Duplication In Intron Research Articles

White horses - non-coding sequences drive premature hair greying and predisposition to melanoma.

The Grey allele in horses is causing premature hair greying and susceptibility to melanoma. The causal mutation is a 4.6 kb tandem duplication in intron 6 of the Syntaxin 17 gene. A recent study demonstrated that the most common allele at the Grey locus (G3) involves three tandem copies of this sequence, whilst a more rare allele (G2) has two tandem copies and the wild-type allele (G1) only one copy. The G3 allele is causing fast greying and high incidence of skin melanoma, whereas the G2 allele is causing slow greying and no obvious increase in melanoma incidence. Further somatic copy number expansion has been documented in melanoma tissue from Grey horses. Functional studies showed that this intronic sequence acts as a weak melanocyte-specific enhancer that becomes substantially stronger by the copy number expansion. The Grey mutation is associated with upregulated expression of both Syntaxin 17 and the neighbouring NR4A3 gene in Grey horse melanomas. It is still an open question which of these genes is most important for the phenotypic effects or if causality is due to the combined effect of upregulation of both genes. Interestingly, RNAseq data in the Human Protein Atlas give support for a possible role of NR4A3 because it is particularly upregulated in human skin cancer, and it belongs to a cluster of genes associated with skin cancer and melanin biosynthesis. The Grey mutation and its association with melanoma provide a possibility to study the path to tumour development in numerous Grey horses carrying exactly the same predisposing mutation.

HOXC10 intronic duplication is associated with unsealed skull and crest in crested chicken with cerebral hernia

The genetic basis and developmental mechanism of unsealed skull in crested chicken with cerebral hernia remain unclear. Here, a genomic region including six HOXC genes was mapped by bulked segregant analysis (BSA) in a crested chicken resource population. A 195-bp intronic tandem duplication was further confirmed in the HOXC10 gene. HOXC genes, particularly HOXC10, were expressed ectopically in fetal skin and meningeal tissues of crested chicken with cerebral hernia, indicating its impact on the cranial mesenchymal tissues that drive the development of scalp skin, frontal bone, and meninges. The restricted expansion of frontal bone progenitors labeled with anti-RUNX2 antibody in the supraorbital mesenchyme of the fetal head implied abnormal migration, which contributed to the formation of the unsealed skull. This study suggests that HOXC genes were potent drivers for the abnormalities of the head crest and unsealed skull observed in crested chicken with cerebral hernia.

A Mild Case of Autosomal Recessive Osteopetrosis Masquerading as the Dominant Form Involving Homozygous Deep Intronic Variations in the CLCN7 Gene

Osteopetrosis is a heterogeneous group of rare hereditary diseases characterized by increased bone mass of poor quality. Autosomal-dominant osteopetrosis type II (ADOII) is most often caused by mutation of the CLCN7 gene leading to impaired bone resorption. Autosomal recessive osteopetrosis (ARO) is a more severe form and is frequently accompanied by additional morbidities. We report an adult male presenting with classical clinical and radiological features of ADOII. Genetic analyses showed no amino-acid-converting mutation in CLCN7 but an apparent haploinsufficiency and suppression of CLCN7 mRNA levels in peripheral blood mononuclear cells. Next generation sequencing revealed low-frequency intronic homozygous variations in CLCN7, suggesting recessive inheritance. In silico analysis of an intronic duplication c.595-120_595-86dup revealed additional binding sites for Serine- and Arginine-rich Splicing Factors (SRSF), which is predicted to impair CLCN7 expression. Quantitative backscattered electron imaging and histomorphometric analyses revealed bone tissue and material abnormalities. Giant osteoclasts were present and additionally to lamellar bone, and abundant woven bone and mineralized cartilage were observed, together with increased frequency and thickness of cement lines. Bone mineralization density distribution (BMDD) analysis revealed markedly increased average mineral content of the dense bone (CaMean T-score + 10.1) and frequency of bone with highest mineral content (CaHigh T-score + 19.6), suggesting continued mineral accumulation and lack of bone remodelling. Osteocyte lacunae sections (OLS) characteristics were unremarkable except for an unusually circular shape. Together, our findings suggest that the reduced expression of CLCN7 mRNA in osteoclasts, and possibly also osteocytes, causes poorly remodelled bone with abnormal bone matrix with high mineral content. This together with the lack of adequate bone repair mechanisms makes the material brittle and prone to fracture. While the skeletal phenotype and medical history were suggestive of ADOII, genetic analysis revealed that this is a possible mild case of ARO due to deep intronic mutation.

