Intronic Deletion Research Articles

The genomic architecture of NLRP7 is Alu rich and predisposes to disease-associated large deletions.

NLRP7 is a major gene responsible for recurrent hydatidiform moles. Here, we report 11 novel NLRP7 protein truncating variants, of which five deletions of more than 1-kb. We analyzed the transcriptional consequences of four variants. We demonstrate that one large homozygous deletion removes NLRP7 transcription start site and results in the complete absence of its transcripts in a patient in good health besides her reproductive problem. This observation strengthens existing data on the requirement of NLRP7 only for female reproduction. We show that two other variants affecting the splice acceptor of exon 6 lead to its in-frame skipping while another variant affecting the splice donor site of exon 9 leads to an in-frame insertion of 54 amino acids. Our characterization of the deletion breakpoints demonstrated that most of the breakpoints occurred within Alu repeats and the deletions were most likely mediated by microhomology events. Our data define a hotspot of Alu instability and deletions in intron 5 with six different breakpoints and rearrangements. Analysis of NLRP7 genomic sequences for repetitive elements demonstrated that Alu repeats represent 48% of its intronic sequences and these repeats seem to have been inserted into the common NLRP2/7 primate ancestor before its duplication into two genes.

Gene-Based Markers for the Tomato Yellow Leaf Curl Virus Resistance Gene Ty-3

The viral disease induced by Tomato yellow leaf curl virus (TYLCV) reduces tomato (Solanum lycopersicum) yield significantly in tropical and subtropical regions. A number of loci, including Ty-1 to Ty-5, conferring resistance to TYLCV have been described and introgressed into modern tomato cultivars. The availability of molecular markers linked to these genes would expedite the introgression of TYLCV resistance into commercial cultivars. In the present study, we developed gene-based markers linked to the Ty-3 gene using a segregating population derived from a cross between the TYLCV-resistant line S. lycopersicum ‘A45’ and the susceptible line S. lycopersicum ‘A39’. Agrobacterium-mediated screening was used to test TYLCV resistance of plants in the segregating population, and the resistance was evaluated by a visual scoring method and polymerase chain reaction analysis. By comparing sequences of the Ty-3 genes of the resistant and susceptible lines, two high-resolution melting (HRM) markers (Ty3-HRM1 and Ty3-HRM2) and one sequence characterized amplified region (SCAR) marker (Ty3-SCAR1) were developed. The HRM markers were based on single nucleotide polymorphisms at the 13th exon and the 15th intron, whereas the SCAR marker was based on a 246-bp deletion in the 16th intron. These gene-based markers will be useful tools for marker-assisted selection in breeding programs to improve TYLCV resistance of tomato.

Molecular analysis of the LDLR gene in coronary artery disease patients from the Indian population

BackgroundCardiovascular disease is a leading cause of mortality in Indian population. Mutations in LDLR, APOB and PCSK9 genes may lead to Familial Hypercholesterolemia, an autosomal dominant disorder which in turn leads to cardiovascular diseases. The primary objective of this study is to analyze these genes in CAD patients of Indian population. MethodsA total of 30 patients were selected out of 300 CAD patients based on UK-Simon Broome criteria from South India. The gDNA was isolated by organic extraction method and the exons and exon-intron boundaries of LDLR gene, APOB (exon 26) and PCSK9 (exon 7) were screened by PCR-high resolution melt analysis. The amplicons showing shift in melting pattern were sequenced to find out the variation. ResultsThis study reports three novel variations, an intronic deletion c.694+8_694+18del in intron 4, a synonymous variation c.966 C>T [p. (N322=)] in exon 7 and a deletion insertion c.1399_1340delinsTA [p. (T467Y)] in exon 10, two recurrent variations c.862G>A [p. (E288K)] in exon 6 and a splice site variation c.1845+2T>C in exon–intron junction of exon 12 in LDLR gene and PCSK9 gene had c.1180+17C>T change in intron 7. However there are no pathogenic variations in APOB and PCSK9 genes in Indian population. In silico analysis predicted all the variations as pathogenic except the synonymous variation. ConclusionThis report adds five new variations to the spectrum of LDLR variations in Indian population. This study also suggests that UK Simon Broom criteria can be followed to categorize FH patients in Indian population.

