A novel class of oxazepine-based anti-cancer agents induces cell death in primary human CLL cells and efficiently reduces tumor growth in Eµ-TCL1 mice through the JNK/STAT4/p66Shc axis.

Survival and expansion of malignant B cells in chronic lymphocytic leukemia (CLL) are highly dependent both on intrinsic defects in the apoptotic machinery and on the interactions with cells and soluble factors in the lymphoid microenvironment. The adaptor protein p66Shc is a negative regulator of antigen receptor signaling, chemotaxis and apoptosis whose loss in CLL B cells contributes to their extended survival and poor prognosis. Hence, the identification of compounds that restore p66Shc expression and function in malignant B cells may pave the way to a new therapeutic approach for CLL. Here we show that a novel oxazepine-based compound (OBC-1) restores p66Shc expression in primary human CLL cells by promoting JNK-dependent STAT4 activation without affecting normal B cells. Moreover, we demonstrate that the potent pro-apoptotic activity of OBC-1 in human leukemic cells directly correlates with p66Shc expression levels and is abrogated when p66Shc is genetically deleted. Preclinical testing of OBC-1 and the novel analogue OBC-2 in Eµ-TCL1 tumor-bearing mice resulted in a significantly longer overall survival and a reduction of the tumor burden in the spleen and peritoneum. Interestingly, OBCs promote leukemic cell mobilization from the spleen to the blood, which correlates with upregulation of sphingosine-1-phosphate receptor expression. In summary, our work identifies OBCs as a promising class of compounds that, by boosting p66Shc expression through the activation of the JNK/STAT4 pathway, display dual therapeutic effects for CLL intervention, namely the ability to mobilize cells from secondary lymphoid organs and a potent pro-apoptotic activity against circulating leukemic cells.

Complexity index in sarcoma and genomic grade index gene signatures in rhabdomyosarcoma of pediatric and adult ages.

BACKGROUND: Rhabdomyosarcoma (RMS), the most frequent soft-tissue sarcoma in childhood, shows extensive heterogeneity in histology, site and age of onset, clinical course, and prognosis. Adolescents and young adults (AYA) with RMS form a subgroup of patients whose survival lacks behind that of children while diagnosed with histologically similar tumors. PROCEDURES: A 67-gene prognostic signature related to chromosome integrity, mitotic control, and genome complexity in sarcomas (CINSARC) is considered a powerful tool for identifying tumors with a highly metastatic potential. With this study, we investigated the prognostic value of CINSARC signature on a cohort of 48 pediatric (PEDs) and AYAs-RMS. RESULTS: CINSARC resulted not significantly correlated with age, suggesting other determinants to be responsible for that difference in survival. It remained a significant prognostic variable in both the groups of PEDs and AYAs. Also, genomic grade index signature was tested on the same cohort and showed very similar results with CINSARC. CONCLUSIONS: Our study showed that CINSARC correlated with outcome in RMS patients and may be potentially considered a tool to predict outcome, and so stratify RMS patients.

Ectopic ILT3 controls BCR-dependent activation of Akt in B-cell chronic lymphocytic leukemia.

The high proportion of long-term nonprogressors among chronic lymphocytic leukemia (CLL) patients suggests the existence of a regulatory network that restrains the proliferation of tumor B cells. The identification of molecular determinants composing such network is hence fundamental for our understanding of CLL pathogenesis. Based on our previous finding establishing a deficiency in the signaling adaptor p66Shc in CLL cells, we undertook to identify unique phenotypic traits caused by this defect. Here we show that a lack of p66Shc shapes the transcriptional profile of CLL cells and leads to an upregulation of the surface receptor ILT3, the immunoglobulin-like transcript 3 that is normally found on myeloid cells. The ectopic expression of ILT3 in CLL was a distinctive feature of neoplastic B cells and hematopoietic stem cells, thus identifying ILT3 as a selective marker of malignancy in CLL and the first example of phenotypic continuity between mature CLL cells and their progenitors in the bone marrow. ILT3 expression in CLL was found to be driven by Deltex1, a suppressor of antigen receptor signaling in lymphocytes. Triggering of ILT3 inhibited the activation of Akt kinase upon B-cell receptor (BCR) stimulation. This effect was achieved through the dynamic coalescence of ILT3, BCRs, and phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 into inhibitory clusters at the cell surface. Collectively, our findings identify ILT3 as a signature molecule of p66Shc deficiency in CLL and indicate that ILT3 may functionally contribute to a regulatory network controlling tumor progression by suppressing the Akt pathway.

