ß-catenin attenuation leads to up-regulation of activating NKG2D ligands and tumor regression in BrafV600E-driven thyroid cancer cells.

Introduction: BRAFV600E mutations frequently occur in papillary thyroid cancer (PTC). ß-catenin, encoded by CTNNB1, is a key downstream component of the canonical Wnt signaling pathway and is often overexpressed in PTC. BRAFV600E-driven PTC tumors rely on Wnt/ß-catenin signaling to sustain growth and progression. Methods: In the present study, we investigated the tumorigenicity of thyroid cancer cells derived from BRAFV600E PTC mice following Ctnnb1 ablation (BVE-Ctnnb1null). Results: Remarkably, the tumorigenic potential of BVE-Ctnnb1null tumor cells was lost in nude mice. Global gene expression analysis of BVE-Ctnnb1null tumor cells showed up-regulation of NKG2D receptor activating ligands (H60a, H60b, H60c, Raet1a, Raet1b, Raet1c, Raet1d, Raet1e, and Ulbp1) and down-regulation of inhibitory MHC class I molecules H-2L and H-2K2 in BVE-Ctnnb1null tumor cells. In vitro cytotoxicity assay demonstrated that BVE-Ctnnb1wt tumor cells were resistant to NK cell-mediated cytotoxicity, whereas BVE-Ctnnb1null tumor cells were sensitive to NK cell-mediated killing. Furthermore, the overexpression of any one of these NKG2D ligands in the BVE-Ctnnb1wt cell line resulted in a significant reduction of tumor growth in nude mice. Conclusions: Our results indicate that active ß-catenin signaling inhibits NK cell-mediated immune responses against thyroid cancer cells. Targeting the ß-catenin signaling pathway may have significant therapeutic benefits for BRAF-mutant thyroid cancer by not only inhibiting tumor growth but also enhancing host immune surveillance.

Established and candidate transthyretin amyloidosis variants identified in the Saudi population by data mining.

BACKGROUND: Familial transthyretin (TTR) amyloidosis (ATTR) is an autosomal dominant disease with significant phenotypic heterogeneity. Its prevalence in Saudi Arabia has not previously been investigated. An existing exome variant database of Saudi individuals, sequenced to globally investigate rare diseases in the population, was mined for TTR variants and filtered for missense mutations resulting in single amino acid changes. A total of 13,906 Saudi exomes from unrelated individuals were analyzed blindly. RESULTS: Three TTR variants known to be associated with ATTR amyloidosis were identified. Additionally, three novel TTR mutations were identified. Structural analysis of the three novel variants suggests that at least two could be amyloidogenic. The most common variant associated with amyloidosis was p.Val142Ile (allele frequency 0.001). Further investigation of these variants and their translation to clinical practice may help to diagnose, monitor, and manage patients with ATTR amyloidosis. CONCLUSION: Multiple TTR variants potentially associated with systemic ATTR amyloidosis were identified in the Saudi population. Early diagnosis and intervention, facilitated by familial genetic testing of patients with ATTR amyloidosis, may benefit in the management of this disease. Early diagnosis could be enhanced through inclusion of ATTR variants in existing population-based screening programs.

ß-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAFV600E-Driven Thyroid Cancer Animal Model.

BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). ß-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAF V600E-driven tumors have been speculated to rely on Wnt/ß-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of ß-catenin in BrafV600E -driven thyroid cancer in a transgenic mouse model. In Braf V600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of ß-catenin was observed in thyroid tumors. In Braf V600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFß pathways and loss of epithelial-mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual ß-catenin/KDM4A inhibitor PKF118-310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo These findings indicate that ß-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/ß-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.

Mutation spectrum of EXT1 and EXT2 in the Saudi patients with hereditary multiple exostoses.