Functional Dissection of an Alternatively Spliced Herpesvirus Gene by Splice Site Mutagenesis.

Herpesviruses have large and complex DNA genomes. The largest among the herpesviruses, those of the cytomegaloviruses, include over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult, as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here, we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement for each isoform in MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87 isoform, but not the p33, p36, and p38 isoforms, is essential for viral replication in cell culture. Moreover, the presence of one of the two medium-size isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes. Herpesviruses include up to 170 genes in their DNA genomes. The functions of most viral gene products remain poorly defined. The construction of viral gene knockout mutants has thus been an important tool for functional analysis of viral proteins. However, this strategy is of limited use when viral gene transcripts are alternatively spliced, leading to the expression of several proteins from a single gene. In this study, we showed, as a proof of principle, that each protein product of an alternatively spliced gene can be eliminated individually by splice site mutagenesis. Mutant viruses lacking individual protein products displayed different phenotypes, demonstrating that the products of alternatively spliced genes have nonredundant functions.

Assessment of PAX6 alleles in 66 families with aniridia.

We report on PAX6 alleles associated with a clinical diagnosis of classical aniridia in 81 affected individuals representing 66 families. Allelic variants expected to affect PAX6 function were identified in 61 families (76 individuals). Ten cases of sporadic aniridia (10 families) had complete (8 cases) or partial (2 cases) deletion of the PAX6 gene. Sequence changes that introduced a premature termination codon into the open reading frame of PAX6 occurred in 47 families (62 individuals). Three individuals with sporadic aniridia (three families) had sequence changes (one deletion, two run-on mutations) expected to result in a C-terminal extension. An intronic deletion of unknown functional significance was detected in one case of sporadic aniridia (one family), but not in unaffected relatives. Within these 61 families, single nucleotide substitutions accounted for 30/61 (49%), indels for 23/61 (38%), and complete deletion of the PAX6 locus for 8/61 (13%). In five cases of sporadic aniridia (five families), no disease-causing mutation in the coding region was detected. In total, 23 unique variants were identified that have not been reported in the Leiden Open Variation Database (LOVD) database. Within the group assessed, 92% had sequence changes expected to reduce PAX6 function, confirming the primacy of PAX6 haploinsufficiency as causal for aniridia.

Discrepancy in compliance between the clinical and genetic diagnosis of choroidal hypoplasia in Danish Rough Collies and Shetland Sheepdogs.

Collie eye anomaly (CEA) is a congenital, inherited ocular disorder which is widespread in herding breeds. Clinically, the two major lesions associated with CEA are choroidal hypoplasia (CH) and coloboma, and both lesions are diagnosed based on ophthalmological examination. A 7.8-kb intronic deletion in the gene encoding non-homologous end-joining factor 1 (NHEJ1) has been reported to be the causative mutation underlying CH when present in the homozygous state. In this study, we have investigated the compliance between the clinical and genetic diagnosis of CH in the Danish Rough Collie and Shetland Sheepdog populations. Our results show that the deletion in NHEJ1 is not predictive for CH in the Danish Rough Collie population, whereas the clinical and genetic diagnosis is in accordance with each other in the Shetland Sheepdog population. Based on these results, it can be concluded that the intronic deletion in NHEJ1 is not the causative mutation but, rather, a marker linked to the locus underlying the trait in some populations but linked to both the wild-type and CH-causing locus in most dogs in the Danish Rough Collie population.

Extracellular matrix protein 1 gene (ECM1) mutations in nine Iranian families with lipoid proteinosis.

Background & objectives:Lipoid proteinosis (LP) is an autosomal recessive disease. Clinical characteristics of this disease are hoarse voice, scarring of the skin, brain calcifications, and eyelid papules (moniliform blepharosis). Mutations in the ECM1 gene on 1q21.2 are responsible for this disease. This study was conducted to investigate the mutation spectrum of ECM1 gene in nine Iranian families having at least one LP patient diagnosed clinically.Methods:The entire ECM1 gene was screened using PCR and direct sequencing in nine Iranian families with 12 suspected LP patients who were referred to the clinic, along with their parents and siblings. Thirty healthy individuals were included as controls.Results:In only one patient a homozygous G>A transition at nucleotide c.806 in exon 7 was detected. A G>A substitution at nucleotide 1243 in exon 8 that changes glycine (GGT) to serine (AGT) was observed in most of our patients. Furthermore, in one patient there was a change in the sequence of intron 8, the A>T transition in nucleotide 4307. In addition, in two cases (one patient and one healthy mother with affected child) there was a C (4249) deletion in intron 8.Interpretation & conclusions:Our results indicate that although mutation in ECM1gene is responsible for lipoid proteinosis, it is likely that this is not the only gene causing this disease and probably other genes may be involved in the pathogenesis of the LP disease.

