP17 indeed correlated with aneuploidy of the tumour (91.1%; p < 0.001). Our results indicate that CEP17 gain is not due to isolated polysomy, but rather due to widespread aneuploidy with gain of multiple chromosomes. As aneuploidy is associated with poor clinical outcome, irrespective of tumour grade, this could improve future therapeutic decision making.

Genomic mid-range inhomogeneity correlates with an abundance of RNA secondary structures.

BACKGROUND: Genomes possess different levels of non-randomness, in particular, an inhomogeneity in their nucleotide composition. Inhomogeneity is manifest from the short-range where neighboring nucleotides influence the choice of base at a site, to the long-range, commonly known as isochores, where a particular base composition can span millions of nucleotides. A separate genomic issue that has yet to be thoroughly elucidated is the role that RNA secondary structure (SS) plays in gene expression. RESULTS: We present novel data and approaches that show that a mid-range inhomogeneity (~30 to 1000 nt) not only exists in mammalian genomes but is also significantly associated with strong RNA SS. A whole-genome bioinformatics investigation of local SS in a set of 11,315 non-redundant human pre-mRNA sequences has been carried out. Four distinct components of these molecules (5'-UTRs, exons, introns and 3'-UTRs) were considered separately, since they differ in overall nucleotide composition, sequence motifs and periodicities. For each pre-mRNA component, the abundance of strong local SS (< -25 kcal/mol) was a factor of two to ten greater than a random expectation model. The randomization process preserves the short-range inhomogeneity of the corresponding natural sequences, thus, eliminating short-range signals as possible contributors to any observed phenomena. CONCLUSION: We demonstrate that the excess of strong local SS in pre-mRNAs is linked to the little explored phenomenon of genomic mid-range inhomogeneity (MRI). MRI is an interdependence between nucleotide choice and base composition over a distance of 20-1000 nt. Additionally, we have created a public computational resource to support further study of genomic MRI.

Human centromeric alphoid domains are periodically homogenized so that they vary substantially between homologues. Mechanism and implications for centromere functioning.

Sequence analysis of alphoid repeats from human chromosomes 17, 21 and 13 reveals recurrent diagnostic variant nucleotides. Their combinations define haplotypes, with higher order repeats (HORs) containing identical or closely-related haplotypes tandemly arranged into separate domains. The haplotypes found on homologues can be totally different, while HORs remain 99.8% homogeneous both intrachromosomally and between homologues. These results support the hypothesis, never before demonstrated, that unequal crossovers between sister chromatids accumulate to produce homogenization and amplification into tandem alphoid repeats. I propose that the molecular basis of this involves the diagnostic variant nucleotides, which enable pairing between HORs with identical or closely-related haplotypes. Domains are thus periodically renewed to maintain high intrachromosomal and interhomologue homogeneity. The capacity of a domain to form an active centromere is maintained as long as neither retrotransposons nor significant numbers of mutations affect it. In the presented model, a chromosome with an altered centromere can be transiently rescued by forming a neocentromere, until a restored, fully-competent domain is amplified de novo or rehomogenized through the accumulation of unequal crossovers.

Chromosome 17q21 Genes ORMDL3 and GSDMB in Asthma and Immune Diseases.

Chromosome 17q21 contains a cluster of genes including ORMDL3 and GSDMB, which have been highly linked to asthma in genome-wide association studies. ORMDL3 is localized to the endoplasmic reticulum and regulates downstream pathways including sphingolipids, metalloproteases, remodeling genes, and chemokines. ORMDL3 inhibits serine palmitoyl-CoA transferase, the rate-limiting enzyme for sphingolipid biosynthesis. In addition, ORMDL3 activates the ATF6α branch of the unfolded protein response which regulates SERCA2b and IL-6, pathways of potential importance to asthma. The SNP-linking chromosome 17q21 to asthma is associated with increased ORMDL3 and GSDMB expression. Mice expressing either increased levels of human ORMDL3, or human GSDMB, have an asthma phenotype characterized by increased airway responsiveness and increased airway remodeling (increased smooth muscle and fibrosis) in the absence of airway inflammation. GSDMB regulates expression of 5-LO and TGF-ß1 which are known pathways involved in the pathogenesis of asthma. GSDMB is one of four members of the GSDM family (GSDMA, GSDMB, GSDMC, and GSDMD). GSDMD (located on chromosome 8q24 and not linked to asthma) has emerged as a key mediator of pyroptosis. GSDMD is a key component of the NLPR3 inflammasome and is required for its activation. GSDMD undergoes proteolytic cleavage by caspase-1 to release its N-terminal fragment, which in turn mediates pyroptosis and IL-1ß secretion. Chromosome 17q21 has not only been linked to asthma but also to type 1 diabetes, inflammatory bowel disease, and primary biliary cirrhosis suggesting that future insights into the biology of genes located in this region will increase our understanding of these diseases.

Lymphocyte Activation Dynamics Is Shaped by Hereditary Components at Chromosome Region 17q12-q21.

