Recurrence, submicroscopic complexity, and potential clinical relevance of copy gains detected by array CGH that are shown to be unbalanced insertions by FISH.

Insertions occur when a segment of one chromosome is translocated and inserted into a new region of the same chromosome or a non-homologous chromosome. We report 71 cases with unbalanced insertions identified using array CGH and FISH in 4909 cases referred to our laboratory for array CGH and found to have copy-number abnormalities. Although the majority of insertions were non-recurrent, several recurrent unbalanced insertions were detected, including three der(Y)ins(Y;18)(q?11.2;p11.32p11.32)pat inherited from parents carrying an unbalanced insertion. The clinical significance of these recurrent rearrangements is unclear, although the small size, limited gene content, and inheritance pattern of each suggests that the phenotypic consequences may be benign. Cryptic, submicroscopic duplications were observed at or near the insertion sites in two patients, further confounding the clinical interpretation of these insertions. Using FISH, linear amplification, and array CGH, we identified a 126-kb duplicated region from 19p13.3 inserted into MECP2 at Xq28 in a patient with symptoms of Rett syndrome. Our results demonstrate that although the interpretation of most non-recurrent insertions is unclear without high-resolution insertion site characterization, the potential for an otherwise benign duplication to result in a clinically relevant outcome through the disruption of a gene necessitates the use of FISH to determine whether copy-number gains detected by array CGH represent tandem duplications or unbalanced insertions. Further follow-up testing using techniques such as linear amplification or sequencing should be used to determine gene involvement at the insertion site after FISH has identified the presence of an insertion.

Experience using a rapid assay for aneuploidy and microdeletion/microduplication detection in over 2,900 prenatal specimens.

BACKGROUND: While microarray testing can identify chromosomal abnormalities missed by karyotyping, its prenatal use is often avoided in low-risk pregnancies due to the possible identification of variants of uncertain significance (VOUS). METHODS: We tested 2,970 prenatal samples of all referral indications using a rapid BACs-on-Beads-based assay with probes for sex chromosomes, common autosomal aneuploidies, and 20 microdeletion/microduplication syndromes, designed as an alternative to microarray in low-risk pregnancies and an alternative to rapid aneuploidy testing in pregnancies also undergoing microarray analysis. RESULTS: Interpretable results were obtained in 2,940 cases (99.0%), with 89% receiving results in 1 day. Aneuploidies were detected in 7.3% and partial chromosome abnormalities in 0.45% (n = 13), including 5 referred for maternal age, abnormal maternal serum screen, or isolated ultrasound markers. The added detection above karyotype was 1 in 745 in lower-risk cases with normal ultrasounds or isolated ultrasound markers/increased nuchal measurements and 1 in 165 for fetuses with structural/growth abnormalities. Neither false negatives nor false positives were found within test limitations. Female polyploidy could not be detected, while polyploidies with Y chromosomes were suspected and confirmed through additional analysis. CONCLUSION: When combined with karyotyping, this assay provides increased interrogation of specific chromosomal regions, while limiting VOUS identification.

Eight Domains of Pediatrician Wellness: A Stakeholder Informed Model.

BACKGROUND: Physician wellness is important to health care systems and quality patient care. There has been limited research clarifying the physician wellness construct. We aimed to develop a stakeholder-informed model of pediatrician wellness. METHODS: We performed a group concept mapping (GCM) study to create a model of pediatrician wellness. We followed the four main steps of GCM and recruited pediatricians at multiple sites and on social media. During brainstorming, pediatricians individually responded to a prompt to generate ideas describing the concept of pediatrician wellness. Second, pediatricians sorted the list of brainstormed ideas into conceptually similar groups and rated them on importance. Sorted data were analyzed to create maps showing each idea as a point, with lines around groups of points to create clusters of wellness. Mean importance scores for each cluster were calculated and compared using pattern match. RESULTS: Pediatricians in this study identified eight clusters of wellness: 1) Experiencing belonging and support at work, 2) Alignment in my purpose, my work, and my legacy, 3) Feelings of confidence and fulfillment at work, 4) Skills and mindset for emotional well-being, 5) Harmony in personal, professional, and community life, 6) Time and resources to support holistic sense of self, 7) Work boundaries and flexibility, and 8) Organizational culture of inclusion and trust. There were no significant differences in mean cluster rating score; the highest rated cluster was Harmony in personal, professional and community life (3.62). CONCLUSION: Pediatricians identified eight domains of wellness, spanning professional and personal life, work, and individual factors.