Germline copy number variation analysis in Finnish families with hereditary prostate cancer

The inherited factors that predispose individuals to prostate cancer (PrCa) remain largely unknown. The aim of this study was to identify germline copy number variants (CNVs) in Finnish individuals that could contribute to an increased PrCa risk. Genome-wide CNV screening was performed by analyzing single nucleotide polymorphisms from 105 PrCa patients and 37 unaffected relatives, representing 31 Finnish hereditary PrCa (HPC) families. The CNVs that aggregated in affected individuals and overlapped with genes implicated in cancer were validated using quantitative PCR in 189 index patients from Finnish HPC families and in 476 controls. An intronic deletion (14.7 kb) in the EPHA3 gene coding for class A ephrin receptor was observed in 11.6% of PrCa patients and in 6.1% of controls. The deletion associated with an increased PrCa risk (P = 0.018, OR = 2.06, 95%CI = 1.18-3.61). Although incomplete segregation with affection status was observed, the results show that the deletion was overrepresented in PrCa patients (56.1%) when compared to unaffected male relatives (31.2%). Interestingly, PrCa-specific mortality was higher among EPHA3 deletion carriers (24.3%) than among patients with a normal EPHA3 copy number (3.4%). This study is the first investigation of the contribution of germline CNVs to HPC susceptibility in Finland. A novel association between the EPHA3 deletion and PrCa risk was observed and, if confirmed, screening for this variant may aid in risk stratification among HPC patients.

MG-123 Exonic and intronic NRXN1 deletions: Novel genotype-phenotype correlations

Objectives To 1) further characterise the extended phenotype of exonic NRXN1 deletions and 2) systematically evaluate the pathogenicity of intronic NRXN1 deletions in order to help inform clinical diagnostic practice. Methods We examined clinically ascertained cases from three Canadian cytogenetic laboratories for exonic NRXN1 deletions. Referring physicians completed a clinical checklist to identify major lifetime features and illnesses. One lab’s clinically referred cases were available to investigate the prevalence of intronic NRXN1 deletions in comparison to 15,254 controls. Genome-wide CNV data from high-resolution microarrays were investigated for the presence of additional rare variants. Results We identified 41 (0.21%) exonic NRXN1 deletions among 19,263 clinically referred cases, an over eight-fold increase compared to controls (p NRXN1 deletion cases included nine adults with comorbid intellectual disability and a psychiatric illness, movement disorders, automutilation, sleep disorders, and obsessions and preoccupations. The prevalence of congenital anomalies was low. The prevalence of intronic NRXN1 deletions did not differ between clinical cases (19/6,022; 0.32%) and controls (55/15,524; 0.35%). Additional pathogenic rare variants were four times more common in intronic (n = 6/19; 31.6%) compared to exonic (n = 3/41; 7.3%) NRXN1 deletion cases. Conclusions The expression of exonic NRXN1 deletions is primarily neuropsychiatric and may be associated with comorbid intellectual disability and a psychiatric illness in adulthood. To our knowledge this is the first study to demonstrate that the majority of intronic NRXN1 deletions by themselves are unlikely to cause clinical phenotypes, however further study on their potential functional impact is needed.