p66Shc deficiency in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia enhances leukemogenesis by altering the chemokine receptor landscape.

The Shc family adaptor p66Shc acts as a negative regulator of proliferative and survival signals triggered by the B-cell receptor and, by enhancing the production of reactive oxygen species, promotes oxidative stress-dependent apoptosis. Additionally, p66Shc controls the expression and function of chemokine receptors that regulate lymphocyte traffic. Chronic lymphocytic leukemia cells have a p66Shc expression defect which contributes to their extended survival and correlates with poor prognosis. We analyzed the impact of p66Shc ablation on disease severity and progression in the Eµ-TCL1 mouse model of chronic lymphocytic leukemia. We showed that Eµ-TCL1/p66Shc-/- mice developed an aggressive disease that had an earlier onset, occurred at a higher incidence and led to earlier death compared to that in Eµ-TCL1 mice. Eµ-TCL1/p66Shc-/- mice displayed substantial leukemic cell accumulation in both nodal and extranodal sites. The target organ selectivity correlated with upregulation of chemokine receptors whose ligands are expressed therein. This also applied to chronic lymphocytic leukemia cells, where chemokine receptor expression and extent of organ infiltration were found to correlate inversely with these cells' level of p66Shc expression. p66Shc expression declined with disease progression in Eµ-TCL1 mice and could be restored by treatment with the Bruton tyrosine kinase inhibitor ibrutinib. Our results highlight p66Shc deficiency as an important factor in the progression and severity of chronic lymphocytic leukemia and underscore p66Shc expression as a relevant therapeutic target.

The systemic amyloid precursor transthyretin (TTR) behaves as a neuronal stress protein regulated by HSF1 in SH-SY5Y human neuroblastoma cells and APP23 Alzheimer's disease model mice.

Increased neuronal synthesis of transthyretin (TTR) may favorably impact on Alzheimer's disease (AD) because TTR has been shown to inhibit Aß aggregation and detoxify cell-damaging conformers. The mechanism whereby hippocampal and cortical neurons from AD patients and APP23 AD model mice produce more TTR is unknown. We now show that TTR expression in SH-SY5Y human neuroblastoma cells, primary hippocampal neurons and the hippocampus of APP23 mice, is significantly enhanced by heat shock factor 1 (HSF1). Chromatin immunoprecipitation (ChIP) assays demonstrated occupation of TTR promoter heat shock elements by HSF1 in APP23 hippocampi, primary murine hippocampal neurons, and SH-SY5Y cells, but not in mouse liver, cultured human hepatoma (HepG2) cells, or AC16 cultured human cardiomyocytes. Treating SH-SY5Y human neuroblastoma cells with heat shock or the HSF1 stimulator celastrol increased TTR transcription in parallel with that of HSP40, HSP70, and HSP90. With both treatments, ChIP showed increased occupancy of heat shock elements in the TTR promoter by HSF1. In vivo celastrol increased the HSF1 ChIP signal in hippocampus but not in liver. Transfection of a human HSF1 construct into SH-SY5Y cells increased TTR transcription and protein production, which could be blocked by shHSF1 antisense. The effect is neuron specific. In cultured HepG2 cells, HSF1 was either suppressive or had no effect on TTR expression confirming the differential effects of HSF1 on TTR transcription in different cell types.