Single nucleotide polymorphisms (SNPs) located in the chromosome region 17q12-q21 are risk factors for asthma. Particularly, there are cis-regulatory haplotypes within this region that regulate differentially the expression levels of ORMDL3, GSDMB and ZPBP2 genes. Remarkably, ORMDL3 has been shown to modulate lymphocyte activation parameters in a heterologous expression system. In this context, it has been shown that Th2 and Th17 cytokine production is affected by SNPs in this region. Therefore, we aim to assess the impact of hereditary components within region 17q12-q21 on the activation profile of human T lymphocytes, focusing on the haplotype formed by allelic variants of SNPs rs7216389 and rs12936231. We measured calcium influx and activation markers, as well as the proliferation rate upon T cell activation. Haplotype-dependent differences in mRNA expression levels of IL-2 and INF-γ were observed at early times after activation. In addition, the allelic variants of these SNPs impacted on the extent of calcium influx in resting lymphocytes and altered proliferation rates in a dose dependent manner. As a result, the asthma risk haplotype carriers showed a lower threshold of saturation during activation. Finally, we confirmed differences in activation marker expression by flow cytometry using phytohemagglutinin, a strong polyclonal stimulus. Altogether, our data suggest that the genetic component of pro-inflammatory pathologies present in this chromosome region could be explained by different T lymphocyte activation dynamics depending on individual allelic heredity.

Molecular genetic alterations of chromosome 17 and p53 nuclear overexpression in human bladder cancer.

We set out to define the alterations of chromosome 17 in human bladder tumors and to correlate p53 nuclear over-expression with 17p deletions in those neoplasms. We studied 60 bladder tumors by restriction fragment-length polymorphism analysis directed at five different loci on chromosome 17. The same tumors were studied with a panel of mouse monoclonal antibodies (PAb1801, PAb240, and PAb1620) to mutant and wild-type p53 proteins using immunohistochemistry. Deletion of 17p correlated with grade (p = 0.039), stage (p = 0.004), and the presence of vascular invasion (p = 0.056). None of the pathologic parameters correlated with 17q deletions. p53 nuclear overexpression correlated with grade (p = 0.027), stage (p = 0.008), vascular invasion (p = 0.021), and the presence of nodal metastases (p = 0.007). In superficial (Ta) lesions, 17p was not deleted, whereas 55% of T1 and T2-T4 tumors showed a loss of heterozygosity. Mutations of p53 as detected by immunohistochemistry were seen in superficial as well as invasive tumors, whereas loss of heterozygosity was seen only in invasive tumors. A strong correlation was found between the presence of mutation and the loss of heterozygosity of the remaining allele (p = 0.0003). Additional follow-up and further studies are required to better define the role of p53 nuclear overexpression and 17p deletions as markers of tumor progression in human bladder cancer.

Identification of novel subregions of LOH in gastric cancer and analysis of the HIC1 and TOB1 tumor suppressor genes in these subregions.

Previously, we identified 3 overlapping regions showing loss of heterozygosity (LOH, R(1)-R(3) from 11 to 30 cM) on chromosome 17 in 45 primary gastric cancers (GCs). The data indicated the presence of tumor suppressor genes (TSGs) on chromosome 17 involved in GC. Among the putative TSGs in these regions, HIC1 (in SR(1)) and TOB1 (in SR(3)) remain to be examined in GC. By immunohistochemistry (IHC), methylation-specific PCR (MSP) and western blot, we evaluated the expression and regulation status for HIC1 and TOB1 protein in GC. We narrowed down the deletion intervals on chromosome 17 and defined five smaller LOH subregions, SR(1)-SR(5) (0.54 to 3.42 cM), in GC. We found that HIC1 had downregulated expression in 86% (91/106) and was methylated in 87% (26/30) of primary GCs. Of the primary GCs showing downregulation of HIC1 protein, 75% (18/24) had methylated HIC1 gene. TOB1 was either absent or expressed at reduced levels in 75% (73/97) of the GC samples. In addition, a general reduction was found in total and the ratio of unphosphorylated to phosphorylated TOB1 protein levels in the differentiated GC cell lines. Further analysis revealed significant simultaneous downregulation of both HIC1 and TOB1 protein in GC tissue microarray samples (67%, 52/78) and in primary GCs (65%, 11/17). These results indicate that silencing of HIC1 and TOB1 expression is a common occurrence in GC and may contribute to the development and progression of the disease.

Relationship between the 17q21 locus and adult asthma in a Czech population.

Several whole-genome association studies have shown a significant link between childhood asthma and the 17q12 chromosome region. We selected tagging single nucleotide polymorphisms (SNPs) in the ORMDL3 gene (17q12) to investigate gene variability in relation to adult allergic asthma and asthma/atopy traits in a Czech Caucasian population of adults. We conducted a case-control association study comprising 668 unrelated subjects (337 asthmatic and 331 control subjects). Four selected SNPs (rs17608925, rs12603332, rs8076131, and rs3169572) were genotyped using the TaqMan SNP Genotyping Assays. The single locus analysis showed only a borderline association between rs3169572 variant and asthma (p = 0.030, p(corr) > 0.05). However, seven different haplotypes were identified; among them, the TTAA haplotype was marginally associated with asthma (p = 0.045, p(corr) > 0.05) and TCAG haplotype was significantly associated with asthma in males (p = 0.009, p(corr) < 0.05, odds ratio = 1.48, 95% confidence interval = 1.10-2.00). In addition, associations between the ORMDL3 genotypes and the total IgE level (p = 0.05, p(corr) > 0.05) and hypersensitivity to the pollen (p = 0.007, p(corr) < 0.05) were established. However, no relationship between ORMDL3 SNPs and the pulmonary functions was found (p > 0.05). These findings suggest that the genetic variability in the 17q21 region may be one of the risk factors also for adult asthma, especially in male individuals.