ROBO1 deletion as a novel germline alteration in breast and colorectal cancer patients

Despite one third of breast (BC) and colorectal cancer (CRC) cases having a hereditary component, only a small proportion can be explained by germline mutations. The aim of this study was to identify potential genomic alterations related to cancer predisposition. Copy number variations (CNVs) were interrogated in 113 unrelated cases fulfilling the criteria for hereditary BC/CRC and presenting non-pathogenic mutations in BRCA1, BRCA2, MLH1, MSH2, TP53, and CHEK2 genes. An identical germline deep intronic deletion of ROBO1 was identified in three index patients using two microarray platforms (Agilent 4x180K and Affymetrix CytoScan HD). The ROBO1 deletion was confirmed by quantitative PCR (qPCR). Six relatives were also evaluated by CytoScan HD Array. Genomic analysis confirmed a co-segregation of the ROBO1 deletion with the occurrence of cancer in two families. Direct sequencing revealed no pathogenic ROBO1 point mutations. Transcriptomic analysis (HTA 2.0, Affymetrix) in two breast carcinomas from a single patient revealed ROBO1 down-expression with no splicing events near the intronic deletion. Deeper in silico analysis showed several enhancer regions and a histone methylation mark in the deleted region. The ROBO1 deletion in a putative transcriptional regulatory region, its down-expression in tumor samples, and the results of the co-segregation analysis revealing the presence of the alteration in affected individuals suggest a pathogenic effect of the ROBO1 in cancer predisposition.

Mutations in Known Monogenic High Bone Mass Loci Only Explain a Small Proportion of High Bone Mass Cases.

ABSTRACTHigh bone mass (HBM) can be an incidental clinical finding; however, monogenic HBM disorders (eg, LRP5 or SOST mutations) are rare. We aimed to determine to what extent HBM is explained by mutations in known HBM genes. A total of 258 unrelated HBM cases were identified from a review of 335,115 DXA scans from 13 UK centers. Cases were assessed clinically and underwent sequencing of known anabolic HBM loci: LRP5 (exons 2, 3, 4), LRP4 (exons 25, 26), SOST (exons 1, 2, and the van Buchem's disease [VBD] 52‐kb intronic deletion 3′). Family members were assessed for HBM segregation with identified variants. Three‐dimensional protein models were constructed for identified variants. Two novel missense LRP5 HBM mutations ([c.518C>T; p.Thr173Met], [c.796C>T; p.Arg266Cys]) were identified, plus three previously reported missense LRP5 mutations ([c.593A>G; p.Asn198Ser], [c.724G>A; p.Ala242Thr], [c.266A>G; p.Gln89Arg]), associated with HBM in 11 adults from seven families. Individuals with LRP5 HBM (∼prevalence 5/100,000) displayed a variable phenotype of skeletal dysplasia with increased trabecular BMD and cortical thickness on HRpQCT, and gynoid fat mass accumulation on DXA, compared with both non‐LRP5 HBM and controls. One mostly asymptomatic woman carried a novel heterozygous nonsense SOST mutation (c.530C>A; p.Ser177X) predicted to prematurely truncate sclerostin. Protein modeling suggests the severity of the LRP5‐HBM phenotype corresponds to the degree of protein disruption and the consequent effect on SOST‐LRP5 binding. We predict p.Asn198Ser and p.Ala242Thr directly disrupt SOST binding; both correspond to severe HBM phenotypes (BMD Z‐scores +3.1 to +12.2, inability to float). Less disruptive structural alterations predicted from p.Arg266Cys, p.Thr173Met, and p.Gln89Arg were associated with less severe phenotypes (Z‐scores +2.4 to +6.2, ability to float). In conclusion, although mutations in known HBM loci may be asymptomatic, they only account for a very small proportion (∼3%) of HBM individuals, suggesting the great majority are explained by either unknown monogenic causes or polygenic inheritance. © 2015 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

OR14 Characterization of HLA class I new alleles with insertions and deletions in exons and introns

Recent advances in DNA sequencing technologies, the so-called Next Generation Sequencing technologies (NGS), introduced breakthroughs in deciphering the complex genetic information in all living species in large scale and affordable level. Since October 2014, we have been routinely using PacBio for HLA class I typing for resolving exon shuffling ambiguities and the new alleles. We have performed more than 5000 HLA-ABC 1 kb amplicon sequencing and typing on PacBio platform. Recently we also introduced full gene length typing −3.5 kb-8 exons and seven introns as routine typing method for the new alleles that have been detected but were not be able to be sequenced in isolation, especially the ones with insertions and deletion. In this study we will summarize characterization of several hundred new alleles with insertions and deletions in exons and introns using PacBio platform.

