WTX Mutations Research Articles

MDB-31. PRELIMINARY STUDY ON BIOLOGICAL SIGNIFICANCE OF WTX G435D MUTATION IN MEDULLOBLASTOMA

Abstract BACKGROUND: Previously, we reported a case of familial medulloblastoma without hereditary tumor syndrome. Through analysis of the whole genome sequencing of the family members, we found a common germline WTX (c.1863C>T, p.Gly435Asp) mutation in this family: cytosine (C) at position 63411863 on X chromosome was replaced by thymine (T), and the corresponding amino acid was glycine Gly (G) at position 435, which was converted to aspartic acid Asp (D). We conduct this study to explore the biological significance of this mutation in medulloblastoma cell lines. METHODS: Medulloblastoma cells (cell lines and primary cells) with stable WTX (c.1863C > T) hybrid mutation were contstrcuted by lentivirus transfection. The effects of WTXG435D on proliferation, invasion and chemosensitivity of medulloblastoma cells were investigated in vitro and in vivo. RESULTS: Proliferation and invasion ability of WTXG435D medulloblastoma cell were significantly weaker than WTX WT cells, and they were more sensitive to radiation and chemotherapy(cisplatin, vincristine) treatment in vitro. In vivo, animal experiments also showed that intracranial in situ tumors formed by WTXG435D medulloblastoma cells were more sensitive to radiotherapy and chemotherapy than in situ tumors formed by WTXWT ones, and the survival was longer. CONCLUSION: WTX (c.1863C>T, p. Gly435Asp) mutation could reduce the proliferation and invasion ability of medulloblastoma cells, and enhance the sensitivity of radiotherapy and chemotherapy. Which could be a potential therapeutic target for further investigation.

Somatic, Genetic and Epigenetic Changes in Nephrogenic Rests and Their Role in the Transformation to Wilms Tumors, a Systematic Review.

To review somatic genetic changes in nephrogenic rests (NR), which are considered to be precursor lesions of Wilms tumors (WT). This systematic review is written according to the PRISMA statement. PubMed and EMBASE were systematically searched for articles in the English language studying somatic genetic changes in NR between 1990 and 2022. Twenty-three studies were included in this review, describing 221 NR of which 119 were pairs of NR and WT. Single gene studies showed mutations in WT1 and WTX, but not CTNNB1 to occur in both NR and WT. Studies investigating chromosomal changes showed loss of heterozygosity of 11p13 and 11p15 to occur in both NR and WT, but loss of 7p and 16q occurred in WT only. Methylome-based studies found differential methylation patterns between NR, WT, and normal kidney (NK). Over a 30-year time frame, few studies have addressed genetic changes in NR, likely hampered by technical and practical limitations. A limited number of genes and chromosomal regions have been implicated in the early pathogenesis of WT, exemplified by their occurrence in NR, including WT1, WTX, and genes located at 11p15. Further studies of NR and corresponding WT are urgently needed.

Genetic changes associated with relapse in favorable histology Wilms tumor: A Children’s Oncology Group AREN03B2 study

SummaryOver the last decade, sequencing of primary tumors has clarified the genetic underpinnings of Wilms tumor but has not affected therapy, outcome, or toxicity. We now sharpen our focus on relapse samples from the umbrella AREN03B2 study. We show that over 40% of relapse samples contain mutations in SIX1 or genes of the MYCN network, drivers of progenitor proliferation. Not previously seen in large studies of primary Wilms tumors, DIS3 and TERT are now identified as recurrently mutated. The analysis of primary-relapse tumor pairs suggests that 11p15 loss of heterozygosity (and other copy number changes) and mutations in WT1 and MLLT1 typically occur early, but mutations in SIX1, MYCN, and WTX are late developments in some individuals. Most strikingly, 75% of relapse samples had gain of 1q, providing strong conceptual support for studying circulating tumor DNA in clinical trials to better detect 1q gain earlier and monitor response.

WT1-Mutant Wilms Tumor Progression Is Associated With Diverting Clonal Mutations of CTNNB1.

