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Hydatidiform mole (HM) is an aberrant human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. HM has two morphological types, complete (CHM) and partial (PHM), and non-recurrent ones have three genotypic types, androgenetic monospermic, androgenetic dispermic, and triploid dispermic. Most available studies on risk factors predisposing to different types of HM and their malignant transformation mainly suffer from the lack of comprehensive genotypic analysis of large cohorts of molar tissues combined with accurate postmolar hCG follow-up. Moreover, 10–20% of patients with one HM have at least one non-molar miscarriage, which is higher than the frequency of two pregnancy losses in the general population (2–5%), suggesting a common genetic susceptibility to HM and miscarriages. However, the underlying causes of the miscarriages in these patients are unknown. Here, we comprehensively analyzed 204 HM, mostly from patients referred to the Quebec Registry of Trophoblastic Diseases and for which postmolar hCG monitoring is available, and 30 of their non-molar miscarriages. We revisited the risk of maternal age and neoplastic transformation across the different HM genotypic categories and investigated the presence of chromosomal abnormalities in their non-molar miscarriages. We confirm that androgenetic CHM is more prone to gestational trophoblastic neoplasia (GTN) than triploid dispermic PHM, and androgenetic dispermic CHM is more prone to high-risk GTN and choriocarcinoma (CC) than androgenetic monospermic CHM. We also confirm the association between increased maternal age and androgenetic CHM and their malignancies. Most importantly, we demonstrate for the first time that patients with an HM and miscarriages are at higher risk for aneuploid miscarriages [83.3%, 95% confidence interval (CI): 0.653–0.944] than women with sporadic (51.5%, 95% CI: 50.3–52.7%, p value = 0.0003828) or recurrent miscarriages (43.8%, 95% CI: 40.7–47.0%, p value = 0.00002). Our data suggest common genetic female germline defects predisposing to HM and aneuploid non-molar miscarriages in some patients.

Purpose Current diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, genome sequencing (GS) can detect all genomic pathogenic variant types on a single platform. Here we evaluate copy-number variant (CNV) calling as part of a clinically accredited GS test. Methods We performed analytical validation of CNV calling on 17 reference samples, compared the sensitivity of GS-based variants with those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis of GS-based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases. Results We found that CNV calls from GS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed, all of which were confirmed by an orthogonal approach. The pipeline also enabled discovery of a uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of select CNVs enabled breakpoint level resolution of genomic rearrangements and phasing of de novo CNVs. Conclusion Robust identification of CNVs by GS is possible within a clinical testing environment.

Recessive mutations in FRAS1-related extracellular matrix 1 (FREM1) are associated with two rare genetic disorders, Manitoba-oculo-tricho-anal (MOTA) and bifid nose with or without anorectal and renal anomalies (BNAR). Fraser syndrome is a more severe disorder that shows phenotypic overlap with both MOTA and anorectal and renal anomalies and results from mutations in FRAS1, FREM2 and GRIP1. Heterozygous missense mutations in FREM1 were reported in association with isolated trigonocephaly with dominant inheritance and incomplete penetrance. Moreover, large deletions encompassing FREM1 have been reported in association with a syndromic form of trigonocephaly and were designated as trigonocephaly type 2. Trigonocephaly results from premature closure of the metopic suture and typically manifests as a form of nonsyndromic craniosynostosis. We report on 20 patients evaluated for developmental delay and without abnormal metopic suture. Chromosomal microarray analysis revealed heterozygous FREM1 deletions in 18 patients and in 4 phenotypically normal parents. Two patients were diagnosed with MOTA and had homozygous FREM1 deletions. Therefore, although our results are consistent with the previous reports of homozygous deletions causing MOTA, we report no association between heterozygous FREM1 deletions and trigonocephaly in this cohort.

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Androgenetic complete hydatidiform moles are human pregnancies with no embryos and affect 1 in every 1,400 pregnancies. They have mostly androgenetic monospermic genomes with all the chromosomes originating from a haploid sperm and no maternal chromosomes. Androgenetic complete hydatidiform moles were described in 1977, but how they occur has remained an open question. We identified bi-allelic deleterious mutations in MEI1, TOP6BL/C11orf80, and REC114, with roles in meiotic double-strand breaks formation in women with recurrent androgenetic complete hydatidiform moles. We investigated the occurrence of androgenesis in Mei1-deficient female mice and discovered that 8% of their oocytes lose all their chromosomes by extruding them with the spindles into the first polar body. We demonstrate that Mei1−/− oocytes are capable of fertilization and 5% produce androgenetic zygotes. Thus, we uncover a meiotic abnormality in mammals and a mechanism for the genesis of androgenetic zygotes that is the extrusion of all maternal chromosomes and their spindles into the first polar body. © 2018 American Society of Human Genetics