Collie eye anomaly: a review

Collie eye anomaly (CEA) is an inherited congenital visual impairment with heterogeneous signs. The first symptoms are already visible in the early embryo. Among the most affected breeds are Collies and Shetland Sheepdogs but the disease has spread to different breeds depending on the country of origin. Dogs affected with this disease share a 7.8 kb deletion in intron 4 of the NHEJ1 gene. Inheritance of this disease is autosomal reces - sive with incomplete penetrance. Thanks to a commercially available genetic test breeders can identify genetically affected recessive homozygotes and clinically healthy but genetic carriers of the mutation and thus select healthy parents for the next generation of dogs. However, the exact cause of the disease is not known and it is not known whether the causative mutation influences the occurrence of some other diseases (e.g. immunodeficiencies).

De novo deleterious genetic variations target a biological network centered on Aβ peptide in early-onset Alzheimer disease.

We hypothesized that de novo variants (DNV) might participate in the genetic determinism of sporadic early-onset Alzheimer disease (EOAD, onset before 65 years). We investigated 14 sporadic EOAD trios first by array-comparative genomic hybridization. Two patients carried a de novo copy number variation (CNV). We then performed whole-exome sequencing in the 12 remaining trios and identified 12 non-synonymous DNVs in six patients. The two de novo CNVs (an amyloid precursor protein (APP) duplication and a BACE2 intronic deletion) and 3/12 non-synonymous DNVs (in PSEN1, VPS35 and MARK4) targeted genes from a biological network centered on the Amyloid beta (Aβ) peptide. We showed that this a priori-defined genetic network was significantly enriched in amino acid-altering DNV, compared with the rest of the exome. The causality of the APP de novo duplication (which is the first reported one) was obvious. In addition, we provided evidence of the functional impact of the following three non-synonymous DNVs targeting this network: the novel PSEN1 variant resulted in exon 9 skipping in patient's RNA, leading to a pathogenic missense at exons 8-10 junction; the VPS35 missense variant led to partial loss of retromer function, which may impact neuronal APP trafficking and Aβ secretion; and the MARK4 multiple nucleotide variant resulted into increased Tau phosphorylation, which may trigger enhanced Aβ-induced toxicity. Despite the difficulty to recruit Alzheimer disease (AD) trios owing to age structures of the pedigrees and the genetic heterogeneity of the disease, this strategy allowed us to highlight the role of de novo pathogenic events, the putative involvement of new genes in AD genetics and the key role of Aβ network alteration in AD.

Identification and distribution of VERNALIZATION1 alleles in Chinese barley (Hordeum vulgare) germplasm

VERNALIZATION1 (VRN1) is a central regulator of the vernalization requirement, which affects the yield and geographical distribution of Triticeae crops through regulation of the flowering time. The first intron (Intron I) of VRN1 contains putative cis-elements that mediate repression of VRN1 expression. Allelic variations in Intron I are closely associated with increased VRN1 expression in vernalization-responsive accessions in the absence of vernalization. In this study, diagnostic molecular markers were developed to distinguish the main 12 known alleles, including a new allele, of barley (Hordeum vulgare) VRN1 (HvVRN1), and to investigate HvVRN1 alleles in 373 barley varieties from major agroecological regions in China. The new allele, HvVRN1-11, with a 3172-bp deletion in Intron I of HvVRN1, was identified in a Chinese barley landrace. Analysis of eight polymorphic sites flanking major variations in Intron I of HvVRN1 classified the 12 HvVRN1 alleles into five distinct groups. Compared with modern commercial barley, landraces possessed greater genetic diversity and contained the majority of HvVRN1 alleles, especially in the winter barley regions. HvVRN1 diversity in terms of allele number reduced significantly (P < 0.01) along with higher growing latitudes, but increased in accessions carrying the winter HvVRN2 allele. Wild-type HvVRN1 and the HvVRN1-8 allele showed the most widespread distribution in most regions whereas HvVRN1-1 & HvVRN1-8, and HvVRN1-5 are predominant in the Inner Mongolian Plateau spring barley region and the Northeast Plain spring barley region, respectively, with relatively higher latitudes. Compared with landraces, the frequency of HvVRN1-8 decreased while wild-type HvVRN1 increased significantly (P < 0.01) in commercial varieties. Our study classified HvVRN1 genetic diversity in a large collection of Chinese germplasm and provided a tool for identifying HvVRN1 alleles in barley breeding populations.