WT1-mutant Wilms tumors exhibit a high rate of concomitant CTNNB1 mutations, associated with activated Wnt signaling. Here, we show by laser and manual microdissection of different histologic cell types from 6 WT1-mutant tumor samples that 1 patient's tumor can contain up to 4 distinct mutations in CTNNB1 and/or WTX. Consecutive sections may also harbor different CTNNB1 mutations. The variability of activating CTNNB1 mutations demonstrates the multifocal nature of WT1-mutant Wilms tumors. As multiple independent tumors can occur in patients with constitutional WT1 mutations, they need to be surveyed more closely for tumor development.

Preclinical evaluation of XPO1 inhibition in Wilms tumors.

3580 Background: XPO1 is a nuclear export protein that selectively transports tumor and growth regulatory proteins out of the nucleus, thereby effectively inhibiting their function. We previously utilized the Virtual Inference of Protein-activity by Enriched Regulon analysis (VIPER) algorithm to discover that malignant rhabdoid tumors were dependent upon XPO1 inhibition and then evaluated a preclinical cohort using selinexor (KPT-330), the first-in-class selective inhibitor of nuclear export, to demonstrate that XPO1 inhibition was sufficient to cause cell cycle arrest, apoptosis, and disease control in multiple cell line and patient derived xenograft (PDXs) models. Our subsequent analysis revealed that the most common childhood kidney tumor, Wilms tumor, has even high higher inferred activity of XPO1 than rhabdoid tumors leading to our hypothesis that XPO1 inhibition is an effective therapeutic strategy to treat Wilms tumors. Methods: A panel of 9 Wilms tumor cell lines and 3 Wilms tumor PDXs were genomically characterized and tested to evaluate the pre-clinical efficacy of XPO1 inhibition in Wilms tumors. Results: Proliferation rate, increased XPO1 protein expression, and loss of function mutations in TP53 correlated with in vitro Wilms tumor cell line sensitivity to selinexor. Evaluation of co-segregation of all single nucleotide variant changes using with inferred activity of XPO1 on VIPER in all TGCA tumors demonstrates a strong association with TP53 alterations. XPO1 inhibition was effective in all Wilms tumor models tested, most significantly in MSKREN-57196, a favorable histology Wilms tumor PDX with somatic 1q gain as well as WTX and MYCN mutations, as well as in MSKREN-31827, a diffusely anaplastic TP53 mutant Wilms tumor PDX. Eltanexor (KPT-8602) is an XPO1 inhibitor with decreased CNS penetration and an improved toxicity profile; this drug was tested in these in vivo models and found to be at least as effective as selinexor. Conclusions: Somatic 1q gain in favorable histology Wilms tumors and TP53 mutations in diffusely anaplastic Wilms tumors have a particularly poor prognosis in the relapsed setting. Our study demonstrates that XPO1 inhibition may provide a rational therapeutic option to treat such high-risk Wilms tumors. Future clinical trials evaluating XPO1 inhibitors should evaluate its efficacy in children with relapsed Wilms tumors.

Zebrafish Wtx is a negative regulator of Wnt signaling but is dispensable for embryonic development and organ homeostasis.

The X-chromosomally linked gene WTX is a human disease gene and a member of the AMER family. Mutations in WTX are found in Wilms tumor, a form of pediatric kidney cancer and in patients suffering from OSCS (Osteopathia striata with cranial sclerosis), a sclerosing bone disorder. Functional data suggest WTX to be an inhibitor of the Wnt/β-catenin signaling pathway. Deletion of Wtx in mouse leads to perinatal death, impeding the analysis of its physiological role. To gain insights into the function of Wtx in development and homeostasis we have used zebrafish as a model and performed both knockdown and knockout studies using morpholinos and transcription activator-like effector nucleases (TALENs), respectively. Wtx knockdown led to increased Wnt activity and embryonic dorsalization. Also, wtx mutants showed a transient upregulation of Wnt target genes in the context of caudal fin regeneration. Surprisingly, however, wtx as well as wtx/amer2/amer3 triple mutants developed normally, were fertile and did not show any anomalies in organ maintenance. Our data show that members of the zebrafish wtx/amer gene family, while sharing a partially overlapping expression pattern do not compensate for each other. This observation demonstrates a remarkable robustness during development and regeneration in zebrafish.