GRIN2B predicts attention problems among disadvantaged children.

It is well established that adversities and GRIN2B (coding an N-methyl-D-aspartate receptor subunit) are independently associated with behavioral and cognitive impairments in childhood. However, a high proportion of children exposed to adversities have good, long-term outcomes. We hypothesized that among children exposed to adversities, GRIN2B variants would predict the worst cognitive and behavioral outcomes. 6 single nucleotide polymorphisms of GRIN2B were genotyped in 625 children aged 6-11 years from an Italian community-based sample. The interacting effect of GRIN2B variants with 4 measures of adversities [low socioeconomic status (SES), preterm delivery, maternal smoking during pregnancy, and absence of breastfeeding] was investigated upon blindly assessed cognitive abilities (vocabulary, block design, digit spans of Wechsler's Intelligence Scale, and Rey complex figure) and parents-rated behavioral problems (Child Behavior Checklist/6-18). Rs2268119 × SES interaction (Hotelling's Trace = 0.07; F(12,1154) = 3.53; p = 0.00004) influenced behavior, with more attention problems among children in the 'either A/T or T/T genotype and low SES' group, compared to all other groups. This interaction effect was not significant in an independent, replication sample of 475 subjects from an Italian community-based sample. GRIN2B variants predict children with the worst outcome in attention functioning among children exposed to low SES. Our findings, if replicated, could help in the identification of children with the highest risk and may prompt cost-effective preventive/treatment strategies.

Methylation and silencing of miRNA-124 by EVI1 and self-renewal exhaustion of hematopoietic stem cells in murine myelodysplastic syndrome.

By expressing EVI1 in murine bone marrow (BM), we previously described a myelodysplastic syndrome (MDS) model characterized by pancytopenia, dysmegakaryopoiesis, dyserythropoiesis, and BM failure. The mice invariably died 11-14 months after BM transplantation (BMT). Here, we show that a double point mutant EVI1-(1+6Mut), unable to bind Gata1, abrogates the onset of MDS in the mouse and re-establishes normal megakaryopoiesis, erythropoiesis, BM function, and peripheral blood profiles. These normal features were maintained in the reconstituted mice until the study was ended at 21 months after BMT. We also report that EVI1 deregulates several genes that control cell division and cell self-renewal. In striking contrast, these genes are normalized in the presence of the EVI1 mutant. Moreover, EVI1, but not the EVI1 mutant, seemingly deregulates these cellular processes by altering miRNA expression. In particular, the silencing of miRNA-124 by DNA methylation is associated with EVI1 expression, but not that of the EVI1 mutant, and appears to play a key role in the up-regulation of cell division in murine BM cells and in the hematopoietic cell line 32Dcl3. The results presented here demonstrate that EVI1 induces MDS in the mouse through two major pathways, both of which require the interaction of EVI1 with other factors: one, results from EVI1-Gata1 interaction, which deregulates erythropoiesis and leads to fatal anemia, whereas the other occurs by interaction of EVI1 with unidentified factors causing perturbation of the cell cycle and self-renewal, as a consequence of silencing miRNA-124 by EVI1 and, ultimately, ensues in BM failure.

Pleiotropic effects of DCDC2 and DYX1C1 genes on language and mathematics traits in nuclear families of developmental dyslexia.

Converging evidence indicates that developmental problems in oral language and mathematics can predate or co-occur with developmental dyslexia (DD). Substantial genetic correlations have been found between language, mathematics and reading traits, independent of the method of sampling. We tested for association of variants of two DD susceptibility genes, DCDC2 and DYX1C1, in nuclear families ascertained through a proband with DD using concurrent measurements of language and mathematics in both probands and siblings by the Quantitative Transmission Disequilibrium Test. Evidence for significant associations was found between DCDC2 and 'Numerical Facts' (p value = 0.02, with 85 informative families, genetic effect = 0.57) and between 'Mental Calculation' and DYX1C1 markers -3GA (p value = 0.05, with 40 informative families, genetic effect = -0.67) and 1249GT (p value = 0.02, with 49 informative families, genetic effect = -0.65). No statistically significant associations were found between DCDC2 or DYX1C1 and language phenotypes. Both DCDC2 and DYX1C1 DD susceptibility genes appear to have a pleiotropic role on mathematics but not language phenotypes.