Natural variation of the expression pattern of the segmentation gene even-skipped in melanogaster

The evolution of canalized traits is a central question in evolutionary biology. Natural variation in highly conserved traits can provide clues about their evolutionary potential. Here we investigate natural variation in a conserved trait—even-skipped (eve) expression at the cellular blastoderm stage of embryonic development in Drosophila melanogaster. Expression of the pair-rule gene eve was quantitatively measured in three inbred lines derived from a natural population of D. melanogaster. One line showed marked differences in the spacing, amplitude and timing of formation of the characteristic seven-striped pattern over a 50-min period prior to the onset of gastrulation. Stripe 5 amplitude and the width of the interstripe between stripes 4 and 5 were both reduced in this line, while the interstripe distance between stripes 3 and 4 was increased. Engrailed expression in stage 10 embryos revealed a statistically significant increase in the length of parasegment 6 and a decrease in the length of parasegments 8 and 9. These changes are larger than those previously reported between D. melanogaster and D. pseudoobscura, two species that are thought to have diverged from a common ancestor over 25 million years ago. This line harbors a rare 448 bp deletion in the first intron of knirps (kni). This finding suggested that reduced Kni levels caused the deviant eve expression, and indeed we observed lower levels of Kni protein at early cycle 14A in L2 compared to the other two lines. A second of the three lines displayed an approximately 20% greater level of expression for all seven eve stripes. The three lines are each viable and fertile, and none display a segmentation defect as adults, suggesting that early-acting variation in eve expression is ameliorated by developmental buffering mechanisms acting later in development. Canalization of the segmentation pathway may reduce the fitness consequences of genetic variation, thus allowing the persistence of mutations with unexpectedly strong gene expression phenotypes.

Intronic deletions of tva receptor gene decrease the susceptibility to infection by avian sarcoma and leukosis virus subgroup A.

The group of avian sarcoma and leukosis virus (ASLV) in chickens contains six highly related subgroups, A to E and J. Four genetic loci, tva, tvb, tvc and tvj, encode for corresponding receptors that determine the susceptibility to the ASLV subgroups. The prevalence of ASLV in hosts may have imposed strong selection pressure toward resistance to ASLV infection, and the resistant alleles in all four receptor genes have been identified. In this study, two new alleles of the tva receptor gene, tvar5 and tvar6, with similar intronic deletions were identified in Chinese commercial broilers. These natural mutations delete the deduced branch point signal within the first intron, disrupting mRNA splicing of the tva receptor gene and leading to the retention of intron 1 and introduction of premature TGA stop codons in both the longer and shorter tva isoforms. As a result, decreased susceptibility to subgroup A ASLV in vitro and in vivo was observed in the subsequent analysis. In addition, we identified two groups of heterozygous allele pairs which exhibited quantitative differences in host susceptibility to ASLV-A. This study demonstrated that defective splicing of the tva receptor gene can confer genetic resistance to ASLV subgroup A in the host.

Identification of a novel MET mutation in high-grade glioma resulting in an auto-active intracellular protein.

MET has gained interest as a therapeutic target for a number of malignancies because of its involvement in tumorigenesis, invasion and metastasis. At present, a number of inhibitors, both antibodies against MET or its ligand hepatocyte growth factor, and small molecule MET tyrosine kinase inhibitors are in clinical trials. We here describe a novel variant of MET that is expressed in 6 % of high-grade gliomas. Characterization of this mutation in a glioma cell line revealed that it consists of an intronic deletion, resulting in a splice event connecting an intact splice donor site in exon 6 with the next splice acceptor site being that of exon 9. The encoded protein lacks parts of the extracellular IPT domains 1 and 2, encoded by exons 7 and 8, resulting in a novel pseudo-IPT and is named METΔ7−8. METΔ7−8 is located predominantly in the cytosol and is constitutively active. The auto-activating nature of METΔ7−8, in combination with a lack of transmembrane localization, renders METΔ7−8 not targetable using antibodies, although the protein is efficiently deactivated by MET-specific tyrosine kinase inhibitors. Testing of MET-expressing tumors for the presence of this variant may be important for treatment decision making.