Burden and Profile of Somatic Mutation in Duodenal Adenomas from Patients with Familial Adenomatous- and MUTYH-associated Polyposis

Purpose: Duodenal polyposis and cancer are important causes of morbidity and mortality in familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP). This study aimed to comprehensively characterize somatic genetic changes in FAP and MAP duodenal adenomas to better understand duodenal tumorigenesis in these disorders.Experimental Design: Sixty-nine adenomas were biopsied during endoscopy in 16 FAP and 10 MAP patients with duodenal polyposis. Ten FAP and 10 MAP adenomas and matched blood DNA samples were exome sequenced, 42 further adenomas underwent targeted sequencing, and 47 were studied by array comparative genomic hybridization. Findings in FAP and MAP duodenal adenomas were compared with each other and to the reported mutational landscape in FAP and MAP colorectal adenomas.Results: MAP duodenal adenomas had significantly more protein-changing somatic mutations (P = 0.018), truncating mutations (P = 0.006), and copy number variants (P = 0.005) than FAP duodenal adenomas, even though MAP patients had lower Spigelman stage duodenal polyposis. Fifteen genes were significantly recurrently mutated. Targeted sequencing of APC, KRAS, PTCHD2, and PLCL1 identified further mutations in each of these genes in additional duodenal adenomas. In contrast to MAP and FAP colorectal adenomas, neither exome nor targeted sequencing identified WTX mutations (P = 0.0017).Conclusions: The mutational landscapes in FAP and MAP duodenal adenomas overlapped with, but had significant differences to those reported in colorectal adenomas. The significantly higher burden of somatic mutations in MAP than FAP duodenal adenomas despite lower Spigelman stage disease could increase cancer risk in the context of apparently less severe benign disease. Clin Cancer Res; 23(21); 6721-32. ©2017 AACR.

Clinical, Pathologic, and Genetic Features of Wilms Tumors With WTX Gene Mutation.

Clinical and pathologic features of patients with WTX-mutated Wilms tumor (WT) have not been studied in detail. We characterize the clinical and pathologic findings in WT with WTX abnormalities and provide comparison with WT without WTX mutation. Clinical, gross, and microscopic features in 35 patients with WT were examined. Karyotype was examined in a subset of cases. All cases had been previously analyzed for WTX, WT1, and CTNNB1 aberrations via array comparative genomic hybridization; OncoMap 4 high throughput genotyping was performed on 18 cases. Eleven tumors had WTX abnormality. No significant differences were identified between patients with mutated versus nonmutated WTX with respect to gender (45% versus 33% male), age (mean 3.9 versus 4.1 years), tumor size (mean 12.7 cm versus 12.8 cm), anaplasia (9% versus 12%), rhabdomyoblastic differentiation (18% versus 8%), cartilage differentiation (9% versus 4%), mucinous epithelial differentiation (9% versus 4%), nephrogenic rests (28% versus 21%), or relapse rate (11% versus 25%). Mutations in KRAS, MYC, and PIK3R1 were restricted to WTX-mutated WT, mutations in AKT, CKDN2A, EFGR, HRAS, MET, and RET were restricted to WT without WTX mutation, and mutations in BRAF, CTTNB1, NRAS, PDGFRA, and STK11 were seen in both groups. Our study revealed no clinical or pathologic distinctions between WT with and without WTX abnormality. This similarity lends support to the concept of a common tumorigenic pathway between WT with aberrant WTX and those without.