Establishment of a human induced pluripotent stem cell line, KMUGMCi001-A, from a patient bearing a heterozygous c.772 + 3_772 + 4dup mutation in the ACVRL1 gene leading Telangiectasia, hereditary hemorrhagic, type 2 (HHT2)

Telangiectasia, hereditary hemorrhagic, type 2 (HHT2) is a rare autosomal dominant disease caused by a mutated ACVRL1 gene (Letteboer et al., 2005). The peripheral blood mononuclear cells (PBMCs) from a patient carrying a heterozygous 2 bp duplication in intron 6 of the ACVRL1 gene, NG_009549.1(NM_000020.2):c.772 + 3_772 + 4dup, were reprogrammed using episomal vectors. The inserted mutation in ACVRL1 will causes the abnormal splicing, which will be associated with HHT2. The cell line will enable proper in vitro disease modelling of HHT2(Roman and Hinck, 2017).

Decoding the Role of DVL1 in Intracranial Meningioma.

In the search for molecular candidates for targeted meningioma therapies, increasing attention has been paid to the role of signaling pathways in the development and progression of intracranial meningiomas. Although it is well known that the Wnt signaling pathway is involved in meningioma progression, the role of its central mediator, DVL1, is still unclear. In order to investigate the influence of DVL1 gene alterations on the progression of human intracranial meningioma, we focused on its central PDZ domain, which is responsible for DVL interaction with the Fzd receptor and the phosphorylation of DVL mediated through the casein kinases CK1 and CK2. A genetic analysis of genomic instability revealed the existence of microsatellite instability in 9.09% and the loss of heterozygosity in 6.06% of the samples. The sequencing of the PDZ gene region showed repetitive deletions of two bases located in intron 7 and exon 8, and a duplication in intron 8 in most samples, with different outcomes on the biological function of the DVL1 protein. Immunohistochemistry revealed that the nuclear expression of DVL1 was significantly correlated with a higher expression of active β-catenin (p = 0.029) and a higher meningioma grade (p = 0.030), which leads to the conclusion that it could be used as biomarker for meningioma progression and the activation of the Wnt signaling pathway.

DPF3, A Putative Candidate Gene For Melanoma Etiopathogenesis in Gray Horses

Melanoma prevalence in gray horses reaches up to 50% and more. Several studies have documented a genetic melanoma predisposition which is referred to the 4.6 kb duplication in intron 6 of STX17 and its surrounding haplotype. However, the genetic background and mechanisms responsible for differences in etiopathogenesis of equine dermal melanomatosis still remain unknown. In the current study, we performed a genome wide association analysis in 141 Lipizzan horses and subsequently identified one candidate gene on chromosome 24 putatively involved in melanoma pathogenesis in gray horses. The associated SNP was located in the intronic region of DPF3, a gene which is involved in humans in cell growth, proliferation, apoptosis and motility of cancer cells. The replication study in 1210 horses from seven breeds demonstrated, that the G/G genotype of the DPF3 associated SNP exhibits putative melanoma suppression effects. As a conclusion DPF3 represents a candidate gene, which might play an essential role for gray horses coping with high genetic melanoma related tumor load.