Tumorial: Video Tutorials Produced by Young Patients on the Youth Project to Voice Their Experiences.

Purpose: Adolescent and young adult patients of the Milan's Youth Project developed a new communication project aimed at young people with cancer: in a series of video tutorials called Tumorial, they talk about their day-to-day experiences, offering "survival tricks" to their peers. Methods: The project was developed during group meetings taking place every week in a dedicated room near the ward. Each meeting focused on a single topic and was led by the patients themselves, who talked about their experiences; staff members moderated the discussion and took notes, which was used as script for a video, recorded by one patient as a spokesperson. All the videos had English subtitles. Results: The project was implemented between March 2018 and June 2019 and involved 53 teenagers and young adults (15-27 years), 33 receiving treatment and 20 in follow-up when the project began. There were 23 video tutorials produced in all, with various topics, for example, school, sex, hair, privacy, social networks, fuck-ups to avoid, scars, ward companions. The videos are published on the Youth Project's YouTube channel (https://www.youtube.com/channel/UCR0EVeYMAjgJlN95tSc_iPA). Conclusion: This innovative approach to communication in the world of oncological disease in the young can be a useful tool as part of their course of care. It appears of great importance considering that social networks-and YouTube in particular-frequently provide unreliable or useless information. In making the project, patients told their innermost feelings, promoting cohesion among them. Patients and caregivers developed the project together in a significant example of cooperation.

EVI1 recruits the histone methyltransferase SUV39H1 for transcription repression.

EVI1 is an oncoprotein inappropriately expressed in acute myeloid leukemia and myelodysplastic syndrome cells. In vitro studies indicate that diverse biological properties can be attributed to this protein. Its role in leukemogenesis is still unclear but it is thought that overall EVI1 can act mostly as a transcription repressor through its interaction with a subset of histone deacetylases. Studies with histone deacetylase inhibitors have however indicated that EVI1-mediated repression can be only partially rescued by deacetylase inhibitor drugs, suggesting that additional chromosomal modifications might occur to induce gene repression by EVI1. To investigate whether histone methylation contributes to the repressive potential of EVI1, we examined a potential association between EVI1, the histone methyltransferase (HMT) SUV39H1, and methyltransferase activity in vitro. We find that EVI1 directly interacts with SUV39H1 and that the proteins form an active complex with methyltransferase activity in vitro. Our data indicate that SUV39H1 enhances the transcription repressive potential of EVI1 in vivo. We suggest that EVI1 affects promoters' activity in two different pathways, by association with histone deacetylases and by recruiting chromatin-modifying enzymes to impose a heterochromatin-like structure establishing a lasting transcription repression.

Src Family Kinases Regulate Interferon Regulatory Factor 1 K63 Ubiquitination following Activation by TLR7/8 Vaccine Adjuvant in Human Monocytes and B Cells.