Clinical, pathologic, and genetic features of Wilms tumors with WTX gene mutation

Clinical and pathologic features of patients with WTX-mutated Wilms tumor (WT) have not been studied in detail. We characterize the clinical and pathologic findings in WT with WTX abnormalities and provide comparison with WT without WTX mutation. Clinical, gross, and microscopic features in 35 patients with WT were examined. Karyotype was examined in a subset of cases. All cases had been previously analyzed for WTX, WT1, and CTNNB1 aberrations via array comparative genomic hybridization; OncoMap 4 high throughput genotyping was performed on 18 cases. Eleven tumors had WTX abnormality. No significant differences were identified between patients with mutated versus nonmutated WTX with respect to gender (45 versus 33% male), age (mean 3.9 versus 4.1 years), tumor size (mean 12.7 versus 12.8 cm), anaplasia (9 versus 12%), rhabdomyoblastic differentiation (18 versus 8%), cartilage differentiation (9 versus 4%), mucinous epithelial differentiation (9 versus 4%), nephrogenic rests (28 versus 21%), or relapse rate (11 versus 25%). Mutations in KRAS, MYC, and PIK3R1 were restricted to WTX-mutated WT, mutations in AKT, CKDN2A, EFGR, HRAS, MET, and RET were restricted to WT without WTX mutation, and mutations in BRAF, CTTNB1, NRAS, PDGFRA, and STK11 were seen in both groups. Our study revealed no clinical or pathologic distinctions between WT with and without WTX abnormality. This similarity lends support to the concept of a common tumorigenic pathway between WT with aberrant WTX and those without.

Adenoma development in familial adenomatous polyposis and MUTYH-associated polyposis: somatic landscape and driver genes.

Familial adenomatous polyposis (FAP) and MUTYH‐associated polyposis (MAP) are inherited disorders associated with multiple colorectal adenomas that lead to a very high risk of colorectal cancer. The somatic mutations that drive adenoma development in these conditions have not been investigated comprehensively. In this study we performed analysis of paired colorectal adenoma and normal tissue DNA from individuals with FAP or MAP, sequencing 14 adenoma whole exomes (eight MAP, six FAP), 55 adenoma targeted exomes (33 MAP, 22 FAP) and germline DNA from each patient, and a further 63 adenomas by capillary sequencing (41 FAP, 22 MAP). With these data we examined the profile of mutated genes, the mutational signatures and the somatic mutation rates, observing significant diversity in the constellations of mutated driver genes in different adenomas, and loss‐of‐function mutations in WTX (9%; p < 9.99e‐06), a gene implicated in regulation of the WNT pathway and p53 acetylation. These data extend our understanding of the early events in colorectal tumourigenesis in the polyposis syndromes. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Osteopathia striata congenita with cranial sclerosis and intellectual disability due to contiguous gene deletions involving the WTX locus

Osteopathia striata congenita with cranial sclerosis (OSCS) is a skeletal dysplasia caused by germline deletions of or truncating point mutations in the X-linked gene WTX (FAM123B, AMER1). Females present with longitudinal striations of sclerotic bone along the long axis of long bones and cranial sclerosis, with a high prevalence of cleft palate and hearing loss. Intellectual disability or neurodevelopmental delay is not observed in females with point mutations in WTX leading to OSCS. One female has been described with a deletion spanning multiple neighbouring genes suggesting that deletion of some neighbouring loci may result in abnormal neurodevelopment. In this cohort of 13 females with OSCS resulting from deletions of WTX, a relationship is observed where deletion of ARHGEF9 and/or MTMR8 in conjunction with WTX results in an additional neurodevelopmental phenotype whereas deletion of ASB12 along with WTX is associated with a good neurodevelopmental prognosis.