BACKGROUND: Hereditary Multiple Exostoses (HME), also known as Multiple Osteochondromas (MO) is a rare genetic disorder characterized by multiple benign cartilaginous bone tumors, which are caused by mutations in the genes for exostosin glycosyltransferase 1 (EXT1) and exostosin glycosyltransferase 2 (EXT2). The genetic defects have not been studied in the Saudi patients. AIM OF STUDY: We investigated mutation spectrum of EXT1 and EXT2 in 22 patients from 17 unrelated families. METHODS: Genomic DNA was extracted from peripheral leucocytes. The coding regions and intron-exon boundaries of both EXT1 and EXT2 genes were screened for mutations by PCR-sequencing analysis. Gross deletions were analyzed by MLPA analysis. RESULTS: EXT1 mutations were detected in 6 families (35%) and 3 were novel mutations: c.739G > T (p. E247*), c.1319delG (p.R440Lfs*4), and c.1786delA (p.S596Afs*25). EXT2 mutations were detected in 7 families (41%) and 3 were novel mutations: c.541delG (p.D181Ifs*89), c.583delG (p.G195Vfs*75), and a gross deletion of approximately 10 kb including promoter and exon 1. Five patients from different families had no family history and carried de novo mutations (29%, 5/17). No EXT1 and EXT2 mutations were found in the remaining four families. In total, EXT1 and EXT2 mutations were found in 77% (13/17) of Saudi HME patients. CONCLUSION: EXT1 and EXT2 mutations contribute significantly to the pathogenesis of HME in the Saudi population. In contrast to high mutation rate in EXT 1 (65%) and low mutation rate in EXT2 (25%) in other populations, the frequency of EXT2 mutations are much higher (41%) and comparable to that of EXT1 among Saudi patients. De novo mutations are also common and the six novel EXT1/EXT2 mutations further expands the mutation spectrum of HME.

Mutation of SGK3, a Novel Regulator of Renal Phosphate Transport, Causes Autosomal Dominant Hypophosphatemic Rickets.

CONTEXT: Hypophosphatemic rickets (HR) is a group of rare hereditary renal phosphate wasting disorders caused by mutations in PHEX, FGF23, DMP1, ENPP1, CLCN5, SLC9A3R1, SLC34A1, or SLC34A3. OBJECTIVE: A large kindred with 5 HR patients was recruited with dominant inheritance. The study was undertaken to investigate underlying genetic defects in HR patients. DESIGN: Patients and their family members were initially analyzed for PHEX and FGF23 mutations using polymerase chain reaction sequencing and copy number analysis. Exome sequencing was subsequently performed to identify novel candidate genes. RESULTS: PHEX and FGF23 mutations were not detected in the patients. No copy number variation was observed in the genome using CytoScan HD array analysis. Mutations in DMP1, ENPP1, CLCN5, SLC9A3R1, SLC34A1, or SLC34A3 were also not found by exome sequencing. A novel c.979-96 T>A mutation in the SGK3 gene was found to be strictly segregated in a heterozygous pattern in patients and was not present in normal family members. The mutation is located 1 bp downstream of a highly conserved adenosine branch point, resulted in exon 13 skipping and in-frame deletion of 29 amino acids, which is part of the protein kinase domain and contains a Thr-320 phosphorylation site that is required for its activation. Protein tertiary structure modelling showed significant structural change in the protein kinase domain following the deletion. CONCLUSIONS: The c.979-96 T>A splice mutation in the SGK3 gene causes exon 13 skipping and deletion of 29 amino acids in the protein kinase domain. The SGK3 mutation may cause autosomal dominant HR.

Pattern Recognition Receptor Polymorphisms as Predictors of Oxaliplatin Benefit in Colorectal Cancer.

BACKGROUND: Constitutional loss of function (LOF) single nucleotide polymorphisms (SNPs) in pattern recognition receptors FPR1, TLR3, and TLR4 have previously been reported to predict oxaliplatin benefit in colorectal cancer. Confirmation of this association could substantially improve patient stratification. METHODS: We performed a retrospective biomarker analysis of the Short Course in Oncology Therapy (SCOT) and COIN/COIN-B trials. Participant status for LOF variants in FPR1 (rs867228), TLR3 (rs3775291), and TLR4 (rs4986790/rs4986791) was determined by genotyping array or genotype imputation. Associations between LOF variants and disease-free survival (DFS) and overall survival (OS) were analyzed by Cox regression, adjusted for confounders, using additive, dominant, and recessive genetic models. All statistical tests were two-sided. RESULTS: Our validation study populations included 2929 and 1948 patients in the SCOT and COIN/COIN-B cohorts, respectively, of whom 2728 and 1672 patients had functional status of all three SNPs determined. We found no evidence of an association between any SNP and DFS in the SCOT cohort, or with OS in either cohort, irrespective of the type of model used. This included models for which an association was previously reported for rs867228 (recessive model, multivariable-adjusted hazard ratio [HR] for DFS in SCOT = 1.19, 95% confidence interval [CI] = 0.99 to 1.45, P = .07; HR for OS in COIN/COIN-B = 0.92, 95% CI = 0.63 to 1.34, P = .66), and rs4986790 (dominant model, multivariable-adjusted HR for DFS in SCOT = 0.86, 95% CI = 0.65 to 1.13, P = .27; HR for OS in COIN/COIN-B = 1.08, 95% CI = 0.90 to 1.31, P = .40). CONCLUSION: In this prespecified analysis of two large clinical trials, we found no evidence that constitutional LOF SNPs in FPR1, TLR3, or TLR4 are associated with differential benefit from oxaliplatin. Our results suggest these SNPs are unlikely to be clinically useful biomarkers.