Prospective use of barley spike/flower homeotic single and double mutants for ornamental purposes

Barley and other cereals only in small portions are used as ornamentals for their attractive spike structure and other features, and thus, there is a need to enhance approaches for the enrichment of their ornamental genetic resources. Homeotic Hooded (Hd) mutants, caused by a 305-bp duplication in intron IV of the BKn3 gene and characterized by ectopic flowers instead of awns, are attractive ornamental plants. However, it has been proposed that the hybridization of Hd mutants with other homeotic mutants may widen resources of attractive forms. Two groups of lines were studied: those resulting from the hybridization of Hooded in cv. Colsess II with other Hd mutants and those resulting from the hybridization of Hd with other homeotic mutant tweaky spike2 (tw2). tw2 has lodicules that are irregularly transformed to reproductive organs and specific spike shapes. Attractive lines were selected, propagated and studied in distinct generations (corresponding to F9–15) for decorative quality and quantitative characters. Variations of vegetation time, weight of grains, resistance to lodging and diseases, protein and starch contents are presented. In total, 231 lines were analysed. Not all selected attractive lines possess the mutant BKn3 allele. Wider and more attractive spectra were revealed when different types of mutants, Hd and tw2, were hybridized. More attractive lines were selected among the offspring of tw2 × Lh and tw2× Br. Several lines are also valuable for high protein content.

Spectrum of SMPD1 mutations in Asian‐Indian patients with acid sphingomyelinase (ASM)‐deficient Niemann–Pick disease

Acid sphingomyelinase (ASM)-deficient Niemann-Pick disease is an autosomal recessive lysosomal storage disorder caused by biallelic mutations in the SMPD1 gene. To date, around 185 mutations have been reported in patients with ASM-deficient NPD world-wide, but the mutation spectrum of this disease in India has not yet been reported. The aim of this study was to ascertain the mutation profile in Indian patients with ASM-deficient NPD. We sequenced SMPD1 in 60 unrelated families affected with ASM-deficient NPD. A total of 45 distinct pathogenic sequence variants were found, of which 14 were known and 31 were novel. The variants included 30 missense, 4 nonsense, and 9 frameshift (7 single base deletions and 2 single base insertions) mutations, 1 indel, and 1 intronic duplication. The pathogenicity of the novel mutations was inferred with the help of the mutation prediction software MutationTaster, SIFT, Polyphen-2, PROVEAN, and HANSA. The effects of the identified sequence variants on the protein structure were studied using the structure modeled with the help of the SWISS-MODEL workspace program. The p. (Arg542*) (c.1624C>T) mutation was the most commonly identified mutation, found in 22% (26 out of 120) of the alleles tested, but haplotype analysis for this mutation did not identify a founder effect for the Indian population. To the best of our knowledge, this is the largest study on mutation analysis of patients with ASM-deficient Niemann-Pick disease reported in literature and also the first study on the SMPD1 gene mutation spectrum in India. © 2016 Wiley Periodicals, Inc.

Frequency of gray coat color in native Chinese horse breeds.

Gray horses are born colored, and they then gradually lose their hair pigmentation. Tremendous progress has been made in identifying the genes responsible for graying with age in horses in recent years. Results show that gray coat color in horses is caused by a 4.6-kb duplication in intron 6 of the syntaxin 17 gene (STX17), which constitutes a cis-acting-regulatory mutation. However, little is known about the gray phenotype in native Chinese horses. This study was conducted to explore the frequency distribution of the gray mutation in native Chinese horse breeds. A total of 489 samples from 14 native Chinese horse breeds were genotyped for the STX17 duplication using a simplified conventional PCR-based method. The results show that the gray mutation was present in 12 native Chinese horse breeds, except the Balikun and Guanzhong breeds. The Chakouyi and Hequ breeds had the highest frequency of the gray mutation (P(G) = 0.367 and P(G) = 0.274, respectively). There was no significant geographical difference in the distribution of gray coat color across native Chinese horse breeds. Our results suggest that gray is a common coat color in Chinese horses.

Constitutive activation of the ERK pathway in melanoma and skin melanocytes in Grey horses.

BackgroundConstitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses.MethodsGrey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting.ResultsWe found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway. The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases. Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses.ConclusionsThese findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this pathway during melanomagenesis. These findings are consistent with the universal importance of the ERK pathway in melanomagenesis and may have valuable implications for human melanoma research.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-857) contains supplementary material, which is available to authorized users.