What's already known about this topic? Current professional guidelines regarding the use of chromosomal microarray analysis (CMA) versus karyotyping in prenatal diagnosis support CMA over karyotype only when fetal structural abnormalities are present.Examination of the clinical utility of these guidelines, given advances in microarray technology and prenatal screening, is largely unaddressed. What does this study add? This study demonstrates the diagnostic superiority of CMA by SNP microarray compared with karyotyping for prenatal diagnosis, regardless of the clinical indication for testing., Objective The American College of Obstetricians and Gynecologists (ACOG) and Society for Maternal‐Fetal Medicine (SMFM) recommend chromosomal microarray analysis (CMA) for prenatal diagnosis in cases with 1 or more fetal structural abnormalities. For patients who elect prenatal diagnosis and have a structurally normal fetus, either microarray or karyotype is recommended. This study evaluates the frequency of clinically significant chromosomal abnormalities (CSCA) that would have been missed if all patients offered the choice between CMA and karyotyping chose karyotyping. Methods A total of 3223 prenatal samples undergoing CMA were evaluated. Cases were categorized into 2 groups: those that met ACOG guidelines for CMA versus those that met ACOG guidelines for either CMA or karyotype. Results Of the 3223 cases, 1475 (45.8%) met ACOG recommendations for CMA, and 1748 (54.2%) met recommendations for either CMA or karyotype. In patients who could have elected either CMA or karyotype, 2.5% had CSCA that would have been missed if the patient had elected to pursue karyotype. Conclusion This study suggests that 2.5% of patients will have a CSCA that may be missed if the guidelines continue to suggest that CMA and karyotyping have equivalent diagnostic value for patients without a fetal structural abnormality.

PurposeCurrent diagnostic testing for genetic disorders involves serial use of specialized assays spanning multiple technologies. In principle, whole genome sequencing (WGS) has the potential to detect all genomic mutation types on a single platform and workflow. Here we sought to evaluate copy number variant (CNV) calling as part of a clinically accredited WGS test.MethodsUsing a depth-based copy number caller we performed analytical validation of CNV calling on a reference panel of 17 samples, compared the sensitivity of WGS-based variants to those from a clinical microarray, and set a bound on precision using orthogonal technologies. We developed a protocol for family-based analysis, annotation, filtering, visualization of WGS based CNV calls, and deployed this across a clinical cohort of 79 rare and undiagnosed cases.ResultsWe found that CNV calls from WGS are at least as sensitive as those from microarrays, while only creating a modest increase in the number of variants interpreted (~10 CNVs per case). We identified clinically significant CNVs in 15% of the first 79 cases analyzed. This pipeline also enabled identification of cases of uniparental disomy (UPD) and a 50% mosaic trisomy 14. Directed analysis of some CNVs enabled break-point level resolution of genomic rearrangements and phasing ofde-novoCNVs.ConclusionRobust identification of CNVs by WGS is possible within a clinical testing environment, and further developments will bring improvements in resolution of smaller and more complex CNVs.

WOS: 000440567300011, PubMed ID: 29463882, Hydatidiform mole is an aberrant human pregnancy characterized by early embryonic arrest and excessive trophoblastic proliferation. Recurrent hydatidiform moles are defined by the occurrence of at least two hydatidiform moles in the same patient. Fifty to eighty percent of patients with recurrent hydatidiform moles have biallelic pathogenic variants in NLRP7 or KHDC3L. However, in the remaining patients, the genotypic types of the moles are unknown. We characterized 80 new hydatidiform mole tissues, 57 of which were from patients with no mutations in the known genes, and we reviewed the genotypes of a total of 123 molar tissues. We also reviewed mutation analysis in 113 patients with recurrent hydatidiform moles. While all hydatidiform moles from patients with biallelic NLRP7 or KHDC3L mutations are diploid biparental, we demonstrate that those from patients without mutations are highly heterogeneous and only a small minority of them are diploid biparental (8%). The other mechanisms that were found to recur in patients without mutations are diploid androgenetic monospermic (24%) and triploid dispermic (32%); the remaining hydatidiform moles were misdiagnosed as moles due to errors in the analyses and/or their unusual mechanisms. We compared three parameters of genetic susceptibility in patients with and without mutations and show that patients without mutations are mostly from non-familial cases, have fewer reproductive losses, and more live births. Our data demonstrate that patients with recurrent hydatidiform moles and no mutations in the known genes are, in general, different from those with mutations; they have a milder genetic susceptibility and/or a multifactorial etiology underlying their recurrent hydatidiform moles. Categorizing these patients according to the genotypic types of their recurrent hydatidiform moles may facilitate the identification of novel genes for this entity., Reseau Quebecois en Reproduction; McGill Faculty of Medicine; RI-MUHC Desjardins Studentship in Child Health Research; CRRD; Canadian Institute of Health ResearchCanadian Institutes of Health Research (CIHR) [MOP-86546, POP-122897, MOP-130364], NMPN was supported by fellowships from Reseau Quebecois en Reproduction, McGill Faculty of Medicine, RI-MUHC Desjardins Studentship in Child Health Research, and CRRD. Yassemine Khawajkie was supported by a CRRD trainee fellowship. RS is supported by the Canadian Institute of Health Research (MOP-86546, POP-122897, and MOP-130364).