Genome-wide association study and meta-analysis in Northern European populations replicate multiple colorectal cancer risk loci.

Genome-wide association studies have been successful in elucidating the genetic basis of colorectal cancer (CRC), but there remains unexplained variability in genetic risk. To identify new risk variants and to confirm reported associations, we conducted a genome-wide association study in 1,701 CRC cases and 14,082 cancer-free controls from the Finnish population. A total of 9,068,015 genetic variants were imputed and tested, and 30 promising variants were studied in additional 11,647 cases and 12,356 controls of European ancestry. The previously reported association between the single-nucleotide polymorphism (SNP) rs992157 (2q35) and CRC was independently replicated (p = 2.08 × 10-4 ; OR, 1.14; 95% CI, 1.06-1.23), and it was genome-wide significant in combined analysis (p = 1.50 × 10-9 ; OR, 1.12; 95% CI, 1.08-1.16). Variants at 2q35, 6p21.2, 8q23.3, 8q24.21, 10q22.3, 10q24.2, 11q13.4, 11q23.1, 14q22.2, 15q13.3, 18q21.1, 20p12.3 and 20q13.33 were associated with CRC in the Finnish population (false discovery rate < 0.1), but new risk loci were not found. These results replicate the effects of multiple loci on the risk of CRC and identify shared risk alleles between the Finnish population isolate and outbred populations.

LGMD1D myopathy with cytoplasmic and nuclear inclusions in a Saudi family due to DNAJB6 mutation.

Autosomal dominant LGMD1D has been described in multiple families in Asia, Europe, and USA. However, to the best of our knowledge, no cases of LGMD1D have been reported among native Bedouin Saudi families. Fifty Saudi families with LGMD were analyzed and the causative underlying genes were studied utilizing genome wide linkage, homozygosity mapping, and neurological gene panel. We identified one family of a Bedouin origin with LGMD1D. Two patients had progressive proximal and distal weakness, dysphagia, and respiratory symptoms. Creatinine kinase was normal. Muscle biopsy showed marked variation in myofibers size with scattered angular atrophic fiber, necrotic fibers, and myophagocytosis, with red-rimmed vacuoles depicting a sarcoplasmic body. Heterozygous c.C287T (p.P96L) variant in exon 5 of DNAJB6 (NM_005494) gene was found. This change is localized within glycine and phenylalanine rich domain and alter an amino acid residue. Our findings will expand on the existing genotypic and phenotypic spectrum of this disorder and aid in elucidating hidden mechanisms implicated in LGMD1D.

Pro-inflammatory fatty acid profile and colorectal cancer risk: A Mendelian randomisation analysis.

BACKGROUND: While dietary fat has been established as a risk factor for colorectal cancer (CRC), associations between fatty acids (FAs) and CRC have been inconsistent. Using Mendelian randomisation (MR), we sought to evaluate associations between polyunsaturated (PUFA), monounsaturated (MUFA) and saturated FAs (SFAs) and CRC risk. METHODS: We analysed genotype data on 9254 CRC cases and 18,386 controls of European ancestry. Externally weighted polygenic risk scores were generated and used to evaluate associations with CRC per one standard deviation increase in genetically defined plasma FA levels. RESULTS: Risk reduction was observed for oleic and palmitoleic MUFAs (OROA = 0.77, 95% CI: 0.65-0.92, P = 3.9 × 10-3; ORPOA = 0.36, 95% CI: 0.15-0.84, P = 0.018). PUFAs linoleic and arachidonic acid had negative and positive associations with CRC respectively (ORLA = 0.95, 95% CI: 0.93-0.98, P = 3.7 × 10-4; ORAA = 1.05, 95% CI: 1.02-1.07, P = 1.7 × 10-4). The SFA stearic acid was associated with increased CRC risk (ORSA = 1.17, 95% CI: 1.01-1.35, P = 0.041). CONCLUSION: Results from our analysis are broadly consistent with a pro-inflammatory FA profile having a detrimental effect in terms of CRC risk.

