NOVEL VDR MUTATIONS IN PATIENTS WITH VITAMIN D-DEPENDENT RICKETS TYPE 2A: A MILD DISEASE PHENOTYPE CAUSED BY A NOVEL CANONICAL SPLICE-SITE MUTATION.

Objective: Vitamin D-dependent rickets type 2A (VDDR2A) is a rare autosomal recessive disorder caused by mutations in the vitamin D receptor gene (VDR), leading to end-organ resistance to 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). The objective of this study was to investigate VDR mutations in 11 patients from 8 Turkish-Arab families. Methods: All coding exons and intron-exon boundaries of the VDR gene were amplified by polymerase chain reaction from peripheral leukocyte DNA and sequenced. The effect of splice-site mutations on mRNA splicing was evaluated by a customized VDR mini-gene assay. Results: Homozygous VDR mutations were found in all the patients, including four novel mutations: c.473G>T (p.R158L), c.1-4A>G (IVS3-2A>G), c.755+1G>T, and c.352_356delGACAG (p.D118Sfs*7). The c.1-4A>G mutation was located in the canonical splice acceptor site and 4 base pairs from the original ATG start codon. The mutation resulted in both complete (60% of transcripts) and partial exon 4 skipping (15% of transcripts). The latter was due to activation of a cryptic splice acceptor site and did not disrupt the open reading frame. Both c.755+1G>T and c.352_356delGACAG resulted in frameshifts and a premature stop codon. Clinically, all the patients required continued treatment, except for patient IV-3, who presented with alopecia, hypocalcemia, and increased 1,25(OH)2D3 at 1.5 years of age as a result of the c.1-4A>G mutation. He stopped taking medication at 6 years of age and still maintained normal height and biochemical profile. Conclusion: We have identified four novel VDR mutations. Although canonical splice-site mutations cause premRNA splicing errors that usually lead to a severe disease phenotype, mild disease can also occur due to activation of a cryptic splice site. Abbreviations: 1,25(OH)2D3 = 1,25-dihydroxyvitamin D3 (calcitriol); 25OHD3 = 25-hydroxyvitamin D3; PCR = polymerase chain reaction; PTH = parathyroid hormone; VDDR2A = vitamin D-dependent rickets type 2A; VDR = vitamin D receptor.

Phenotype heterogeneity of congenital adrenal hyperplasia due to genetic mosaicism and concomitant nephrogenic diabetes insipidus in a sibling.

BACKGROUND: Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder caused by mutations in the CYP21A2. Congenital nephrogenic diabetes insipidus (NDI) is a rare X-linked recessive or autosomal recessive disorder caused by mutations in either AVPR2 or AQP2. Genotype-phenotype discordance caused by genetic mosaicism in CAH patients has not been reported, nor the concomitant CAH and NDI. CASE PRESENTATION: We investigated a patient with concomitant CAH and NDI from a consanguineous family. She (S-1) presented with clitoromegaly at 3 month of age, and polydipsia and polyuria at 13 month of age. Her parents and two elder sisters (S-2 and S-3) were clinically normal, but elevated levels of serum 17-hydroxyprogesterone (17-OHP) were observed in the mother and S-2. The coding region of CYP21A2 and AQP2 were analyzed by PCR-sequencing analysis to identify genetic defects. Two homozygous CYP21A2 mutations (p.R357W and p.P454S) were identified in the proband and her mother and S-2. The apparent genotype-phenotype discordance was due to presence of small amount of wild-type CYP21A2 alleles in S-1, S-2, and their mother's genome, thus protecting them from development of classic form of 21OHD (C21OHD). A homozygous AQP2 mutation (p.A147T) was also found in the patient. The patient was treated with hydrocortisone and hydrochlorothiazide. Her symptoms were improved with normal laboratory findings. The clitoromegaly is persisted. CONCLUSIONS: Genetic mosaicism is a novel mechanism contributing to the genotype-phenotype discordance in 21OHD and small percentage of wild-type CYP21A2 alleles may be sufficient to prevent phenotype development. This is a first report of concurrent 21OHD and NDI caused by simultaneous homozygous CYP21A2 and AQP2 mutations.

Mutational analysis of PHEX, FGF23 and CLCN5 in patients with hypophosphataemic rickets.