Pediatric Resident Well-being: A Group Concept Mapping Study.

OBJECTIVE: Pediatric residency programs invest substantial resources in supporting resident well-being. However, no pediatric resident well-being conceptual model exists to guide interventions. This study aimed to understand how a diverse stakeholder sample conceptualized well-being. METHODS: We used group concept mapping methodology. We sent a brainstorming survey to pediatric residents and program leaders at 24 US residencies with the prompt, "The experience of well-being for resident physicians includes…" Participants at 4 residencies sorted well-being ideas conceptually and rated idea importance. We performed multidimensional scaling and hierarchical cluster analysis to develop cluster maps. Using participant feedback and a consensus-driven process, we determined best cluster representation. We used pattern matching to compare domain ratings between subgroups. RESULTS: In brainstorming, 136 residents and 22 program leaders from 22 residency programs generated 97 unique ideas. Ideas were sorted and rated by 33 residents, 14 program leaders. Eight domains aligning with 4 resident roles were identified. Domains were: 1) positive, safe, and diverse culture; 2) unity and connection; 3) professional fulfillment and mindset; 4) personal health and life satisfaction; 5) professional development and recognition; 6) schedule protections and downtime; 7) work systems and benefits; 8) proactive and compassionate leadership. Domains aligned with the following roles: 1) individual, 2) colleague, 3) employee, 4) emerging pediatrician. Residents placed higher value on schedule protections and downtime than program leaders, P < .05. CONCLUSIONS: Pediatric resident well-being may be conceptualized as inter-related domains corresponding with various resident roles. Participants aligned on many well-being priorities but differed regarding work schedules.

MEF2C Haploinsufficiency features consistent hyperkinesis, variable epilepsy, and has a role in dorsal and ventral neuronal developmental pathways.

MEF2C haploinsufficiency syndrome is an emerging neurodevelopmental disorder associated with intellectual disability, autistic features, epilepsy, and abnormal movements. We report 16 new patients with MEF2C haploinsufficiency, including the oldest reported patient with MEF2C deletion at 5q14.3. We detail the neurobehavioral phenotype, epilepsy, and abnormal movements, and compare our subjects with those previously reported in the literature. We also investigate Mef2c expression in the developing mouse forebrain. A spectrum of neurofunctional deficits emerges, with hyperkinesis a consistent finding. Epilepsy varied from absent to severe, and included intractable myoclonic seizures and infantile spasms. Subjects with partial MEF2C deletion were statistically less likely to have epilepsy. Finally, we confirm that Mef2c is present both in dorsal primary neuroblasts and ventral gamma-aminobutyric acid(GABA)ergic interneurons in the forebrain of the developing mouse. Given interactions with several key neurodevelopmental genes such as ARX, FMR1, MECP2, and TBR1, it appears that MEF2C plays a role in several developmental stages of both dorsal and ventral neuronal cell types.

Medical Student Well-being Outcomes After a Novel Shared Meal and Resiliency Skills Course.

INTRODUCTION: Medical student well-being is a major problem. The authors aimed to assess well-being outcomes 6-months after a novel extracurricular shared meal and resiliency course. METHODS: We implemented the course during 3 academic years (2018-2020). Participants received surveys assessing resilience, perspective-taking, self-compassion, and empathy at 4 timepoints. We used linear mixed effects models to assess changes from baseline to post-course assessments for the 3-year aggregate and pre-COVID and early-COVID time periods. RESULTS: One week and 6 months post-course, resilience, perspective-taking, and self-compassion scores improved (P < 0.01). Notably, resilience changed significantly only during early-COVID (P < 0.01), not pre-COVID (P = 0.16). For scores with evidence-based interpretation cut-offs, no clinical changes occurred. DISCUSSION: Several well-being measures statistically improved post-course but did not change clinically. Qualitative studies may better capture meaningful well-being outcome impact.