Chromosomal microarray analysis (CMA) is currently considered first-tier testing in pediatric care and prenatal diagnosis owing to its high diagnostic sensitivity for chromosomal imbalances. The aim of this study was to determine the efficacy and diagnostic power of CMA in both fresh and formalin-fixed paraffin-embedded (FFPE) samples of products of conception (POCs). Over a 44-month period, 8,118 consecutive samples were received by our laboratory for CMA analysis. This included both fresh (76.4%) and FFPE samples (22.4%), most of which were ascertained for recurrent pregnancy loss and/or spontaneous abortion (83%). The majority of samples were evaluated by a whole-genome single-nucleotide polymorphism (SNP)-based array (81.6%); the remaining samples were evaluated by array-comparative genomic hybridization (CGH). A successful result was obtained in 7,396 of 8,118 (91.1%), with 92.4% of fresh tissue samples and 86.4% of FFPE samples successfully analyzed. Clinically significant abnormalities were identified in 53.7% of specimens (3,975 of 7,396), 94% of which were considered causative. Analysis of POC specimens by karyotyping fails in 20–40% of cases. SNP-based CMA is a robust platform, with successful results obtained in >90% of cases. SNP-based CMA can identify aneuploidy, polyploidy, whole-genome homozygosity, segmental genomic imbalances, and maternal cell contamination, thus maximizing sensitivity and decreasing false-negative results. Understanding the etiology of fetal loss enables clarification of recurrence risk and assists in determining appropriate management for future family planning. Genet Med 19 1, 83–89.

We evaluated a patient with mild intellectual disability, obesity, overgrowth, and dysmorphic features. Array comparative genomic hybridization (aCGH) analysis showed a single copy number increase of a BAC clone in the 11p15.4 region. Oligonucleotide aCGH refined the duplication to approximately 2.29 megabases (Mb) in size. Testing the parents revealed that the father, who had learning disabilities and overgrowth, also had the 11p15.4 duplication, and the mother had a normal microarray. In addition, the patient's brother and grandmother all share clinical features with the proband and tested positive for the duplication. The duplicated region (Chr11:6,934,067-9,220,605) encompasses 29 genes, including the ZNF214 gene, which has been postulated to play a role in Beckwith-Wiedemann syndrome [Alders et al., 2000]. This three-generation pedigree outlines features of a novel microduplication syndrome.

Acute myelomonocytic leukemia with eosinophilia is commonly associated with pericentric inversions of chromosome 16, involving the core binding factor beta gene ( CBFB ) on 16q22 and the myosin heavy chain gene ( MYH11 ) on 16p13. The inv(16)(p13q22) results in a fusion gene comprising the 5′CBFB gene and the 3′MYH11 gene on the short arm of chromosome 16. The fusion gene interferes with the normal transcription of the CBFA/CBFB heterodimer and disrupts myeloid differentiation. The inv(16) is associated with a good prognosis. The inv(16) with deletion of the 3′CBFB region of the gene is a very rare occurrence. Although the number of cases is small, inv(16) with a deleted 3′CBFB seems to be associated with a poorer prognosis than that generally associated with inv(16). Our patient was a 30-year-old man with newly diagnosed acute myeloid leukemia who was found to have a CBFB–MYH11 fusion by reverse transcriptase–polymerase chain reaction. The high blast count and lack of differentiation were not typical for this entity and suggested clonal progression. The initial karyotype by conventional cytogenetic analysis, in all metaphases examined, was 46,XY,del(7)(q32),del(16)(q22). Fluorescence in situ hybridization analysis with a dual-color, break-apart probe corresponding to the CBFB gene locus (Abbott, Des Plaines, IL) showed a derivative chromosome 16 resulting from an inversion of the CBFB gene with a deletion of the 3′CBFB probe region. Oligonucleotide array comparative genetic hybridization analysis was performed on this patient's diagnostic bone marrow DNA referenced to a normal male control DNA by using the DNA array Heme Profile (CombiMatrix Diagnostics, Irvine, CA) microarray. This analysis showed a 1.2 Mb loss of 16q22.1, which did not include loss of the 3′CBFB gene locus, but rather sequences distal to this locus. The DNA array Heme Profile results illustrate the importance of microarray in the correct identification of abnormalities that will affect prognosis.