Cyp24a1 Attenuation Limits Progression of BrafV600E -Induced Papillary Thyroid Cancer Cells and Sensitizes Them to BRAFV600E Inhibitor PLX4720.

CYP24A1, the primary inactivating enzyme for vitamin D, is often overexpressed in human cancers, potentially neutralizing the antitumor effects of calcitriol, the active form of vitamin D. However, it is unclear whether CYP24A1 expression serves as a functional contributor versus only a biomarker for tumor progression. In this study, we investigated the role of CYP24A1 on malignant progression of a murine model of BrafV600E -induced papillary thyroid cancer (PTC). Mice harboring wild-type Cyp24a1 (BVECyp24a1-wt) developed PTC at 5 weeks of age. Mice harboring a homozygous deletion of Cyp24a1 (BVECyp24a1-null) exhibited a 4-fold reduction in tumor growth. Notably, we found the tumorigenic potential of BVECyp24a1-null-derived tumor cells to be nearly abolished in immunocompromised nude mice. This phenotype was associated with downregulation of the MAPK, PI3K/Akt, and TGFß signaling pathways and a loss of epithelial-mesenchymal transition (EMT) in BVECyp24a1-null cells, associated with downregulation of genes involved in EMT, tumor invasion, and metastasis. While calcitriol treatment did not decrease cell proliferation in BVECyp24a1-null cells, it strengthened antitumor responses to the BRAFV600E inhibitor PLX4720 in both BVECyp24a1-null and BVECyp24a1-wt cells. Our findings offer direct evidence that Cyp24a1 functions as an oncogene in PTC, where its overexpression activates multiple signaling cascades to promote malignant progression and resistance to PLX4720 treatment. Cancer Res; 77(8); 2161-72. ©2017 AACR.

Mendelian randomisation implicates hyperlipidaemia as a risk factor for colorectal cancer.

While elevated blood cholesterol has been associated with an increased risk of colorectal cancer (CRC) in observational studies, causality is uncertain. Here we apply a Mendelian randomisation (MR) analysis to examine the potential causal relationship between lipid traits and CRC risk. We used single nucleotide polymorphisms (SNPs) associated with blood levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) as instrumental variables (IV). We calculated MR estimates for each risk factor with CRC using SNP-CRC associations from 9,254 cases and 18,386 controls. Genetically predicted higher TC was associated with an elevated risk of CRC (odds ratios (OR) per unit SD increase = 1.46, 95% confidence interval [CI]: 1.20-1.79, p = 1.68 × 10-4 ). The pooled ORs for LDL, HDL, and TG were 1.05 (95% CI: 0.92-1.18, p = 0.49), 0.94 (95% CI: 0.84-1.05, p = 0.27), and 0.98 (95% CI: 0.85-1.12, p = 0.75) respectively. A genetic risk score for 3-hydoxy-3-methylglutaryl-coenzyme A reductase (HMGCR) to mimic the effects of statin therapy was associated with a reduced CRC risk (OR = 0.69, 95% CI: 0.49-0.99, p = 0.046). This study supports a causal relationship between higher levels of TC with CRC risk, and a further rationale for implementing public health strategies to reduce the prevalence of hyperlipidaemia.

A first-line diagnostic assay for limb-girdle muscular dystrophy and other myopathies.