Age at cancer onset in germline TP53 mutation carriers: association with polymorphisms in predicted G-quadruplex structures

Germline TP53 mutations predispose to multiple cancers defining Li-Fraumeni/Li-Fraumeni-like syndrome (LFS/LFL), a disease with large individual disparities in cancer profiles and age of onset. G-quadruplexes (G4s) are secondary structural motifs occurring in guanine tracks, with regulatory effects on DNA and RNA. We analyzed 85 polymorphisms within or near five predicted G4s in TP53 in search of modifiers of penetrance of LFS/LFL in Brazilian cancer families with (n = 35) or without (n = 110) TP53 mutations. Statistical analyses stratified on family structure showed that cancer tended to occur ~15 years later in mutation carriers who also carried the variant alleles of two polymorphisms within predicted G4-forming regions, rs17878362 (TP53 PIN3, 16 bp duplication in intron 3; P = 0.082) and rs17880560 (6 bp duplication in 3' flanking region; P = 0.067). Haplotype analysis showed that this inverse association was driven by the polymorphic status of the remaining wild-type (WT) haplotype in mutation carriers: in carriers with a WT haplotype containing at least one variant allele of rs17878362 or rs17880560, cancer occurred ~15 years later than in carriers with other WT haplotypes (P = 0.019). No effect on age of cancer onset was observed in subjects without a TP53 mutation. The G4 in intron 3 has been shown to regulate alternative p53 messenger RNA splicing, whereas the biological roles of predicted G4s in the 3' flanking region remain to be elucidated. In conclusion, this study demonstrates that G4 polymorphisms in haplotypes of the WT TP53 allele have an impact on LFS/LFL penetrance in germline TP53 mutation carriers.

Variant ATRX Syndrome with Dysfunction ofATRXandMAGT1Genes

A 0.8 kb intronic duplication in MAGT1 and a single base pair deletion in the last exon of ATRX were identified using a chromosome X-specific microarray and exome sequencing in a family with five males demonstrating intellectual disability (ID) and unusual skin findings (e.g., generalized pruritus). MAGT1 is an Mg²⁺ transporter previously associated with primary immunodeficiency and ID, whereas mutations in ATRX cause ATRX-ID syndrome. In patient cells, the function of ATRX was demonstrated to be abnormal based on altered RNA/protein expression, hypomethylation of rDNA, and abnormal cytokinesis. Dysfunction of MAGT1 was reflected in reduced RNA/protein expression and Mg²⁺ influx. The mutation in ATRX most likely explains the ID, whereas MAGT1 disruption could be linked to abnormal skin findings, as normal magnesium homeostasis is necessary for skin health. This work supports observations that multiple mutations collectively contribute to the phenotypic variability of syndromic ID, and emphasizes the importance of correlating clinical phenotype with genomic and cell function analyses.

Association between p53 Gene Variants and Oral Cancer Susceptibility in Population from Gujarat, West India

p53 gene variants i.e. 16 bp duplication in intron 3, Arg72Pro in exon 4 and G>A in intron 6 have been reported to modulate susceptibility to various malignancies. Therefore, the present study evaluated the role of these p53 polymorphisms in oral cancer susceptibility in a population from Gujarat, West India. Genotype frequencies at the three p53 loci in 110 controls and 79 oral cancer cases were determined by the PCR-RFLP method. Heterozygous individuals at exon 4 showed protection from developing oral cancer. Homozygous wild and heterozygous individuals at intron 3 and those heterozygous at exon 4 in combination appeared to be at lowered risk. Furthermore, carriers of the 16 bp duplication allele at intron 3, proline allele at exon 4 and G allele at intron 6 were protected from oral cancer development. p53 polymorphisms, especially Arg72Pro in exon 4 could significantly modify the risk of oral cancer development in Gujarat, West Indian population.