The human 3q29 microdeletion syndrome is associated with mild facial dysmorphism, developmental delay and variable congenital malformations. We report three new unrelated patients with this syndrome. We also performed in silico RNA binding analysis in silico on the 3q29 critical region genes. Several genes within this genomic region including DLG1 and RNF168 are predicted to bind RNA. While recessive mutations in RNF168 cause RIDDLE syndrome, an immune deficiency and radiosensitivity disorder, the potential impact of heterozygous deletion of RNF168 on patients with the 3q29 deletion syndrome is still unknown.

Expanding noninvasive prenatal testing to include microdeletions and segmental aneuploidy: cause for concern?

The proximal region of the long arm of chromosome 15q11.2-q13 is associated with various neurodevelopmental disorders, including Prader-Willi (PWS) and Angelman (AS) syndromes, autism, and other developmental abnormalities resulting from deletions and duplications. In addition, this region encompasses imprinted genes that cause PWS or AS, depending on the parent-of-origin. This imprinting allows for diagnosis of PWS or AS based on methylation status using methylation sensitive (MS) multiplex ligation dependent probe amplification (MLPA). Maternally derived microduplications at 15q11.2-q13 have been associated with autism and other neuropsychiatric disorders. Multiple methods have been used to determine the parent-of-origin for 15q11.2-q13 microdeletions and microduplications. In the present study, a four-year-old nondysmorphic female patient with developmental delay was found to have ade novo~5 Mb duplication within 15q11.2 by oligonucleotide genomic array. In order to determine the significance of this microduplication to the clinical phenotype, the parent-of-origin needed to be identified. The PWS/AS MS-MLPA assay is generally used to distinguish between deletion and uniparental disomy (UPD) of 15q11.2-q13, resulting in either PWS or AS. However, our study shows that PWS/AS MS-MLPA can also efficiently distinguish the parental origin of duplications of 15q11.2-q13.

Objective To evaluate the growth disorder and phenotype in prepubertal children with Leri-Weill dyschondrosteosis (LWD), a dominantly inherited skeletal dysplasia, and to compare the findings from girls with Turner syndrome (TS). Study design We studied the auxologic and phenotypic characteristics in 34 prepubertal LWD subjects (ages 1 to 10 years; 20 girls, 14 boys) with confirmed short stature homeobox-containing gene ( SHOX ) abnormalities. For comparative purposes, we evaluated similar physical and growth parameters in 76 girls with TS (ages 1 to 19 years) and 24 girls with LWD (ages 1 to 15 years) by using data collected from the postmarketing observational study, GeNeSIS. Results In the clinic sample LWD subjects, height standard deviation score ranged from −5.5 to +0.1 (−2.3 ± 1.3, girls and −1.8 ± 0.6, boys). Wrist changes related to Madelung deformity were present in 18 of 34 (53%) LWD subjects. In comparing the LWD and TS populations in the GeNeSIS sample, Madelung deformity, increased carrying angle, and scoliosis were more prevalent in the LWD population, whereas high arched palate was similarly prevalent in the two populations. Conclusions Short stature is common in both LWD (girls and boys) and TS (girls). Clinical clues to the diagnosis of SHOX haploinsufficiency in childhood include short stature, short limbs, wrist changes, and tibial bowing.

Lymphoid Enhancer Factor-1 (LEF-1) is a member of a family of transcription factors that function as downstream mediators of the Wnt signal transduction pathway. In the absence of Wnt signals, specific LEF/TCF isoforms repress rather than activate gene targets through recruitment of the co-repressor CtBP. Characterization of the full-length human LEF-1 gene locus and its complete set of mRNA products shows that this family member exists as a unique set of alternatively spliced isoforms; none are homologous to TCF-1E/TCF-4E. Therefore LEF-1 is distinct from its TCF family members in that it cannot engage in activities specific to this isoform such as recruitment of the co-repressor CtBP. Expression of alternatively spliced LEF-1 isoforms are driven by a promoter that is highly active in lymphocyte cell lines. Transcription initiates within a TATA-less core promoter region that contains consensus binding sites for Sp1, an E box, an Initiator element and a LEF/TCF binding site, all juxtaposed to the start sites of transcription. The promoter is most active in a B lymphocyte cell line (Raji) in which the endogenous LEF-1 gene is silent, suggesting that the promoter region is actively repressed by a silencing mechanism.