WT1, WTX and CTNNB1 mutation analysis in 43 patients with sporadic Wilms’ tumor

Wilms' tumor (WT) is a heterogeneous neoplasia characterized by a number of genetic abnormalities, involving tumor suppressor genes, oncogenes and genes related to the Wnt signaling pathway. Somatic biallelic inactivation of WT1 is observed in 5-10% of sporadic WT. Somatic mutations in exon 3 of CTNNB1, which encodes β-catenin, were initially observed in 15% of WT. WTX encodes a protein that negatively regulates the Wnt/β-catenin signaling pathway and mediates the binding of WT1. In this study, we screened germline and somatic mutations in selected regions of WT1, WTX and CTNNB1 in 43 WT patients. Mutation analysis of WT1 identified two single-nucleotide polymorphisms, one recurrent nonsense mutation (p.R458X) in a patient with proteinuria but without genitourinary findings of Denys-Drash syndrome (DDS) and one novel missense mutation, p.C428Y, in a patient with Denys-Drash syndrome phenotype. WT1 SNP rs16754A>G (R369R) was observed in 17/43 patients, and was not associated with significant difference in age at diagnosis distribution, or with 60-month overall survival rate. WTX mutation analysis identified five sequence variations, two synonymous substitutions (p.Q1019Q and p.D379D), a non-synonymous mutation (p.F159L), one frameshift mutation (p.157X) and a novel missense mutation, p.R560W. Two sequence variations in CTNNB1 were identified, p.T41A and p.S45C. Overall survival of bilateral cases was significantly lower (p=0.005). No difference was observed when survival was analyzed among patients with WT1 or with WTX mutations. On the other hand, the survival of two patients with the CTNNB1 p.T41A mutation was significantly lower (p=0.000517) than the average.

Osteopathia striata with cranial sclerosis and developmental delay in a male with a mosaic deletion in chromosome region Xq11.2

Osteopathia striata with cranial sclerosis (OSCS) is an X-linked disease caused by mutations involving WTX (FAM123B), a tumor suppressor protein with dual functions. OSCS typically affects females whereas males generally have fetal or neonatal lethality. Surviving affected males have characteristic facial dysmorphisms, skeletal features such as macrocephaly and short stature, neurodevelopmental disabilities and a high prevalence of neuromuscular anomalies. On imaging, hemizygous males display marked cranial and peripheral skeletal sclerosis without the metaphyseal striations that are seen in women with OSCS. Observations of striation in males may be indicative of a somatic mosaic mutation in WTX. To date only two cases of surviving males haves been confirmed with mosaic point mutations in WTX. We report on the first case of a male with a mosaic deletion of the entire WTX gene. We show that a mosaic deletion in a hemizygous male patient can cause a mild phenotype of OSCS, including facial and skull base bone striations, nasal stenosis, conductive hearing loss, global developmental delay, and mild facial dysmorphology without short stature or macrocephaly.

Clinically Relevant Subsets Identified by Gene Expression Patterns Support a Revised Ontogenic Model of Wilms Tumor: A Children's Oncology Group Study

Wilms tumors (WT) have provided broad insights into the interface between development and tumorigenesis. Further understanding is confounded by their genetic, histologic, and clinical heterogeneity, the basis of which remains largely unknown. We evaluated 224 WT for global gene expression patterns; WT1, CTNNB1, and WTX mutation; and 11p15 copy number and methylation patterns. Five subsets were identified showing distinct differences in their pathologic and clinical features: these findings were validated in 100 additional WT. The gene expression pattern of each subset was compared with published gene expression profiles during normal renal development. A novel subset of epithelial WT in infants lacked WT1, CTNNB1, and WTX mutations and nephrogenic rests and displayed a gene expression pattern of the postinduction nephron, and none recurred. Three subsets were characterized by a low expression of WT1 and intralobar nephrogenic rests. These differed in their frequency of WT1 and CTNNB1 mutations, in their age, in their relapse rate, and in their expression similarities with the intermediate mesoderm versus the metanephric mesenchyme. The largest subset was characterized by biallelic methylation of the imprint control region 1, a gene expression profile of the metanephric mesenchyme, and both interlunar and perilobar nephrogenic rests. These data provide a biologic explanation for the clinical and pathologic heterogeneity seen within WT and enable the future development of subset-specific therapeutic strategies. Further, these data support a revision of the current model of WT ontogeny, which allows for an interplay between the type of initiating event and the developmental stage in which it occurs.