CONTEXT: Hypophosphataemic rickets (HR) is a group of rare hereditary renal phosphate wasting disorders caused by mutations in PHEX, FGF23, DMP1, ENPP1, CLCN5 or SLC34A3. OBJECTIVE: To investigate underlying genetic defects in patients with hypophosphataemic rickets. METHODS: We analysed genomic DNA from nine unrelated families for mutations in the entire coding region of PHEX, FGF23, DMP1, ENPP1, CLCN5 or SLC34A3 by PCR sequencing and copy number analysis. RESULTS: A total of 14 patients were studied. PHEX mutations were identified in 12 patients from seven families. Five of them were novel mutations present in eight patients: c.154G>T (p.E52*), c.401_402insGCCAAA (p.Q134_K135insPK), c.1600C>T (p.P534S), g.22016715_22056805del (40-kb deletion including promoter and exons 1-3) and c.2242_2243delCT (p.L748 fs*48). Four patients had previously reported mutations: c.1768+1G>A and c.1807G>A (p.W602*). Novel CLCN5 (c.1205G>A, p.W402*) and FGF23 (c.526C>G, p.R176G) mutations were found in two patients from the remaining two families. Many of the mutations were de novo: c.154G>T and c.2242_2243delCT in PHEX and c.526C>G in FGF23. Furthermore, we characterized the breakpoint of the novel PHEX g.22016715_22056805del and the c.2242_2243delCT, which is 6 bp from the stop codon, resulting in a frameshift and extension of the reading frame by 42 amino acids. CONCLUSIONS: Novel and de novo mutations are frequent and PHEX mutations are still the most common genetic defects in the Turkish population. Gene copy number analysis should be considered in patients with negative results by conventional PCR-based sequencing analysis. The current study further expands the mutation spectrum underlying HR.

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.

Increased CYP24A1 expression is associated with BRAF(V600E) mutation and advanced stages in papillary thyroid carcinoma.

OBJECTIVE: 1α, 25(OH)2 D3 (calcitriol), the active form of vitamin D, has been shown to exert antiproliferative effects in many cancers. Overexpression of CYP24A1, the primary vitamin D-inactivating enzyme, is also observed in a variety of human cancers, thus potentially neutralizing the antitumour effect of 1α, 25(OH)2 D3. This study investigates the expression of CYP24A1 and the effect of BRAF(V600E) on its expression in thyroid cancer. METHODS: We investigated 60 papillary thyroid carcinoma (PTC) specimens for CYP24A1 expression and its association with BRAF mutation and disease progression. CYP24A1 expression was measured by real-time RT-PCR, and BRAF(V600E) mutation was detected by PCR-DNA sequencing analysis. The interaction between BRAF(V600E) and CYP24A1 expression was determined by Western blot analysis and real-time RT-PCR. RESULTS: CYP24A1 expression was increased in PTC as compared to benign multinodular goitre. The expression was further increased in stage III and IV tumours. There is a strong correlation between CYP24A1 overexpression and BRAF(V600E) mutation (P < 0·01). In thyroid cancer cell lines expressing BRAF(V600E) , CYP24A1 expression was significantly higher when compared to those without BRAF(V600E) expression. BRAF(V600E) transgene expression in CAL62 cell line can induce CYP24A1 expression. Furthermore, BRAF(V600E) inhibitor PLX4720 can significantly down-regulate CYP24A1 expression and enhance the antiproliferative effects of calcitriol in thyroid cancer cell lines. CONCLUSION: CYP24A1 overexpression is a poor prognostic indicator for PTC and may reflect BRAF(V600E) mutation and MARK activation. The crosstalk between vitamin D and MAPK signalling pathways results in resistance to calcitriol-mediated antitumour effects, and the resistance can be reversed by BRAF(V600E) inhibitor PLX4720.

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.

Clinical and genetic analysis of patients with vitamin D-dependent rickets type 1A.