Signal transduction by beta-catenin involves its posttranslational stabilization and import to the nucleus where it interacts with transcription factors. Recent implications for beta-catenin signaling in cancer prompted us to examine colon cancer cell lines for the expression of LEF-1, a transcription factor that binds to beta-catenin. The analysis of several cell lines revealed the expression of LEF1 mRNA and a constitutive association of the LEF-1 protein with beta-catenin. In contrast to the colon cells, PC12 and 293 cells did not contain a beta-catenin-LEF-1 complex, even though both proteins were detected in cell lysates. In these cells, the association of endogenous LEF1 and beta-catenin was induced by stimulation with the wnt-1 proto-oncogene. The complex formed following transient stimulation with wnt-1 and also persisted in cells stably expressing wnt-1. Ectopic overexpression of beta-catenin in 293 cells also induced the assembly of the beta-catenin-LEF-1 complex and activated gene transcription from a LEF-1-dependent promotor. Expression of mutant oncogenic forms of beta-catenin identified in cancer cells resulted in higher levels of transcriptional activity. The results suggest that a cancer pathway driven by wnt-1, or mutant forms of beta-catenin, may involve the formation of a persistent transcriptionally active complex of beta-catenin and LEF1.

Recently, 3 unrelated children with a potentially novel 3q26.33–3q27.2 microdeletion syndrome were reported. We now report a new 9 ½ years old Caucasian boy with a 2 Mb deletion of the same genomic region in combination with Klinefelter syndrome. He presented with facial dysmorphism, developmental delay, Asperger syndrome, thrombocytopenia, recurrent infections and hypogammaglobulinemia. The deletion in our patient improves upon the minimum region of the novel 3q26.33–3q27.2 microdeletion, and provides additional insights into the underlying genetic basis of the observed phenotypes. Consistent with two of three previously described patients, our patient also presents with thrombocytopenia, which we postulate is caused by haploinsufficiency of THPO. In addition, haploinsufficiency of LAMP3 , a lymphoid and dendritic cell expressed protein that is implicated in bacterial and viral infections, pulmonary surfactant protein transport and amelogenin degradation, may be a novel cause for the immune deficiency, lung disease and dental abnormalities respectively as seen in these patients.

Chromosomal microarray analysis is now commonly used in clinical practice to identify copy number variants (CNVs) in the human genome. We report our experience with the use of the 105K and 180K oligonucleotide microarrays in 215 consecutive patients referred with either autism or autism spectrum disorders (ASD) or developmental delay/learning disability for genetic services at the University of Kansas Medical Center during the past 4 years (2009–2012). Of the 215 patients [140 males and 75 females (male/female ratio = 1.87); 65 with ASD and 150 with learning disability], abnormal microarray results were seen in 45 individuals (21%) with a total of 49 CNVs. Of these findings, 32 represented a known diagnostic CNV contributing to the clinical presentation and 17 represented non-diagnostic CNVs (variants of unknown significance). Thirteen patients with ASD had a total of 14 CNVs, 6 CNVs recognized as diagnostic and 8 as non-diagnostic. The most common chromosome involved in the ASD group was chromosome 15. For those with a learning disability, 32 patients had a total of 35 CNVs. Twenty-six of the 35 CNVs were classified as a known diagnostic CNV, usually a deletion (n = 20). Nine CNVs were classified as an unknown non-diagnostic CNV, usually a duplication (n = 8). For the learning disability subgroup, chromosomes 2 and 22 were most involved. Thirteen out of 65 patients (20%) with ASD had a CNV compared with 32 out of 150 patients (21%) with a learning disability. The frequency of chromosomal microarray abnormalities compared by subject group or gender was not statistically different. A higher percentage of individuals with a learning disability had clinical findings of seizures, dysmorphic features and microcephaly, but not statistically significant. While both groups contained more males than females, a significantly higher percentage of males were present in the ASD group.

Transcription of the lymphoid enhancer factor-1 (LEF1) gene is aberrantly activated in sporadic colon cancer, whereas this gene is not expressed in the normal adult colon. We have shown previously that promoter 1 of the LEF1 gene is activated by T cell factor (TCF)-beta-catenin complexes in transient transfection assays, suggesting that LEF1 is a target of the Wnt pathway in colon cancer. To further explore the link between LEF1 expression and the Wnt pathway, we studied two response elements in the promoter. Surprisingly we found that the LEF1 promoter is selectively activated by specific isoforms of the LEF/TCF transcription factor family that contain an alternative C-terminal "E" tail. These isoforms, TCF-1E and TCF-4E, activate the promoter in a beta-catenin-dependent manner. We show that a complete E-tail domain is necessary for full activity and delimits residues within two highly conserved peptide motifs within the tail that are required (KKCRARFG; WCXXCRRKKKC). These peptide motifs are not only conserved among the TCF family members but are also found in two newly identified DNA-binding proteins named papillomavirus binding factor and GLUT4 enhancer factor. This study thus identifies a new and unique set of motifs used by the Wnt pathway for target gene regulation.

Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as “Nasu-Hakola disease,” is a globally distributed recessively inherited disease leading to death during the 5th decade of life and is characterized by early-onset progressive dementia and bone cysts. Elsewhere, we have identified PLOSL mutations in TYROBP (DAP12), which codes for a membrane receptor component in natural-killer and myeloid cells, and also have identified genetic heterogeneity in PLOSL, with some patients carrying no mutations in TYROBP. Here we complete the molecular pathology of PLOSL by identifying TREM2 as the second PLOSL gene. TREM2 forms a receptor signaling complex with TYROBP and triggers activation of the immune responses in macrophages and dendritic cells. Patients with PLOSL have no defects in cell-mediated immunity, suggesting a remarkable capacity of the human immune system to compensate for the inactive TYROBP-mediated activation pathway. Our data imply that the TYROBP-mediated signaling pathway plays a significant role in human brain and bone tissue and provide an interesting example of how mutations in two different subunits of a multisubunit receptor complex result in an identical human disease phenotype.

Constitutive activation of the Wnt signaling pathway is a root cause of many colon cancers. Activation of this pathway is caused by genetic mutations that stabilize the beta-catenin protein, allowing it to accumulate in the nucleus and form complexes with any member of the lymphoid enhancer factor (LEF1) and T-cell factor (TCF1, TCF3, TCF4) family of transcription factors (referred to collectively as LEF/TCFs) to activate transcription of target genes. Target genes such as MYC, CCND1, MMP7 and TCF7 (refs. 5-9) are normally expressed in colon tissue, so it has been proposed that abnormal expression levels or patterns imposed by beta-catenin/TCF complexes have a role in tumor progression. We report here that LEF1 is a new type of target gene ectopically activated in colon cancer. The pattern of this ectopic expression is unusual because it derives from selective activation of a promoter for a full-length LEF1 isoform that binds beta-catenin, but not a second, intronic promoter that drives expression of a dominant-negative isoform. beta-catenin/TCF complexes can activate the promoter for full-length LEF1, indicating that in cancer high levels of these complexes misregulate transcription to favor a positive feedback loop for Wnt signaling by inducing selective expression of full-length, beta-catenin-sensitive forms of LEF/TCFs.

The gene mutated in Treacher Collins syndrome, an autosomal dominant disorder of facial development, has recently been cloned. While the function of the predicted protein, Treacle, is unknown, it has been shown to share a number of features with the highly phosphorylated nucleolar phosphoproteins, which play a role in nucleolar-cytoplasmic transport. In the current study, the murine homologue of the Treacher Collins syndrome gene has been isolated and shown to encode a low complexity, serine/alanine-rich protein of 133 kDa. Interspecies comparison indicates that the proteins display 61.5% identity, with the level of conservation being greatest in the regions of acidic/basic amino acid repeats and nuclear localization signals. These features are shared with the nucleolar phosphoproteins. Confirmation that the gene isolated in the current study is orthologous with the Treacher Collins syndrome gene was provided by the demonstration that it mapped to central mouse chromosome 18 in a conserved syntenic region with human chromosome 5q21-q33. Expression analysis in the mouse indicated that the gene was expressed in a wide variety of embryonic and adult tissues. Peak levels of expression in the developing embryo were observed at the edges of the neural folds immediately prior to fusion, and also in the developing branchial arches at the times of critical morphogenetic events. These observations support a role for the gene in the development of the craniofacial complex and provide further evidence that the gene encodes a protein which may be involved in nucleolar-cytoplasmic transport.