Characterization of 17.94, a novel anaplastic Wilms' tumor cell line

Despite considerable advances in understanding the molecular pathogenesis of Wilms' tumor (WT), its cell biology is less well understood, partly due to the paucity of established WT cell lines. We report here the establishment of a new anaplastic WT cell line, 17.94, which expressed NCAM, SALL1, and CITED1-phenotypic features expected of metanephric blastema-derived cells. Treatment of 17.94 cells with 12-O-Tetradecanoylphorbol 13-acetate caused morphological changes, which led to reduced NCAM and SALL1 expression, but expression of vimentin was maintained, indicating a potential for stromal differentiation. The 17.94 cell line contained a TP53 mutation, consistent with the anaplastic histology of the original tumor, but lacked mutations in WT1, WTX, or CTNNB1, which are the other genes involved in WT pathogenesis. The 17.94 cells showed no loss of heterozygosity at 7p, 11p, or 16q; however, DNA hypermethylation was detected at several loci, including the H19 differentially methylated region (indicative of loss of imprinting of IGF2 at 11p15) and at the PCDH@ gene clusters at 5q31. The derivation of the 17.94 cell line should help to further dissect the genetic-epigenetic interactions involved in the pathogenesis of WT.

Stratification of Wilms tumor by genetic and epigenetic analysis

Somatic defects at five loci, WT1, CTNNB1, WTX, TP53 and the imprinted 11p15 region, are implicated in Wilms tumor, the commonest childhood kidney cancer. In this study we analysed all five loci in 120 Wilms tumors. We identified epigenetic 11p15 abnormalities in 69% of tumors, 37% were H19 epimutations and 32% were paternal uniparental disomy (pUPD). We identified mutations of WTX in 32%, CTNNB1 in 15%, WT1 in 12% and TP53 in 5% of tumors. We identified several significant associations: between 11p15 and WTX (P=0.007), between WT1 and CTNNB1 (P less than 0.001), between WT1 and pUPD 11p15 (P=0.01), and a strong negative association between WT1 and H19 epimutation (P less than 0.001). We next used these data to stratify Wilms tumor into three molecular Groups, based on the status at 11p15 and WT1. Group 1 tumors (63%) were defined as 11p15-mutant and WT1-normal; a third also had WTX mutations. Group 2 tumors (13%) were WT1-mutant. They either had 11p15 pUPD or were 11p15-normal. Almost all had CTNNB1 mutations but none had H19 epimutation. Group 3 tumors (25%) were defined as 11p15-normal and WT1-normal and were typically normal at all five loci (P less than 0.001). We also identified a novel clinical association between H19 epimutation and bilateral disease (P less than 0.001). These data provide new insights into the pattern, order, interactions and clinical associations of molecular events in Wilms tumor.

The male phenotype in osteopathia striata congenita with cranial sclerosis

Osteopathia striata with cranial sclerosis (OSCS) is an X-linked disease caused by truncating mutations in WTX. Females exhibit sclerotic striations on the long bones, cranial sclerosis, and craniofacial dysmorphism. Males with OSCS have significant skeletal sclerosis, do not have striations but do display a more severe phenotype commonly associated with gross structural malformations, patterning defects, and significant pre- and postnatal lethality. The recent description of mutations in WTX underlying OSCS has led to the identification of a milder, survivable phenotype in males. Individuals with this presentation can have, in addition to skeletal sclerosis, Hirschsprung disease, joint contractures, cardiomyopathy, and neuromuscular anomalies. A diagnosis of OSCS should be considered in males with macrocephaly, skeletal sclerosis that is most marked in the cranium and the absence of metaphyseal striations. The observation of striations in males may be indicative of a WTX mutation in a mosaic state supporting the contention that this sign in females is indicative of the differential lyonization of cells in the osteoblastic lineage.