Toll-like receptors (TLRs) play a key role in the activation of innate immune cells, in which their engagement leads to production of cytokines and co-stimulatory molecules. TLRs signaling requires recruitment of toll/IL-1R (TIR) domain-containing adaptors, such as MyD88 and/or TRIF, and leads to activation of several transcription factors, such as NF-κB, the AP1 complex, and various members of the interferon regulatory factor (IRF) family, which in turn results in triggering of several cellular functions associated with these receptors. A role for Src family kinases (SFKs) in this signaling pathway has also been established. Our work and that of others have shown that this type of kinases is activated following engagement of several TLRs, and that this event is essential for the initiation of specific downstream cellular response. In particular, we have previously demonstrated that activation of SFKs is required for balanced production of pro-inflammatory cytokines by monocyte-derived dendritic cells after stimulation with R848, an agonist of human TLRs 7/8. We also showed that TLR7/8 triggering leads to an increase in interferon regulatory factor 1 (IRF-1) protein levels and that this effect is abolished by inhibition of SFKs, suggesting a critical role of these kinases in IRF-1 regulation. In this study, we first confirmed the key role of SFKs in TLR7/8 signaling for cytokine production and accumulation of IRF-1 protein in monocytes and in B lymphocytes, two other type of antigen-presenting cells. Then, we demonstrate that TLR7 triggering leads to an increase of K63-linked ubiquitination of IRF-1, which is prevented by SFKs inhibition, suggesting a key role of these kinases in posttranslational regulation of IRF-1 in the immune cells. In order to understand the mechanism that links SFKs activation to IRF-1 K63-linked ubiquitination, we examined SFKs and IRF-1 possible interactors and proved that activation of SFKs is necessary for their interaction with TNFR-associated factor 6 (TRAF6) and promotes the recruitment of both cIAP2 and IRF-1 by TRAF6. Collectively, our data demonstrate that TLR7/8 engagement leads to the formation of a complex that allows the interaction of cIAP2 and IRF-1 resulting in IRF-1 K63-linked ubiquitination, and that active SFKs are required for this process.

p66Shc deficiency enhances CXCR4 and CCR7 recycling in CLL B cells by facilitating their dephosphorylation-dependent release from ß-arrestin at early endosomes.

Neoplastic cell traffic abnormalities are central to the pathogenesis of chronic lymphocytic leukemia (CLL). Enhanced CXC chemokine receptor-4 (CXCR4) and chemokine receptor-7 (CCR7) recycling contributes to the elevated surface levels of these receptors on CLL cells. Here we have addressed the role of p66Shc, a member of the Shc family of protein adaptors the expression of which is defective in CLL cells, in CXCR4/CCR7 recycling. p66Shc reconstitution in CLL cells reduced CXCR4/CCR7 recycling, lowering their surface levels and attenuating B-cell chemotaxis, due to their accumulation in Rab5+ endosomes as serine-phosphoproteins bound to ß-arrestin. This results from the ability of p66Shc to inhibit Ca2+ and PP2B-dependent CXCR4/CCR7 dephosphorylation and ß-arrestin release. We also show that ibrutinib, a Btk inhibitor that promotes leukemic cell mobilization from lymphoid organs, reverses the CXCR4/CCR7 recycling abnormalities in CLL cells by increasing p66Shc expression. These results, identifying p66Shc as a regulator of CXCR4/CCR7 recycling in B cells, underscore the relevance of its deficiency to CLL pathogenesis and provide new clues to the mechanisms underlying the therapeutic effects of ibrutinib.

Heterogeneous molecular mechanisms underlie attenuated familial adenomatous polyposis.

PURPOSE: Familial adenomatous polyposis is a phenotypically heterogeneous disease predisposing to colorectal cancer. It is dominantly transmitted, when associated with the APC gene, and recessively inherited, when associated with MUTYH gene. We searched for APC and MUTYH germline alterations in Italian and Greek patients with attenuated polyposis, a phenotypic variant whose genetic cause remains unknown in many cases. METHODS: We studied 26 unrelated patients (and 16 relatives) with multiple colorectal adenomas (3-100, by endoscopic analysis) that had screened APC mutation-negative by protein truncation test. We searched for APC rearrangements by multiplex ligation-dependent probe amplification and for MUTYH mutations by sequencing. We performed a screening of five MUTYH recurrent pathogenic mutations in 501 Italian and 144 Greek controls. RESULTS: One patient proved to carry an APC whole-gene deletion; 4 of 25 (16%) patients showed biallelic and 3 of 25 (12%) monoallelic MUTYH mutations. In the three heterozygous subjects no pathogenetic variants were found in OGG1, MTH1, APE1, MSH2, and MSH6 genes. Frequency assessment of MUTYH mutations in healthy subjects showed that only Y165C and G382D reach a subpolymorphic frequency. CONCLUSION: Attenuated polyposis patients without "conventional" APC mutations are genetically heterogeneous, and the phenotype is not directly related to the germline defect. Therefore, the families' appropriate management requires an accurate genetic and clinical investigation.