BACKGROUND: Fifty random genetically unstudied families (limb-girdle muscular dystrophy (LGMD)/myopathy) were screened with a gene panel incorporating 759 OMIM genes associated with neurological disorders. Average coverage of the CDS and 10 bp flanking regions of genes was 99 %. All families were referred to the Neurosciences Clinic of King Faisal Specialist Hospital and Research Centre, Saudi Arabia. Patients presented with muscle weakness affecting the pelvic and shoulder girdle. Muscle biopsy in all cases showed dystrophic or myopathic changes. Our main objective was to evaluate a neurological gene panel as a first-line diagnostic test for LGMD/myopathies. RESULTS: Our panel identified the mutation in 76 % of families (38/50; 11 novel). Thirty-four families had mutations in LGMD-related genes with four others having variants not typically associated with LGMD. The majority of cases had recessive inheritance with homoallelic pathogenic variants (97.4 %, 37/38), as expected considering the high rate of consanguinity in the study population. In one case, we detected a heterozygous mutation in DNAJB responsible for LGMD-1E. Our cohort included seven different subtypes of LGMD2. Mutations of DYSF were the most commonly identified cause of disease followed by that in CAPN3 and FKRP. Non-LGMD myopathies were due to mutations in genes associated with congenital disorder of glycosylation (ALG2), rigid spine muscular dystrophy 1 (SEPN1), inclusion body myopathy2/Nonaka myopathy (GNE), and neuropathy (WNK1). Whole exome sequencing (WES) of patients who remained undiagnosed with the neurological panel did not improve our diagnostic yield. CONCLUSIONS: Our neurological panel achieved a high clinical sensitivity (76 %) and is an effective first-line laboratory test in patients with LGMD and other myopathies. This sensitive, cost-effective, and rapid assay significantly assists clinical practice especially in these phenotypically and genetically heterogeneous disorders. Moreover, the application of the American College of Medical Genetics (ACMG) and Association for Molecular Pathology (AMP) guidelines applied in the classification of variant pathogenecity provides a clear interpretation for physicians on the relevance of such findings.

Mendelian randomisation analysis strongly implicates adiposity with risk of developing colorectal cancer.

BACKGROUND: Observational studies have associated adiposity with an increased risk of colorectal cancer (CRC). However, such studies do not establish a causal relationship. To minimise bias from confounding we performed a Mendelian randomisation (MR) analysis to examine the relationship between adiposity and CRC. METHODS: We used SNPs associated with adult body mass index (BMI), waist-hip ratio (WHR), childhood obesity and birth weight as instrumental variables in a MR analysis of 9254 CRC cases and 18 386 controls. RESULTS: In the MR analysis, the odds ratios (ORs) of CRC risk per unit increase in BMI, WHR and childhood obesity were 1.23 (95% CI: 1.02-1.49, P=0.033), 1.59 (95% CI: 1.08-2.34, P=0.019) and 1.07 (95% CI: 1.03-1.13, P=0.018), respectively. There was no evidence for association between birth weight and CRC (OR=1.22, 95% CI: 0.89-1.67, P=0.22). Combining these data with a concurrent MR-based analysis for BMI and WHR with CRC risk (totalling to 18 190 cases, 27 617 controls) provided increased support, ORs for BMI and WHR were 1.26 (95% CI: 1.10-1.44, P=7.7 × 10(-4)) and 1.40 (95% CI: 1.14-1.72, P=1.2 × 10(-3)), respectively. CONCLUSIONS: These data provide further evidence for a strong causal relationship between adiposity and the risk of developing CRC highlighting the urgent need for prevention and treatment of adiposity.

Variation at 2q35 (PNKD and TMBIM1) influences colorectal cancer risk and identifies a pleiotropic effect with inflammatory bowel disease.

To identify new risk loci for colorectal cancer (CRC), we conducted a meta-analysis of seven genome-wide association studies (GWAS) with independent replication, totalling 13 656 CRC cases and 21 667 controls of European ancestry. The combined analysis identified a new risk association for CRC at 2q35 marked by rs992157 (P = 3.15 × 10-8, odds ratio = 1.10, 95% confidence interval = 1.06-1.13), which is intronic to PNKD (paroxysmal non-kinesigenic dyskinesia) and TMBIM1 (transmembrane BAX inhibitor motif containing 1). Intriguingly this susceptibility single-nucleotide polymorphism (SNP) is in strong linkage disequilibrium (r2 = 0.90, D' = 0.96) with the previously discovered GWAS SNP rs2382817 for inflammatory bowel disease (IBD). Following on from this observation we examined for pleiotropy, or shared genetic susceptibility, between CRC and the 200 established IBD risk loci, identifying an additional 11 significant associations (false discovery rate [FDR]) < 0.05). Our findings provide further insight into the biological basis of inherited genetic susceptibility to CRC, and identify risk factors that may influence the development of both CRC and IBD.