Complex Inheritance of Melanoma and Pigmentation of Coat and Skin in Grey Horses

The dominant phenotype of greying with age in horses, caused by a 4.6-kb duplication in intron 6 of STX17, is associated with a high incidence of melanoma and vitiligo-like skin depigmentation. However, the progressive greying and the incidence of melanoma, vitiligo-like depigmentation, and amount of speckling in these horses do not follow a simple inheritance pattern. To understand their inheritance, we analysed the melanoma grade, grey level, vitiligo grade, and speckling grade of 1,119 Grey horses (7,146 measurements) measured in six countries over a 9-year period. We estimated narrow sense heritability (h2), and we decomposed this parameter into polygenic heritability (h2 POLY), heritability due to the Grey (STX17) mutation (h2 STX17), and heritability due to agouti (ASIP) locus (h2 ASIP). A high heritability was found for greying (h2 = 0.79), vitiligo (h2 = 0.63), and speckling (h2 = 0.66), while a moderate heritability was estimated for melanoma (h2 = 0.37). The additive component of ASIP was significantly different from zero only for melanoma (h2 ASIP = 0.02). STX17 controlled large proportions of phenotypic variance (h2 STX17 = 0.18–0.55) and overall heritability (h2 STX17/h2 = 0.28–0.83) for all traits. Genetic correlations among traits were estimated as moderate to high, primarily due to the effects of the STX17 locus. Nevertheless, the correlation between progressive greying and vitiligo-like depigmentation remained large even after taking into account the effects of STX17. We presented a model where four traits with complex inheritance patterns are strongly influenced by a single mutation. This is in line with evidence of recent studies in domestic animals indicating that some complex traits are, in addition to the large number of genes with small additive effects, influenced by genes of moderate-to-large effect. Furthermore, we demonstrated that the STX17 mutation explains to a large extent the moderate to high genetic correlations among traits, providing an example of strong pleiotropic effects caused by a single gene.

Detection of Copy Number Alterations in DNA Repair/Repair-Related Genes for a Diversity of Hematologic Malignancies Identifies Disease-Specific Biomarkers of Potential Clinical Relevance.

AbstractAbstract 2531▪▪This icon denotes a clinically relevant abstractDNA repair and repair-related genes encode molecules that function as critical guardians of the genome, both in terms of chromosome stability and single base integrity. Defects in such functions, both inherited and acquired, represent key factors that contribute to the etiology of cancer (e.g., RB1, TP53 and ATM). Copy number alterations (CNAs) in such genes have been described for one or more hematologic malignancies and disease-specific testing is routinely employed for the detection of these important diagnostic/prognostic markers. Additional DNA repair genes have been implicated as potential factors influencing the onset, progression, and/or chemo-resistance of disease. In the present study, we analyzed 155 DNA repair/repair-related genes for CNAs in 425 cases of hematologic malignancy using microarray analysis. Cases represented nine distinct clinical entities with at least 30 cases per entity. Disorders represented include myelodysplastic syndrome (MDS), chronic lymphocytic leukemia (CLL), myeloproliferative neoplasm (MPN), chronic myeloid leukemia (CML), B-cell and T-cell acute lymphoblastic leukemia (B-ALL, T-ALL), acute myeloid leukemia (AML), non-Hodgkin lymphoma (NHL), and multiple myeloma (MM). An oligo-based microarray platform was employed with higher density coverage for the DNA repair/ repair-related genes (1 probe/0.2–7.0 kb). Results were assessed relative to multiple databases of known benign variation. A proof of principal analysis included RB1, TP53 and ATM, for which genomic alterations and disease preference have been well established. Analysis of RB1 revealed CNAs in 74 cases (4 gains and 70 losses) (17.4% of total) with copy gains uniformly attributable to trisomy 13 in cases of B-ALL. As expected, loss of RB1 typically reflected either monosomy 13 (31/70 seen predominately in MM) or smaller deletions (∼1 Mb, seen in CLL). CNAs in TP53 included 2 gains and 42 losses (10.4% of total), with gains again related to trisomy (exclusively hyperdiploid B-ALL), whereas deletions varied in size and disease association. CNAs in the ATM included 28 gains and 19 losses (11.1% of total). Gains reflecting trisomy correlated with diverse diagnoses, while more focal gains (∼10 Mb) were seen exclusively in B-ALL (3 cases). Not surprisingly, small losses of ATM (<10 Mb) occurred almost exclusively in CLL. Thus, microarray findings for these classic tumor suppressor genes correlated well with disease specificity for alterations submicroscopic in size (<10 Mb).An additional 152 DNA repair/repair-related genes were analyzed and a number of genes showed disease-specific correlations for submicroscopic CNAs. For example, deletion of ERCC5 was seen in B-ALL (2 cases); however, co-deletion of ERCC5 and LIG4 was confined to NHL (3 cases). Smaller deletions involving a number of other genes also occurred specifically in NHL (GTF2H4, POLQ, REV1L, FANCL, BRCA2, and NUDT1). While abnormalities in TERT were infrequent (21 gains and 4 losses) and nearly always associated with aneuploidy, an intragenic duplication of three exons was seen in a single case of diffuse large B-cell lymphoma with a history of failed therapy. Regarding AML, deletion of UBE2V2 was seen in a case exhibiting numerous multi-megabase amplifications, a potential consequence of dysregulated DNA replication. AML-specific CNAs include deletions of RPA2 that always associated with translocation-positive cases (PML/RARA; MLL/MLLT3; MLL/MLLT4), and deletions of APTX (reduced expression correlates with favorable therapy response), as well as an intronic duplication within MGMT (therapy resistance associates with overexpression). Smaller CNAs involving ALKBH3 were seen specifically in MM (3 cases), while deletions of ALKBH5 impacting nearby TOP3A were seen in MDS (2 cases). Additional genes in which recurrent CNAs were associated with multiple disorders included NEIL1, PMS2, ENDOV, CHEK2, and HELQ.The data reveal CNAs in DNA repair and repair related genes; many are recurrent abnormalities that exhibit disease specificity. The findings are consistent with previous data addressing biological function, disease progression and response to therapy. Further study is warranted to validate these alterations as diagnostic/prognostic biomarkers or as predictive biomarkers useful in directing more personalized treatment decisions. They may also represent potential new targets for therapy. Disclosures:Schultz:Signature Genomic Laboratories, PerkinElmer Inc.: Employment. McDaniel:Signature Genomic Laboratories, PerkinElmer Inc.: Employment. Shaffer:Signature Genomic Laboratories, PerkinElmer Inc.: Employment, Equity Ownership. Ballif:Signature Genomic Laboratories, PerkinElmer Inc.: Employment, Equity Ownership.