Functional analysis and case-control study of -160C/A polymorphism in the E-cadherin gene promoter: association with cancer risk.

BACKGROUND: A C-->A polymorphism within the CDH1 (E-cadherin) promoter seems to be associated with a reduced efficiency of gene transcription in vitro. Due to the crucial role of E-cadherin in epithelia, tissue-specific effect of C-->A change on CDH1 transcription was tested and a case-control study was performed on patients affected with epithelial tumors. PATIENTS AND METHODS: The -178/+93 CDH1 region containing either C or A nucleotide was inserted upstream of the Luciferase reporter gene in the pGL-2 vector, and the construct activity was assessed by transient transfection assay in HeLa and HCT116 cells. RESULTS: A significantly lower activity for pGL-2A was found compared to pGL-2C, both in HeLa (54% decrease) and in HCT116 (67% decrease) cells. Genotyping of 246 controls and 505 patients affected with breast, gastric, colorectal, cervical and endometrial cancers demonstrated an association between the A allele and an increased risk of colorectal, gastric and endometrial tumors (1.66-, 1.81- and 2.35-fold, respectively). CONCLUSION: Our data support the notion that the A allele may act as a low-penetrance cancer susceptibility gene.

Expression of the p66Shc protein adaptor is regulated by the activator of transcription STAT4 in normal and chronic lymphocytic leukemia B cells.

p66Shc attenuates mitogenic, prosurvival and chemotactic signaling and promotes apoptosis in lymphocytes. Consistently, p66Shc deficiency contributes to the survival and trafficking abnormalities of chronic lymphocytic leukemia (CLL) B cells. The mechanism of p66shc silencing in CLL B cells is methylation-independent, at variance with other cancer cell types. Here we identify STAT4 as a novel transcriptional regulator of p66Shc in B cells. Chromatin immunoprecipitation and reporter gene assays showed that STAT4 binds to and activates the p66shc promoter. Silencing or overexpression of STAT4 resulted in a co-modulation of p66Shc. IL-12-dependent STAT4 activation caused a coordinate increase in STAT4 and p66Shc expression, which correlated with enhanced B cell apoptosis. Treatment with the STAT4 inhibitor lisofylline reverted partly this effect, suggesting that STAT4 phosphorylation is not essential for but enhances p66shc transcription. Additionally, we demonstrate that CLL B lymphocytes have a STAT4 expression defect which partly accounts for their p66Shc deficiency, as supported by reconstitution experiments. Finally, we show that p66Shc participates in a positive feedback loop to promote STAT4 expression. These results provide new insights into the mechanism of p66Shc expression in B cells and its defect in CLL, identifying the STAT4/IL-12 pathway as a potential therapeutic target in this neoplasia.

Consistent up-regulation of Stat3 Independently of Jak2 mutations in a new murine model of essential thrombocythemia.