IL-12 immunotherapy of Braf(V600E)-induced papillary thyroid cancer in a mouse model.

Papillary thyroid carcinoma (PTC) accounts for >80% thyroid malignancies, and BRAF(V600E) mutation is frequently found in >40% PTC. Interleukin-12 (IL-12) is a proinflammatory heterodimeric cytokine with strong antitumor activity. It is not known whether IL-12 immunotherapy is effective against Braf(V600E)-induced PTC. In the present study, we investigated the effectiveness of IL-12 immunotherapy against Braf(V600E)-induced PTC in LSL-Braf(V600E)/TPO-Cre mice. LSL-Braf(V600E)/TPO-Cre mice were created for thyroid-specific expression of Braf(V600E) under the endogenous Braf promoter, and spontaneous PTC developed at about 5 weeks of age. The mice were subjected to two treatment regimens: (1) weekly intramuscular injection of 50 µg plasmid DNA expressing a single-chain IL-12 fusion protein (scIL-12/CMVpDNA), (2) daily intraperitoneal injection of mouse recombinant IL-12 protein (mrIL-12, 100 ng per day). The role of T cells, natural killer (NK) cells, and transforming growth factor-ß (TGF-ß) in IL-12-mediated antitumor effects was determined by a (51)Cr-release cytotoxicity assay. Tumor size and weight were significantly reduced by either weekly intramuscular injection of scIL-12/CMVpDNA or daily intraperitoneal injection of mrIL-12, and tumor became more localized. Survival was significantly increased when treatment started at 1 week of age as compared with that at the 6 weeks of age. Both NK and CD8(+) T cells were involved in the cytotoxicity against tumor cells and their antitumor activity was significantly reduced in tumor-bearing mice. TGF-ß also inhibited the antitumor activity of NK and CD8(+) T cells. The immune suppression was completely reversed by IL-12 treatment and partially recovered by anti-TGF-ß antibody. We conclude that both IL-12 gene therapy and recombinant protein therapy are effective against PTC. Given that the immune response is significantly suppressed in tumor-bearing mice and can be restored by IL-12, the current study raises a possibility of the application of IL-12 as an adjuvant therapy for thyroid cancer.

KRAS(G12D)-mediated oncogenic transformation of thyroid follicular cells requires long-term TSH stimulation and is regulated by SPRY1.

KRAS(G12D) can cause lung cancer rapidly, but is not sufficient to induce thyroid cancer. It is not clear whether long-term serum thyroid stimulating hormone (TSH) stimulation can promote KRAS(G12D)-mediated thyroid follicular cell transformation. In the present study, we investigated the effect of long-term TSH stimulation in KRAS(G12D) knock-in mice and the role of Sprouty1 (SPRY1) in KRAS(G12D)-mediated signaling. We used TPO-KRAS(G12D) mice for thyroid-specific expression of KRAS(G12D) under the endogenous KRAS promoter. Twenty TPO-KRAS(G12D) mice were given anti-thyroid drug propylthiouracil (PTU, 0.1% w/v) in drinking water to induce serum TSH and 20 mice were without PTU treatment. Equal number of wild-type littermates (TPO-KRAS(WT)) was given the same treatment. The expression of SPRY1, a negative regulator of receptor tyrosine kinase (RTK) signaling, was analyzed in both KRAS(G12D)-and BRAF(V600E)-induced thyroid cancers. Without PTU treatment, only mild thyroid enlargement and hyperplasia were observed in TPO-KRAS(G12D) mice. With PTU treatment, significant thyroid enlargement and hyperplasia occurred in both TPO-KRAS(G12D) and TPO-KRAS(WT) littermates. Thyroids from TPO-KRAS(G12D) mice were six times larger than TPO-KRAS(WT) littermates. Distinct thyroid histology was found between TPO-KRAS(G12D) and TPO-KRAS(WT) mice: thyroid from TPO-KRAS(G12D) mice showed hyperplasia with well-maintained follicular architecture whereas in TPO-KRAS(WT) mice this structure was replaced by papillary hyperplasia. Among 10 TPO-KRAS(G12D) mice monitored for 14 months, two developed follicular thyroid cancer (FTC), one with pulmonary metastasis. Differential SPRY1 expression was demonstrated: increased in FTC and reduced in papillary thyroid cancer (PTC). The increased SPRY1 expression in FTC promoted TSH-RAS signaling through PI3K/AKT pathway whereas downregulation of SPRY1 by BRAF(V600E) in PTC resulted in both MAPK and PI3K/AKT activation. We conclude that chronic TSH stimulation can enhance KRAS(G12D)-mediated oncogenesis, leading to FTC. SPRY1 may function as a molecular switch to control MAPK signaling and its downregulation by BRAF(V600E) favors PTC development.