CONTEXT: Vitamin D-dependent rickets type 1A (VDDR-IA, OMIM 264700) is a rare autosomal recessive disorder and is caused by mutations in the CYP27B1 gene. OBJECTIVES: We aim to investigate CYP27B1 mutation in seven patients from four separate families and characterize the genotype-phenotype correlation. METHODS: The entire coding region of the CYP27B1 gene was sequenced, and genotype-phenotype correlation among patients was assessed. RESULTS: Sequencing analysis identified biallelic CYP27B1 mutations in all patients and monoallelic mutations in their parents. One patient from the first family was compound heterozygous for c.1166G>A (p.Arg389His) and a novel nonsense mutation c.1079 C>A (p.Ser360*). Two patients from the second family were homozygous for a novel splice donor site mutation in intron 1 (c.195 + 2 T>G), causing partial retention of the intron and a shift in the reading frame. Both novel mutations lead to the complete loss of vitamin D1α-hydroxylase activity. Four patients from families 3 and 4 were homozygous for a previously reported duplication mutation in exon 8 (1319-1325dupCCCACCC, Phe443Profs*24). Interestingly, one patient who was presented with severe hypocalcaemia and seizures at 4 months of age as a result of Phe443Profs*24 has improved spontaneously since 11 years of age and does not need regular treatment. Her laboratory tests showed normal serum calcium and 1,25(OH)(2) D after refusing to take medication for 12 months. CONCLUSIONS: There is a good genotype-phenotype correlation in VDDR-IA. However, some patients may recover from the loss of CYP27B1 function, probably due to 1α-hydroxylase activity exerted by a non-CYP27B1 enzyme.

Molecular characterization of a novel p.R118C mutation in the insulin receptor gene from patients with severe insulin resistance.

CONTEXT: Mutations of the insulin receptor gene (INSR) can cause genetic syndromes associated with severe insulin resistance. OBJECTIVES: We aimed to analyse INSR mutations in Saudi patients with severe insulin resistance. DESIGN: Ten patients with Type A insulin resistance syndrome from five unrelated Saudi families were investigated. The entire coding region of INSR was sequenced. The founder effect was assessed by microsatellite haplotype analysis. The functional effect of the mutation was investigated by in vitro functional assays. RESULTS: A novel biallelic c.433 C>T (p.R118C) mutation was detected in all patients. The c.433 C>T (p.R118C) sequence variation was not found in 100 population controls. The arginine residue at position 118 is located in the ligand-binding domain of INSR and is highly conserved across species. Microsatellite haplotype analysis of these patients indicated that p.R118C was a founder mutation created approximately 2900 years ago. The wild-type and mutant (R118C) INSR were cloned and expressed in CHO cells for functional analysis. Specific insulin binding to the mutant receptor was reduced by 83% as compared to wild-type (WT), although the mutant receptor was processed and expressed on the cell surface. Insulin-mediated receptor autophosphorylation was also significantly reduced in CHO(R118C) cells. CONCLUSIONS: Biallelic c.433 C>T (p.R118C) mutation of INSR causes significant damage to insulin binding and insulin-mediated signal transduction. p.R118C is a founder mutation frequently present in the Saudi patients with severe insulin resistance.

Insights of Noncanonical Splice-site Variants on RNA Splicing in Patients With Congenital Hypothyroidism.

CONTEXT: Congenital hypothyroidism (CH) is caused by mutations in the genes for thyroid hormone synthesis. In our previous investigation of CH patients, approximately 53% of patients had mutations in either coding exons or canonical splice sites of causative genes. Noncanonical splice-site variants in the intron were detected but their pathogenic significance was not known. OBJECTIVE: This work aims to evaluate noncanonical splice-site variants on pre-messenger RNA (pre-mRNA) splicing of CH-causing genes. METHODS: Next-generation sequencing data of 55 CH cases in 47 families were analyzed to identify rare intron variants. The effects of variants on pre-mRNA splicing were investigated by minigene RNA-splicing assay. RESULTS: Four intron variants were found in 3 patients: solute carrier family 26 member 4 (SLC26A4) c.1544+9C>T and c.1707+94C>T in one patient, and solute carrier family 5 member 5 (SLC5A5) c.970-48G>C and c.1652-97A>C in 2 other patients. The c.1707+94C>T and c.970-48G>C caused exons 15 and 16 skipping, and exon 8 skipping, respectively. The remaining variants had no effect on RNA splicing. Furthermore, we analyzed 28 previously reported noncanonical splice-site variants (4 in TG and 24 in SLC26A4). Among them, 15 variants (~ 54%) resulted in aberrant splicing and 13 variants had no effect on RNA splicing. These data were compared with 3 variant-prediction programs (FATHMM-XF, FATHMM-MKL, and CADD). Among 32 variants, FATHMM-XF, FATHMM-MKL, and CADD correctly predicted 20 (63%), 17 (53%), and 26 (81%) variants, respectively. CONCLUSION: Two novel deep intron mutations have been identified in SLC26A4 and SLC5A5, bringing the total number of solved families with disease-causing mutations to approximately 45% in our cohort. Approximately 46% (13/28) of reported noncanonical splice-site mutations do not disrupt pre-mRNA splicing. CADD provides highest prediction accuracy of noncanonical splice-site variants.