Janus-activated kinase 2 (JAK2) mutations are common in myeloproliferative disorders; however, although they are detected in virtually all polycythemia vera patients, they are found in approximately 50% of essential thrombocythemia (ET) patients, suggesting that converging pathways/abnormalities underlie the onset of ET. Recently, the chromosomal translocation 3;21, leading to the fusion gene AML1/MDS1/EVI1 (AME), was observed in an ET patient. After we forced the expression of AME in the bone marrow (BM) of C57BL/6J mice, all the reconstituted mice died of a disease with symptoms similar to ET with a latency of 8 to 16 months. Peripheral blood smears consistently showed an elevated number of dysplastic platelets with anisocytosis, degranulation, and giant size. Although the AME-positive mice did not harbor Jak2 mutations, the BM of most of them had significantly higher levels of activated Stat3 than the controls. With combined biochemical and biological assays we found that AME binds to the Stat3 promoter leading to its up-regulation. Signal transducers and activators of transcription 3 (STAT3) analysis of a small group of ET patients shows that in about half of the patients, there is STAT3 hyperactivation independently of JAK2 mutations, suggesting that the hyperactivation of STAT3 by JAK2 mutations or promoter activation may be a critical step in development of ET.

Multipopulation analysis of polymorphisms in five mononucleotide repeats used to determine the microsatellite instability status of human tumors.

PURPOSE: Human gastrointestinal tumors with inactivated DNA mismatch repair system (microsatellite instability [MSI] tumors) have distinct molecular and clinicopathologic profiles, and are associated with favorable prognosis. There is evidence suggesting that colorectal cancer patients with MSI tumors respond differently to adjuvant chemotherapy as compared with patients with non-MSI tumors. Finally, determination of the MSI status has clinical application for assisting in the diagnosis of suspected hereditary cases. It is thus becoming increasingly recognized that testing for MSI should be conducted systematically in all human cancers potentially of this type. We recently described a pentaplex polymerase chain reaction of five mononucleotide repeats to establish the MSI status of human tumors, and showed that this assay was 100% sensitive and specific. Moreover, these markers are quasimonomorphic in germline DNA of the white population (ie, individuals of Eurasian origin), and could be used for tumor MSI determination without the requirement for matching normal DNA in this group. PATIENTS AND METHODS: In this study, we analyzed a comparable panel of five mononucleotide markers in germline DNA from 1,206 individuals encompassing 55 different populations worldwide. Results With the exception of two Biaka Pygmies and one San individual for whom three markers showed variant alleles (three cases [0.2%]), the remaining 1,203 individuals showed no alleles of variant size (1,055 cases [87.5%]), or only one (122 cases [10.1%]) or two (26 cases [2.2%]) markers with variant alleles. All 60 MSI tumors investigated display instability in at least four of the five markers. CONCLUSION: We demonstrated that tumor MSI status can be determined using the pentaplex reaction for all human populations without the need for matching normal DNA.

High frequency of MYH gene mutations in a subset of patients with familial adenomatous polyposis.

BACKGROUND & AIMS: Inherited colorectal polyposis has been linked to constitutive mutations of the APC tumor suppressor gene. Recently, germline mutations in the base excision repair gene MYH have been associated with a recessively inherited form of the disease. The aim of this study was to evaluate germline mutation frequencies of both MYH and APC susceptibility genes in Italian patients with attenuated familial adenomatous polyposis. METHODS: The analysis was performed in 14 unrelated patients by using the protein truncation test for APC and genomic DNA sequencing for MYH. RESULTS: Overall, we identified 7 of 14 (50%) mutation carriers. Two patients were heterozygotes for an APC truncating mutation (2 of 14 [14%]), whereas 5 proved to be homozygotes or compound heterozygotes for MYH gene alterations (5 of 14 [36%]). Two MYH missense mutations, Y165C and G382D, already found to be frequent among patients from northern Europe, were also preponderant in our survey. Individuals with APC-associated syndrome showed a dominant family history of polyposis, whereas patients with MYH-associated disease were either apparently sporadic cases or had a family history consistent with recessive inheritance. MYH biallelic mutation carriers were up to 60% (5 of 8) among patients showing at least 30 adenomas and a family history with no vertical transmission of polyposis. CONCLUSIONS: On the basis of our data, patients with attenuated familial adenomatous polyposis with >30 adenomas and no obvious vertical transmission of the disease should be considered for MYH gene testing.