A new GWAS and meta-analysis with 1000Genomes imputation identifies novel risk variants for colorectal cancer.

Genome-wide association studies (GWAS) of colorectal cancer (CRC) have identified 23 susceptibility loci thus far. Analyses of previously conducted GWAS indicate additional risk loci are yet to be discovered. To identify novel CRC susceptibility loci, we conducted a new GWAS and performed a meta-analysis with five published GWAS (totalling 7,577 cases and 9,979 controls of European ancestry), imputing genotypes utilising the 1000 Genomes Project. The combined analysis identified new, significant associations with CRC at 1p36.2 marked by rs72647484 (minor allele frequency [MAF] = 0.09) near CDC42 and WNT4 (P = 1.21 × 10(-8), odds ratio [OR] = 1.21 ) and at 16q24.1 marked by rs16941835 (MAF = 0.21, P = 5.06 × 10(-8); OR = 1.15) within the long non-coding RNA (lncRNA) RP11-58A18.1 and ~500 kb from the nearest coding gene FOXL1. Additionally we identified a promising association at 10p13 with rs10904849 intronic to CUBN (MAF = 0.32, P = 7.01 × 10(-8); OR = 1.14). These findings provide further insights into the genetic and biological basis of inherited genetic susceptibility to CRC. Additionally, our analysis further demonstrates that imputation can be used to exploit GWAS data to identify novel disease-causing variants.

Clinical and genetic features of anoctaminopathy in Saudi Arabia.

OBJECTIVE: Characterization of the phenotypic, pathological, radiological, and genetic findings in 2 Saudi Arabian families with anoctaminopathies, and limb girdle muscular dystrophy type 2L (LGMD2L). METHODS: Over a 2-year period from December 2010 to January 2013, the clinical presentations were analyzed and all genes responsible for limb girdle muscular dystrophy (LGMD) were screened in families seen at King Faisal Specialist Hospital and Research Centre, a tertiary care hospital in Riyadh, Saudi Arabia. Out of 66 families with LGMD, we identified 2 families (3.1%) with anoctaminopathy, ANO5 muscular dystrophy. RESULTS: In the first case, a man presented with asymmetrical calves` muscles weakness and atrophy, which was first noted at age 39. The creatinine kinase (CK) level was >20x normal, muscle biopsy showed necrotizing myopathic changes, and an MRI of the legs showed fatty-tissue replacement to muscle tissue with volume loss involving the gastrocnemius and soleus muscles in an asymmetrical fashion. Minimal disease progression was noted over 18 years of follow up. Exercise induced recurrent rhabdomyolysis was noted over the last 2 years. A novel ANO5 gene mutation (Arg58Trp) was found. In the second family, a male presented at the age of 41 with asymptomatic hyperCkemia and intermittent dyspnea. Over 10 years follow up, he became disabled with muscle cramps, rhabdomyolysis, my oglobinurea, and difficulty ambulating. Muscle biopsy showed necrotizing myopathy and perivascular and interstitial amyloid deposit in skeletal muscle. A homozygous deletion of 11.9 Kb encompassing exon 13 to exon 17 was found in the ANO5 gene. Full cardiac investigations were normal in both patients. CONCLUSION: The prevalence of LGMD2L is approximately 3.1% in a Saudi Arabian native LGMD cohort. Slowly progressive, late onset, and asymmetrical weakness was the salient features in these 2 families. The genetic findings were novel and will add to the spectrum of ANO5 known mutations.