A novel deletion of the MEN1 gene in a large family of multiple endocrine neoplasia type 1 (MEN1) with aggressive phenotype.

CONTEXT: The MEN1 syndrome is associated with parathyroid, pancreatic and pituitary tumours and is caused by mutations in the MEN1 gene. In general, there is no genotype-phenotype correlation. OBJECTIVES: To characterize a large family with MEN1 with aggressive tumour behaviour: malignant pancreatic endocrine tumours were present in five affected subjects and were the presenting features in three subjects. DESIGN: The coding region of MEN1 was sequenced. Gene copy number analysis was performed by multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (aCGH). Loss of heterozygosity (LOH) in tumour tissue was studied by microsatellite analysis. Insulin-like growth factor II (IGF-II) and CDKN1C/p57KIP2 expression were investigated by immunohistochemistry. RESULTS: Mutation screening by conventional PCR sequence analysis of patients' peripheral blood DNA did not reveal any mutation in the MEN1 or CDKN1B gene. Gene copy number analysis by MLPA and aCGH demonstrated a novel monoallelic deletion of 5 kb genomic DNA involving the MEN1 promoter and exons 1 and 2. LOH analysis indicated somatic deletion of maternal chromosome 11, including MEN1 locus (11q13) and 11p15 imprinting control regions (ICR). Methylation analysis of ICR demonstrated ICR1 hypermethylation and ICR2 hypomethylation in the tumour specimens. ICR1 and ICR2 control the expression of IGF-2 and CDKN1C/p57KIP2, respectively. Immunohistochemistry showed that expression of paternally expressed IGF-2 was up-regulated and the maternally expressed CDKN1C/p57KIP2 was lost in the pancreatic endocrine tumours. CONCLUSIONS: Gene copy number analysis by MLPA should be considered in patients with negative conventional mutation screening. Although large MEN1 deletion causes MEN1, disruption of imprinted CDKN1C/p57KIP2 and IGF-2 gene expression may contribute to tumour progression and aggressive phenotype.

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.

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.

Aberrant BRAF splicing as an alternative mechanism for oncogenic B-Raf activation in thyroid carcinoma.

Activating BRAF mutations have recently been reported in 28-83% of papillary thyroid carcinomas (PTCs). However, it is not known whether aberrant BRAF splicing occurs in thyroid carcinoma. To investigate aberrant BRAF splicing and its association with BRAF mutation in thyroid tumours, we studied aberrant BRAF splicing and BRAF mutation from 68 thyroid tumours. BRAF(V600E) mutation was detected in 20 of 43 PTCs and all three anaplastic thyroid carcinomas (ATCs). There is a higher frequency of BRAF mutation in PTC patients with stage III and IV tumours compared with stage I and II. Novel BRAF splicing variants were detected in 12 PTCs, three follicular variants of PTC (FVPTCs), and one ATC, as well as in two thyroid carcinoma cell lines, ARO and NPA. These variants did not have the N-terminal auto-inhibitory domain of wild-type B-Raf, resulting in an in-frame truncated protein that contained only the C-terminal kinase domain and caused constitutive activation of B-Raf. These variants were significantly associated with advanced disease stage and BRAF(V600E) mutation (p < 0.001, Fisher exact test). Furthermore, expression of these variants in NIH3T3 and CHO cells could activate the MAP kinase signalling pathway, transform them in vitro, and induce tumours in nude mice. These data suggest that BRAF splicing variants may function as an alternative mechanism for oncogenic B-Raf activation. Combination of the BRAF(V600E) mutation and its splicing variants may contribute towards disease progression to poorly differentiated thyroid carcinoma.