Haploinsufficiency of SOX5 at 12p12.1 is associated with developmental delays with prominent language delay, behavior problems, and mild dysmorphic features.

SOX5 encodes a transcription factor involved in the regulation of chondrogenesis and the development of the nervous system. Despite its important developmental roles, SOX5 disruption has yet to be associated with human disease. We report one individual with a reciprocal translocation breakpoint within SOX5, eight individuals with intragenic SOX5 deletions (four are apparently de novo and one inherited from an affected parent), and seven individuals with larger 12p12 deletions encompassing SOX5. Common features in these subjects include prominent speech delay, intellectual disability, behavior abnormalities, and dysmorphic features. The phenotypic impact of the deletions may depend on the location of the deletion and, consequently, which of the three major SOX5 protein isoforms are affected. One intragenic deletion, involving only untranslated exons, was present in a more mildly affected subject, was inherited from a healthy parent and grandparent, and is similar to a deletion found in a control cohort. Therefore, some intragenic SOX5 deletions may have minimal phenotypic effect. Based on the location of the deletions in the subjects compared to the controls, the de novo nature of most of these deletions, and the phenotypic similarities among cases, SOX5 appears to be a dosage-sensitive, developmentally important gene.

High-resolution array CGH defines critical regions and candidate genes for microcephaly, abnormalities of the corpus callosum, and seizure phenotypes in patients with microdeletions of 1q43q44.

Microdeletions of 1q43q44 result in a recognizable clinical disorder characterized by moderate to severe intellectual disability (ID) with limited or no expressive speech, characteristic facial features, hand and foot anomalies, microcephaly (MIC), abnormalities (agenesis/hypogenesis) of the corpus callosum (ACC), and seizures (SZR). Critical regions have been proposed for some of the more prominent features of this disorder such as MIC and ACC, yet conflicting data have prevented precise determination of the causative genes. In this study, the largest of pure interstitial and terminal deletions of 1q43q44 to date, we characterized 22 individuals by high-resolution oligonucleotide microarray-based comparative genomic hybridization. We propose critical regions and candidate genes for the MIC, ACC, and SZR phenotypes associated with this microdeletion syndrome. Three cases with MIC had small overlapping or intragenic deletions of AKT3, an isoform of the protein kinase B family. The deletion of only AKT3 in two cases implicates haploinsufficiency of this gene in the MIC phenotype. Likewise, based on the smallest region of overlap among the affected individuals, we suggest a critical region for ACC that contains ZNF238, a transcriptional and chromatin regulator highly expressed in the developing and adult brain. Finally, we describe a critical region for the SZR phenotype which contains three genes (FAM36A, C1ORF199, and HNRNPU). Although ~90% of cases in this study and in the literature fit these proposed models, the existence of phenotypic variability suggests other mechanisms such as variable expressivity, incomplete penetrance, position effects, or multigenic factors could account for additional complexity in some cases.

NF1 microduplications: identification of seven nonrelated individuals provides further characterization of the phenotype.

PURPOSE: Neurofibromatosis, type 1 (NF1) is an autosomal dominant disorder caused by mutations of the neurofibromin 1 (NF1) gene at 17q11.2. Approximately 5% of individuals with NF1 have a 1.4-Mb heterozygous 17q11.2 deletion encompassing NF1, formed through nonallelic homologous recombination (NAHR) between the low-copy repeats that flank this region. NF1 microdeletion syndrome is more severe than NF1 caused by gene mutations, with individuals exhibiting facial dysmorphisms, developmental delay (DD), intellectual disability (ID), and excessive neurofibromas. Although NAHR can also cause reciprocal microduplications, reciprocal NF1 duplications have been previously reported in just one multigenerational family and a second unrelated proband. METHODS: We analyzed the clinical features in seven individuals with NF1 microduplications, identified among 48,817 probands tested in our laboratory by array-based comparative genomic hybridization. RESULTS: The only clinical features present in more than one individual were variable DD/ID, facial dysmorphisms, and seizures. No neurofibromas were present. Three sets of parents were tested: one duplication was apparently de novo, one inherited from an affected mother, and one inherited from a clinically normal father. CONCLUSION: This is the first report comparing the phenotypes of nonrelated individuals with NF1 microduplications. This comparison will allow for further definition of this emerging microduplication syndrome.