Resistance or sensitivity of Wilms’ tumor to anti-FZD7 antibody highlights the Wnt pathway as a possible therapeutic target

Wilms' tumor (WT), the most frequent renal solid tumor in children, has been linked to aberrant Wnt signaling. Herein, we demonstrate that different WTs can be grouped according to either sensitivity or resistance to an antibody (Ab) specific to frizzled7 (FZD7), a Wnt receptor. In the FZD7-sensitive WT phenotype, the Ab induced cell death of the FZD7(+) fraction, which in turn depleted primary WT cultures of their clonogenic and sphere-forming cells and decreased in vivo proliferation and survival on xenografting to the chick chorio-allantoic-membrane. In contrast, FZD7-resistant WT in which no cell death was induced showed a different intra-cellular route of the Ab-FZD7 complex compared with sensitive tumors and accumulation of β-catenin. This coincided with a low sFRP1 and DKK1 (Wnt inhibitors) expression pattern, restored epigenetically with de-methylating agents, and lack of β-catenin or WTX mutations. The addition of exogenous DKK1 and sFRP1 to the tumor cells enabled the sensitization of FZD7-resistant WT to the FZD7 Ab. Finally, although extremely difficult to achieve because of dynamic cellular localization of FZD7, sorting of FZD7(+) cells from resistant WT, showed them to be highly clonogenic/proliferative, overexpressing WT 'stemness' genes, emphasizing the importance of targeting this fraction. FZD7 Ab therapy alone or in combination with Wnt pathway antagonists may have a significant role in the treatment of WT via targeting of a tumor progenitor population.

Inactivation of theAPCGene Is Constant in Adrenocortical Tumors from Patients with Familial Adenomatous Polyposis but Not Frequent in Sporadic Adrenocortical Cancers

In adrenocortical tumors (ACT), Wnt/β-catenin pathway activation can be explained by β-catenin somatic mutations only in a subset of tumors. ACT is observed in patients with familial adenomatous polyposis (FAP) with germline APC mutations, as well as in patients with Beckwith-Wiedemann syndrome with Wilms' tumors reported to have WTX somatic mutations. Both APC and WTX are involved in Wnt/β-catenin pathway regulation and may play a role in ACT tumorigenesis. The aim of this study was to report if APC and WTX may be associated with FAP-associated and sporadic ACT. ACTs from patients with FAP and sporadic adrenocortical carcinomas (ACC) with abnormal β-catenin localization on immunohistochemistry but no somatic β-catenin mutations were studied. APC was analyzed by denaturing high-performance liquid chromatography followed by direct sequencing and by multiplex ligation-dependent probe amplification when allelic loss was suspected. WTX was studied by direct sequencing. Four ACTs were observed in three patients with FAP and were ACC, adrenocortical adenoma, and bilateral macronodular adrenocortical hyperplasia, all with abnormal β-catenin localization. Biallelic inactivation of APC was strongly suggested by the simultaneous existence of somatic and germline alterations in all ACTs. In the 20 sporadic ACCs, a silent heterozygous somatic mutation as well as a rare heterozygous polymorphism in APC was found. No WTX mutations were observed. ACT should be considered a FAP tumor. Biallelic APC inactivation mediates activation of the Wnt/β-catenin pathway in the ACTs of patients with FAP. In contrast, APC and WTX genetic alterations do not play a significant role in sporadic ACC.

Two novel WTX mutations underscore the unpredictability of male survival in osteopathia striata with cranial sclerosis

Osteopathia striata with cranial sclerosis (OMIM ##300373) is an X-linked dominant sclerosing bone dysplasia that presents in females with macrocephaly, cleft palate, mild learning disabilities, sclerosis of the long bones and skull, and longitudinal striations visible on radiographs of the long bones, pelvis, and scapulae. In males this entity is usually associated with foetal or neonatal lethality, because of severe heart defects and/or gastrointestinal malformations, and is often accompanied by bilateral fibula aplasia. Recently, the disease-causing gene was identified as the WTX gene (FAM123B). Initially it was suggested that the mutations in the 5' region of the WTX gene are associated with male lethality. Mutation analysis in individuals of two families diagnosed with OSCS revealed two novel WTX mutations. In one family, the affected male is still alive in his teens. These mutations underline the unpredictability of male survival and suggest that WTX mutations should be considered in cases of male cranial sclerosis, even if striations are not present. An overview of all known mutations and their associated characteristics provide a valuable resource for the molecular analysis of OSCS.