250 Large Deletion in the EPCAM Gene Responsible for the Milder Phenotype of Congenital Tufting Enteropathy

Introduction: A number of point mutations within the EPCAM gene have been found to be responsible for congenital tufting enteropathy (CTE). We previously described a milder phenotype of this condition in a cohort of patients in the Maltese Islands1. EpCAM staining was negative in all patients in this cohort1, suggesting a defective EPCAM gene in the milder phenotype as well. Aims: To identify the underlying genetic abnormality within the EPCAM gene responsible for the milder phenotype of CTE. Methods: In the period 1985 2012, eight Maltese patients with CTE from six unrelated families were retrospectively identified and enrolled. Genomic DNA was extracted from peripheral blood. Primers for all nine exons within the EPCAM gene were designed and optimized. PCR products were amplified, checked on a gel, purified and sequenced. To sequence exon 4 exon 6 region, the PCR product was purified from the gel and ligated in a TA Vector. The ligated products were transformed into DH5α bacteria and cultured on ampicillin-containing agar plates. The cultured DNA was extracted, sequenced and analysed. All DNA sequences were compared with controls. Unaffected family members (parents) and healthy controls were screened for any mutation identified in the affected patients. Results: Genetic analysis of the EPCAM gene in Maltese CTE patients revealed a novel homozygous 1773bp deletion in seven out of eight patients, starting from 1170bp downstream of exon 4, up to 721bp upstream of exon 6, resulting in complete deletion of exon 5 and partial deletion of Intron 4-5 and Intron 5-6. The remaining patient was a heterozygote for the deletion. The mutant homozygous variant resulted in a frameshift, introducing 23 novel amino acids, followed by the formation of a premature stop codon, p.Ala164Metfs*24. This resulted in a predicted truncated protein with the loss of the transmembrane domain and thus the complete lack of EpCAM protein within the intestinal epithelium. Three sets of parents of 3 affected patients were all heterozygous for this deletion. Out of 100 healthy controls tested, 98 were homozygous wild type and two (2%) heterozygous for the deletion. Conclusions: This novel large deletion within the EPCAM gene was found to be responsible for the milder phenotype of CTE in seven Maltese patients. The heterozygous mutation in the remaining patient is likely to be compounded by a second mutation, which is as yet unidentified. The heterozygous mutation of the patients' parents confirms the autosomal recessive pattern of inheritance of CTE. This genetic deletion present in all patients implies a founder effect and we propose this as the first genotype-phenotype correlation in isolated intestinal TE patients.

Rapid Identification of Major QTLs Associated with Rice Grain Weight and Their Utilization

To uncover the genetics of rice grain weight, we constructed an RIL population derived from a cross between a large grain accession M201 and a small size variety JY293. Specific Locus Amplified Fragment Sequencing (SLAF-Seq) technology was used to genotype two bulked DNA pools made from individual DNA of the heaviest 30 lines and the lightest 30 lines according to the 1000 grain weight (TGW). Bulked segregant analysis (BSA) was used to identify SLAFs strongly associated with TGW. Two marker-intensive regions at 24,600,000–24,850,000 bp and 25,000,000–25,350,000 bp on chromosome 3 were identified tightly related to the TGW. Then a linkage map of chromosome 3 was constructed with SSR markers and some SLAF derived single nucleotide polymorphisms (SNPs). Quantitative trait locus (QTL) mapping for TGW, grain length, grain width, and grain thickness revealed one major QTL in the second hot-region and two other minor QTLs for grain weight. These three QTLs displayed hierarchical effects on grain length and grain weight in order of qTGW3.2 (qGL3) qTGW3.1 (GS3) qTGW3.3. Multiple comparisons of means among the eight combinations of 3 QTLs revealed that the lines with two of three QTLs deriving from M201 displayed a large grain weight phenotype (TGW 40.2g, average data of three years) and lines with both qTGW3.1 and qTGW3.3 alleles from M201 (42.5g) had similar grain weight to the qTGW3.2 (40.8g) alone. Two strategies with similar effectiveness were proposed to improve grain weight by marker-assisted selection (MAS). One is to introduce the novel qTGW3.2 allele alone, and the other is to pyramid qTGW3.1 and qTGW3.3 alleles together. One new allele of GS3 (39 bp deletion in intron 1) and two SNPs in coding sequence of qGL3 identified in this study from M201 are useful in pyramiding elite alleles for molecular breeding for improvement of rice yield.