We have previously shown that lymphocytic cells bind to cultured syncytiotrophoblast and that this may be important in the lymphocyte-mediated infection of trophoblast with the human immunodeficiency virus (HIV). Leukocyte-trophoblast adhesion may also have implications for normal trophoblast function. The following experiments were designed to characterize the adhesion systems that mediate the attachment of lymphocytic cells to trophoblast. Adhesion was assayed by labelling lymphocytic MOLT-4, clone 8 cells with the fluorescent marker, calcein-AM, and then incubating them with primary cultures of human syncytiotrophoblast. Adhesion was stimulated by pretreatment of the trophoblast cultures with several cytokines either alone or together. These included tumor necrosis factor-alpha (TNF-alpha), granulocyte/macrophage-colony stimulating factor (GM-CSF), interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma). Stimulation was time- and dose-dependent. In contrast, preincubation of trophoblast cultures with anti-TNF-alpha antibodies for 2 days reduced MOLT adhesion by almost 50%. Preincubation with other anti-cytokine antibodies had no significant effect on adhesion. In other experiments, adhesion was measured in the presence of antibodies to known adhesion molecules. Adhesion was reduced by 50% in the presence of antibodies to alpha 4 integrin or beta 1 integrin. When present together, these antibodies reduced adhesion by almost 85%. Incubation in the presence of antibodies to the very late activation antigen-4 (VLA-4; alpha 4 beta 1 integrin) counter-receptors, VCAM-1 and CS-1, was without effect. Adhesion was also unaffected by antibodies to LFA-1, ICAM-1, ICAM-2, LFA-2, or LFA-3. These results suggest that adhesion is mediated by an adhesion system consisting of lymphocyte VLA-4 (alpha 4 beta 1) and an as yet unidentified counter receptor on trophoblast.

s 471 Chromosomal microarray as a clinical tool for 13q14 deletion subtyping in chronic lymphocytic leukemia Shelly Gunn , Karine Hovanes , Michelle N. Strecker , Xavier Reveles , Rashmi Shah , Lony Lim a CombiMatrix Diagnostics, Irvine, CA, USA; START Center for Cancer Care, San Antonio, TX, USA Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease which has proven amenable to subtyping through genomic microarray analysis. Initially, 13q14 deletions as an isolated finding were associated with a favorable prognosis. However, recent evidence has revealed significant heterogeneity within the 13q14 deletion subtype, thus, there is a clear clinical need for more precise stratification of 13q14 deletions at the genomic level. The minimal deleted region (MDR) includes: DLEU2, DLEU7, MIR15A/MIR16-1 and part of DLEU1. Recent studies suggest that in addition to the MDR, the size of the deletion and the involvement of the nearby tumor suppressor gene RB1 serve as an independent prognostic biomarker for disease progression. Nine individuals in whom a 13q deletion had previously been identified by bacterial artificial chromosome (BAC) array were re-analyzed using the Cancer Cytogenomics Microarray Consortium’s consensus design on an Agilent (Santa Clara, CA) 180K oligonucleotide platform. Based on the size and location of the 13q14 deletion, cases were further classified as having type (smaller, not including RB1) or type II (larger, including RB1) deletions, as described by Ouilette et al. (Cancer Res, 2008). Type I deletions were seen in 77% of cases, and Type II deletions in 22%. There was a single case of a biallelic 13q14 deletion. Deleted regions ranged in size from 1.0 to 9.8 Mb. Unlike older BAC technologies that are subject to limited resolution, the CCMC 180K oligonucleotide array allowed for further clarification of 13q deletion cases, providing valuable clinical and prognostic information for CLL. Conflict of Interest: Dr. Gunn is the medical director at Combimatrix Diagnostics. Copy number variations and loss of heterozygosity identified in myelodysplastic syndrome X. Hu , A. Iqbal , A. Ahmed , G. Raca , X. Xu , D.J. Wolff , R. Burack , D. Mulford , M.M. Li a Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA; University of Wisconsin-Madison, WI, USA; Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid neoplasms with high risk of progression to acute myeloid leukemia (AML). The molecular pathogenic mechanisms that underlie the transformation of MDS to AML are largely unknown. About 50% of MDS patients show no cytogenetic abnormalities, making monitoring disease progress in these patients difficult. We hypothesize that some cryptic genomic copy number variations (CNVs) and regions with copy number neutral loss of heterozygosity (CN-LOH) may be or harbor genetic events responsible for the disease transformation. We studied 80 patients with newly diagnosed MDS to evaluate genomic alterations using a custom designed cancer specific CGH microarray that targets over 500 cancer genes and more than 100 cancer-associated genomic regions. Thirty six MDS cases were studied for CN-LOH using a SNP array or a combined CGH/SNP array platform that detects both CNVs and CN-LOH. CNVs were identified in all patients including 38 patients with normal cytogenetic results. The CNVs were enriched in genomic regions containing cancer genes TP73, CSF1R, NOTCH1, AKT1, BLM, and BUB1B. Twenty-five of the 36 MDS cases showed CN-LOH of 10Mb in 63 genomic regions. Although CN-LOH was distributed throughout the whole genome, 1p and 14q were more frequently involved. These results demonstrate the utility of arrays that detect both CNVs and CN-LOH in cancer analysis. Our study revealed many previously unrecognized CNVs and CN-LOH in patients with MDS, which may play important roles in the transformation of MDS to AML and be or contain biomarkers for cancer diagnosis and/or appropriate targeted therapies. Conflict of Interest: Some of the arrays used in this study were provided by Agilent Technologies.