Smartphone App for In-home Uroflowmetry.

INTRODUCTION: We compared the test results obtained by a novel MenHealth® uroflowmetry app against the standard in-office uroflowmeter. MenHealth uroflowmetry is a smartphone app that analyzes the sound of urine voided into a water-filled toilet. The program calculates maximum and average flow rates as well as volume voided. METHODS: Men over 18 years old were tested. Group 1 included 47 men with symptoms suggesting overactive bladder and/or outlet obstruction. Group 2 included 15 men with no urinary complaints. Each participant conducted a minimum of 10 MenHealth uroflowmetry measurements at home and 2 standard in-office uroflowmeter tests in our office. Maximum and average flow rates and voided volume were recorded. A comparison of averaged results of MenHealth uroflowmetry and in-office uroflowmeter was performed using a Bland-Altman analysis and a Passing-Bablok nonparametric regression analysis. RESULTS: Regression data analysis indicated a very strong correlation between maximum flow rate and average flow rate when comparing MenHealth uroflowmetry to in-office uroflowmeter (Pearson's correlation coefficients of .91 and .92, respectively). Insignificant difference in mean maximum and average flow rates for Groups 1 and 2 (< 0.5 ml/second) also proves strong correlation between the 2 methods and accuracy of MenHealth uroflowmetry. CONCLUSIONS: Data obtained by a novel MenHealth uroflowmetry app is equivalent to results from a standard in-office uroflowmeter for men with and without voiding symptoms. MenHealth uroflowmetry permits repetitive measurements in a more comfortable, "at home" setting, which provides a more comprehensive analysis, a clearer, nuanced picture of the patient's pathophysiology, and a lesser chance of misdiagnosis.

Upregulation of TRAG3 gene in urothelial carcinoma of the bladder.

Conventional chemotherapy is commonly used for advanced stages of bladder cancer with modest success and high morbidity. Identifying markers of resistance will allow clinicians to tailor treatment to a specific patient population. T24-tumorigenic cell line was grown orthotopically in nude mice and monitored using bioluminescence imaging and microcomputed tomography until they developed metastases. Stable sublines were then developed from primary bladder (T24-P), lung (T24-L) and bone (T24-B) tissues. Chromosomal analysis and DNA microarray were used to characterize these sublines. Real-time quantitative polymerase chain reaction and immunohistochemistry were used for validation. Epigenetic modifiers were used to study gene regulation. The cell viability was quantified with MTT assay. Chromosomal analysis revealed multiple alterations in metastatic cell lines compared to T24-P. DNA microarray analysis showed that taxol resistance-associated gene (TRAG) 3 was the most upregulated gene. From real-time quantitative polymerase chain reaction and immunohistochemistry, TRAG3 was significantly higher in T24-L and T24-B than T24-P. TRAG3 gene expression is likely controlled by DNA methylation but not histone acetylation. Interestingly, T24-B and T24-L cells were more resistant than T24-P to treatment with antimicrotubule agents such as docetaxel, paclitaxel and vinblastine. TRAG3 mRNA expression was higher in 20% of patients with ≤ pT2 (n = 10) and 60% of patients with ≥ pT3 (n = 20) compared to normal adjacent tissue (p = 0.05). In addition, the median TRAG3 expression was 6.7-fold higher in ≥ pT3 tumors compared to ≤ pT2 tumors. Knowing the status of TRAG3 expression could help clinicians tailor treatment to a particular patient population that could benefit from treatment, while allocating patients with resistant tumors to new experimental therapies.

Copy number variants of schizophrenia susceptibility loci are associated with a spectrum of speech and developmental delays and behavior problems.