Abstract B91: Assosiation of tumor supressor gene p53 with lung cancer in Pakistani population.

Abstract Lung cancer is the most common cause of cancer related death in men and second most common in women, after breast cancer. It is known to be responsible for 1.3 million deaths worldwide annually. The p53 gene, is a tumor suppressor gene, i.e. its activity stops the formation of tumors. The gene has been mapped to chromosome 17. In the cell, p53 protein binds DNA, which in turn stimulates another gene to produce a protein called p21 that interacts with a cell division-stimulating protein (cdk2). When p21 is complexed with cdk2 the cell cannot pass through to the next stage of cell division. In present study we check Association of P53 polymorphism with lung cancer in Pakistani population. Polymorphisms in human P53 gene (16bp duplication in intron 3) were studied in patients with lung cancer and controls. Three types of genotypes were seen, homozygous insertion (1\1), homozygous deletion (2\2) and heterozygous insertion/deletion (1/2). Frequency of 1/2 allele was 0.2 in controls and 0.32 in lung cancer patients. This difference is statistically significant (P = 0.053, 95 CI). Our study further showed that smoking increases the risk of lung cancer. Study of polymorphism in p53 gene will help in understanding the patho-physiology of the lung cancer and will open new fronts in future therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B91.

Identification of a melanocyte‐specific, microphthalmia‐associated transcription factor‐dependent regulatory element in the intronic duplication causing hair greying and melanoma in horses

Greying with age in horses is an autosomal dominant trait, characterized by hair greying, high incidence of melanoma and vitiligo-like depigmentation. Previous studies have revealed that the causative mutation for this phenotype is a 4.6-kb intronic duplication in STX17 (Syntaxin 17). By using reporter constructs in transgenic zebrafish, we show that a construct containing two copies of the duplicated sequence acts as a strong enhancer in neural crest cells and has subsequent melanophore-specific activity during zebrafish embryonic development whereas a single copy of the duplicated sequence acts as a weak enhancer, consistent with the phenotypic manifestation of the mutation in horses. We further used luciferase assays to investigate regulatory regions in the duplication, to reveal tissue-specific activities of these elements. One region upregulated the reporter gene expression in a melanocyte-specific manner and contained two microphthalmia-associated transcription factor (MITF) binding sites, essential for the activity. Microphthalmia-associated transcription factor regulates melanocyte development, and these binding sites are outstanding candidates for mediating the melanocyte-specific activity of the element. These results provide strong support for the causative nature of the duplication and constitute an explanation for the melanocyte-specific effects of the Grey allele.

Association of TP53 Intron 3, 16 bp Duplication Polymorphism With Esophageal and Gastric Cancer Susceptibility in Kashmir Valley

TP53 is one of the major tumor suppressor genes, which is essential for the preservation of genome integrity. Different polymorphic variants of the p53 gene have been demonstrated for their association with several human malignancies. Of these, 16 base pair (bp) duplication in intron 3 of the TP53 gene (PIN3 Ins16bp, rs17878362) is the most extensively studied variant. However, no studies have, so far, investigated the association of this polymorphism with esophageal and gastric cancers. Thus, we aimed to investigate the association of PIN3 Ins16bp polymorphism with esophageal cancer (EC) and gastric cancer (GC) in Kashmir Valley, a northern part of India, where incidence of these cancers is very high. In addition, we also tested other covariates such as smoking/tea consumption as potential confounding factors. We analyzed DNA samples from a total of 243 patients (135 EC and 108 GC patients) and 195 healthy controls for PIN3 Ins16bp polymorphisms using PCR. Data were statistically analyzed using chi-square test and logistic regression models. Results showed that carriers for the PIN3 Ins16bp allele (A2) were associated with increased risk for both EC (OR = 2.31, 95% CI = 1.08-4.97, p = 0.03) and GC (OR = 2.91, 95% CI = 1.28-6.63, p = 0.01). Also, in a recessive model, our results showed that PIN3 Ins16bp A2A2 allele was conferring significant high risk for both EC (OR = 2.18, 95% CI = 1.03-4.59, p = 0.04) and GC (OR = 2.87, 95% CI = 1.29-6.42, p = 0.010). Although smoking (Hukka) and high consumption of salted tea are significant risk factors for both EC and GC, interaction of PIN3 Ins16bp genotypes with these factors did not further modulate the risk of EC and GC. Determination of PIN3 A2A2 genotype may provide a useful genetic marker in predicating high-risk individuals for the development of EC and GC and an early diagnosis.

Molecular Insight into the Synergism between the Minor Allele of Human Liver Peroxisomal Alanine:Glyoxylate Aminotransferase and the F152I Mutation

Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe(152) variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe(152) variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5'-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200-1400-fold lower. Thus, Phe(152) is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp(108), as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.

Recurrent CNVs Disrupt Three Candidate Genes in Schizophrenia Patients

Schizophrenia is a severe psychiatric disease with complex etiology, affecting approximately 1% of the general population. Most genetics studies so far have focused on disease association with common genetic variation, such as single-nucleotide polymorphisms (SNPs), but it has recently become apparent that large-scale genomic copy-number variants (CNVs) are involved in disease development as well. To assess the role of rare CNVs in schizophrenia, we screened 54 patients with deficit schizophrenia using Affymetrix's GeneChip 250K SNP arrays. We identified 90 CNVs in total, 77 of which have been reported previously in unaffected control cohorts. Among the genes disrupted by the remaining rare CNVs are MYT1L, CTNND2, NRXN1, and ASTN2, genes that play an important role in neuronal functioning but--except for NRXN1--have not been associated with schizophrenia before. We studied the occurrence of CNVs at these four loci in an additional cohort of 752 patients and 706 normal controls from The Netherlands. We identified eight additional CNVs, of which the four that affect coding sequences were found only in the patient cohort. Our study supports a role for rare CNVs in schizophrenia susceptibility and identifies at least three candidate genes for this complex disorder.