Molecular Analysis of CYP27B1 Mutations in Vitamin D-Dependent Rickets Type 1A: c.590G > A (p.G197D) Missense Mutation Causes a RNA Splicing Error.

CONTEXT: Vitamin D-dependent rickets type 1A (VDDR1A) is a rare autosomal recessively inherited disorder due to loss-of-function mutations in the CYP27B1 gene. CYP27B1 encodes an enzyme of 25-hydroxyvitamin D-1α-hydroxylase for converting inactive 25-OHD to biologically active 1,25-(OH)2D. OBJECTIVE: To identify underlying genetic defects in patients with VDDR1A. METHODS: Twelve patients from 7 Turkish and 2 Saudi families were investigated. The coding exons and intron-exon boundaries of the CYP27B1 gene were amplified by Polymerase Chain Reaction (PCR) from peripheral lymphocyte DNA. PCR products were directly sequenced. The consequences of c.590G > A mutation were analyzed by in silico and functional analysis. RESULTS: CYP27B1 mutations were identified in all the patients. Two novel mutations were identified in two separate families: c.171delG (family 7) and c.398_400dupAAT (family 8). The intra-exon deletion of c.171delG resulted in a frameshift and premature stop codon 20 amino acids downstream from the mutation (p.L58Cfs∗20). The intra-exon duplication of c.398_400dupAAT generated a premature stop codon at the mutation site (p.W134∗). A missense c.590G > A (p.G197D) mutation was found in a patient from family 4 and caused a defect in pre-mRNA splicing. As a result, two populations of transcripts were detected: the majority of them with intron 3 retention (83%), and the minority (17%) being properly spliced transcripts with about 16% of wild-type enzymatic activity. The remaining nine patients from six families carried a previously reported c.1319_1325dupCCCACCC (F443Pfs∗24) mutation. Clinically, all the patients need continued calcitriol treatment, which was consistent with inactivation of 25-hydroxy vitamin D1α-hydroxylase activity. CONCLUSION: Two novel frameshift CYP27B1 mutations were identified and predicted to inactivate 25-hydroxyvitamin D-1α-hydroxylase. The loss of enzymatic activity by c.590G > A missense mutation was mainly caused by aberrant pre-mRNA splicing.

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.

Functional analysis of 22 splice-site mutations in the PHEX, the causative gene in X-linked dominant hypophosphatemic rickets.

CONTEXT: X-linked hypophosphatemic rickets (XLH) is caused by inactivating mutations in the PHEX gene and is the most common form of hereditary rickets. The splice-site mutations account for 17% of all reported PHEX mutations. The functional consequence of these splice-site mutations has not been systemically investigated. OBJECTIVE: The current study was undertaken to functionally annotate previously reported 22 splice-site mutations in the PHEX gene. METHODS: PHEX mini-genes with different splice-site mutations were created by site-directed mutagenesis and expressed in HEK293 cells. The mRNA transcripts were analyzed by RT-PCR, cloning, and sequencing. RESULTS: These splicing mutations led to a variety of consequences, including exon skipping, intron retention, and activation of cryptic splice sites. Among 22 splice-site mutations, exon skipping was the most common event accounting for 73% (16/22). Non-canonical splice-site mutations could result in splicing errors to the same extent as canonical splice-site mutations such as c.436+3G>C, c.436+4A>C, c.436+6T>C, c.437-3C>G, c.850-3C>G, c.1080-3C>A, c.1482+5G>C, c.1586+6T>C, c.1645+5G>A, c.1645+6T>C, c.1701-16T>A, c.1768+5G>A, and c.1899+5G>A. Interestingly, non-canonical (c.436+6T>C and c.1586+6T>C) and canonical splice-site mutations (c.1769-1G>C) could generate partial splicing errors (both wild-type and mutant transcripts were detected), resulting in incomplete inactivation of PHEX gene, which may explain the mild disease phenotype reported previously, providing evidence of genotype-phenotype correlation. c.1645C>T (p.R549*) had no impact on pre-mRNA splicing although it is located next to canonical splice donor site GT. CONCLUSIONS: Exon skipping is the most common outcome due to splice-site mutations. Both canonical and non-canonical splice-site mutations can result in either severe or mild RNA splicing defects, contributing to phenotype heterogeneity. Non-canonical splice-site mutations should not be overlooked in genetic screening especially those located within 50 bp from canonical splice site.