The adherence of lymphocytic MOLT-4/clone 8 cells and normal human peripheral blood mononuclear cells (PBMCs) to primary cultures of term human syncytiotrophoblast has been characterized. Adherence was measured using a fluorescence-based assay in which leukocytic cells were labelled with calcein-AM. Adherence of MOLT cells to syncytiotrophoblast increased in a time-dependent fashion up to about 4 h after which adhesion decreased. Adhesion was detectable at 4 degrees C but was greatly reduced compared to that seen at 37 degrees C. Binding increased linearly as the ratio of MOLT cells to trophoblast was increased. Scanning and transmission electron microscopy of MOLT cell-trophoblast cocultures revealed lymphocytes adherent to the free microvillous surface of the syncytiotrophoblast masses. MOLT cells also adhered to cytotrophoblast but the extent of binding was lower than to syncytiotrophoblast. Normal human peripheral blood mononuclear cells adhered to syncytiotrophoblast. Preincubation of trophoblast cells with trypsin in the presence of calcium had no effect on subsequent adhesion of MOLT cells. However, preincubation of trophoblast cells with trypsin in the absence of divalent cations reduced subsequent adhesion. Adhesion of MOLT cells to syncytiotrophoblast was dependent on magnesium and calcium. These results show for the first time that lymphocytic cells adhere to isolated human syncytiotrophoblast and raise the possibility that this may be an important phenomenon in vivo.

Diffuse Large B-cell Lymphoma (DLBCL) accounts for approximately 30% of all lymphoid malignancies. It can be difficult to separate DLBCL from Burkitt lymphoma and plasmablastic myeloma by morphology and flow cytometric immunophenotyping alone. Fluorescent in situ hybridization (FISH) targetgene analysis can assist the differential diagnosis by detection of genetic abnormalities associated with DLBCL. Though no consistent numeric chromosomal abnormalities or translocations are observed as a hallmark for DLBCL, most cases have rearranged immunoglobulin heavy chain (IgH), BCL2 and MYC genes. Standard interphase FISH analysis is widely utilized to detect these genetic rearrangements. However, standard interphase FISH analysis could be falsely negative if the neoplastic cells are limited or if abundant benign cells are present in the background. Thus, the sensitivity to detect genetic abnormalities is low among the cases with a low number of neoplastic cells. Herein we present three DLBCL cases in which interphase FISH analysis was applied on selective cells that are morphologically consistent with neoplastic large lymphocytes. All three cases showed genetic changes in a high percentage of the selected cells. These three cases were originally analyzed for IgH gene rearrangement, IgH/BCL2 and IgH/MYC translocation by using standard non-selected interphase study and showed false negative results. However, by review of the H&E slide and the flow cytometry results prompted a re-evaluation of the FISH studies using selective analysis of neoplastic cells only. Our observations indicate that selective interphase FISH analysis improves the sensitivity of detecting genetic abnormalities in DLBCL. This analysis does require a corporation between hematopathologists and technologists performing interphase FISH studies.

We have previously shown that lymphocytic cells adhere to cultured syncytiotrophoblast and that this may be important in the lymphocyte-mediated infection of trophoblast with the human immunodeficiency virus (HIV). During the course of studies aimed at investigating the role of cell surface carbohydrates in adhesion, it was discovered that a contaminant of commercial fucose-l-phosphate, dicyclohexylamine, inhibited MOLT-trophoblast adhesion. Dicyclohexylamine and the related compounds, cyclohexylamine and hexylamine, inhibited adhesion in a dose-responsive manner with half-maximal inhibition seen at about 4 mM. While the pressor effects of cyclohexylamine, the principal metabolite of cyclamate, are well known, this is the first report of an effect of this and related compounds on cell adhesion activity. The inhibitory effect was reversible and, at concentrations less than 25 mM, did not result in loss of cell viability. Several possible mechanisms of action of cyclohexylamine were examined in an attempt to explain the effect on adhesion. No evidence was found to suggest that the effects of cyclohexylamine were due to inhibition of polyamine synthesis, increase in intracellular Ca2+ concentration or to a lysosomotropic effect. The concentrations of cyclohexylamine used are within the range of plasma concentrations attainable in humans, raising the possibility that the in vitro effects described here may also occur in vivo. The results also suggest that caution should be used in the interpretation of results obtained from experiments where cell adhesion is blocked using exogenous monosaccharides that are in the form of dicyclohexylammonium salts. Appropriate controls must be included or, if possible, sodium, potassium or barium salts should be chosen.