Molecular Analysis of Congenital Hypothyroidism in Saudi Arabia: SLC26A7 Mutation Is a Novel Defect in Thyroid Dyshormonogenesis.

Context: Congenital hypothyroidism (CH) is the most common neonatal endocrine disorder, affecting one in 3000 to 4000 newborns. Since the introduction of a newborn screening program in 1988, more than 300 cases have been identified. The underlying genetic defects have not been systematically studied. Objective: To identify the mutation spectrum of CH-causing genes. Methods: Fifty-five patients from 47 families were studied by next-generation exome sequencing. Results: Mutations were identified in 52.7% of patients (29 of 55) in the following 11 genes: TG, TPO, DUOX2, SLC26A4, SLC26A7, TSHB, TSHR, NKX2-1, PAX8, CDCA8, and HOXB3. Among 30 patients with thyroid dyshormonogenesis, biallelic TG mutations were found in 12 patients (40%), followed by biallelic mutations in TPO (6.7%), SLC26A7 (6.7%), and DUOX2 (3.3%). Monoallelic SLC26A4 mutations were found in two patients, one of them coexisting with two tandem biallelic deletions in SLC26A7. In 25 patients with thyroid dysgenesis, biallelic mutations in TSHR were found in six patients (24%). Biallelic mutations in TSHB, PAX 8, NKX2-1, or HOXB3 were found once in four different patients. A monoallelic CDCA8 mutation was found in one patient. Most mutations were novel, including three TG, two TSHR, and one each in DUOX2, TPO, SLC26A7, TSHB, NKX2-1, PAX8, CDCA8, and HOXB3. SLC26A7 and HOXB3 were novel genes associated with thyroid dyshormonogenesis and dysgenesis, respectively. Conclusions: TG and TSHR mutations are the most common genetic defects in Saudi patients with CH. The prevalence of other disease-causing mutations is low, reflecting the consanguineous nature of the population. SLC26A7 mutations appear to be associated with thyroid dyshormonogenesis.

Clinical and genetic characteristics of 15 families with hereditary hypophosphatemia: Novel Mutations in PHEX and SLC34A3.

BACKGROUND: Hereditary hypophosphatemia is a group of rare renal phosphate wasting disorders. The diagnosis is based on clinical, radiological, and biochemical features, and may require genetic testing to be confirmed. METHODOLOGY: Clinical features and mutation spectrum were investigated in patients with hereditary hypophosphatemia. Genomic DNA of 23 patients from 15 unrelated families were screened sequentially by PCR-sequencing analysis for mutations in the following genes: PHEX, FGF23, DMP1, ENPP1, CLCN5, SLC34A3 and SLC34A1. CytoScan HD Array was used to identify large deletions. RESULTS: Genetic evaluation resulted in the identification of an additional asymptomatic but intermittent hypophosphatemic subject. Mutations were detected in 21 patients and an asymptomatic sibling from 13 families (86.6%, 13/15). PHEX mutations were identified in 20 patients from 12 families. Six of them were novel mutations present in 9 patients: c.983_987dupCTACC, c.1586+2T>G, c.1206delA, c.436+1G>T, c.1217G>T, and g.22,215,887-22,395,767del (179880 bp deletion including exon 16-22 and ZNF645). Six previously reported mutations were found in 11 patients. Among 12 different PHEX mutations, 6 were de novo mutations. Patients with de novo PHEX mutations often had delayed diagnosis and significantly shorter in height than those who had inherited PHEX mutations. Novel compound heterozygous mutations in SLC34A3 were found in one patient and his asymptomatic sister: c.1335+2T>A and c.1639_1652del14. No mutation was detected in two families. CONCLUSIONS: This is the largest familial study on Turkish patients with hereditary hypophosphatemia. PHEX mutations, including various novel and de novo variants, are the most common genetic defect. More attention should be paid to hypophosphatemia by clinicians since some cases remain undiagnosed both during childhood and adulthood.

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.