PURPOSE: : Recently, molecular cytogenetic techniques have identified novel copy number variants in individuals with schizophrenia. However, no large-scale prospective studies have been performed to characterize the broader spectrum of phenotypes associated with such copy number variants in individuals with unexplained physical and intellectual disabilities encountered in a diagnostic setting. METHODS: : We analyzed 38,779 individuals referred to our diagnostic laboratory for microarray testing for the presence of copy number variants encompassing 20 putative schizophrenia susceptibility loci. We also analyzed the indications for study for individuals with copy number variants overlapping those found in six individuals referred for schizophrenia. RESULTS: : After excluding larger gains or losses that encompassed additional genes outside the candidate loci (e.g., whole-arm gains/losses), we identified 1113 individuals with copy number variants encompassing schizophrenia susceptibility loci and 37 individuals with copy number variants overlapping those present in the six individuals referred to our laboratory for schizophrenia. Of these, 1035 had a copy number variant of one of six recurrent loci: 1q21.1, 15q11.2, 15q13.3, 16p11.2, 16p13.11, and 22q11.2. The indications for study for these 1150 individuals were diverse and included developmental delay, intellectual disability, autism spectrum, and multiple congenital anomalies. CONCLUSION: : The results from our study, the largest genotype-first analysis of schizophrenia susceptibility loci to date, suggest that the phenotypic effects of copy number variants associated with schizophrenia are pleiotropic and imply the existence of shared biologic pathways among multiple neurodevelopmental conditions.

Deletions and duplications of developmental pathway genes in 5q31 contribute to abnormal phenotypes.

Although copy number changes of 5q31 have been rarely reported, deletions have been associated with some common characteristics, such as short stature, failure to thrive, developmental delay (DD)/intellectual disability (ID), club feet, dislocated hips, and dysmorphic features. We report on three individuals with deletions and two individuals with duplications at 5q31, ranging from 3.6 Mb to 8.1 Mb and 830 kb to 3.4 Mb in size, respectively. All five copy number changes are apparently de novo and involve several genes that are important in developmental pathways, including PITX1, SMAD5, and WNT8A. The individuals with deletions have characteristic features including DD, short stature, club feet, cleft or high palate, dysmorphic features, and skeletal anomalies. Haploinsufficiency of PITX1, a transcription factor important for limb development, is likely the cause for the club feet, skeletal anomalies, and cleft/high palate, while additional genes, including SMAD5 and WNT8A, may also contribute to additional phenotypic features. Two patients with deletions also presented with corneal anomalies. To identify a causative gene for the corneal anomalies, we sequenced candidate genes in a family with apparent autosomal dominant keratoconus with suggestive linkage to 5q31, but no mutations in candidate genes were found. The duplications are smaller than the deletions, and the patients with duplications have nonspecific features. Although development is likely affected by increased dosage of the genes in the region, the developmental disruption appears less severe than that seen with deletion.

The development of a rapid assay for prenatal testing of common aneuploidies and microdeletion syndromes.

OBJECTIVE: To develop a novel, rapid prenatal assay for pregnancies with high likelihood of normal karyotypes, using BACs-on-Beads(™) technology, a suspension array-based multiplex assay that employs Luminex(®) xMAP(®) technology, for the detection of gains and losses in chromosomal DNA. METHODS: Fifteen relatively common microdeletions were selected that are not detectable, or may be missed, by karyotyping and usually do not present with abnormal ultrasound findings. Chromosomes 13, 18, 21, X, and Y were included. We validated the assay with 430 samples. RESULTS: All microdeletions and aneuploidies were correctly identified, except for a 69,XXX incorrectly identified as a normal female and a male with ∼20% maternal cell contamination (MCC) that could not be distinguished from 69,XXY. MCC became apparent at 20 to 30%. Mosaicism was identified at 30 to 35% abnormal cells. CONCLUSION: We have developed an alternative to fluorescence in situ hybridization (FISH) aneuploidy screening and microarray analysis in otherwise normal pregnancies undergoing invasive testing. We demonstrated that the assay will detect all microdeletions and aneuploidies of regions covered on the assay. We developed analytical software that displays results for well-characterized syndromes but not abnormalities of unclear clinical significance. This assay is likely to be preferred by women seeking testing beyond routine karyotyping but who desire more information than provided by aneuploidy FISH.

Loss of Blm enhances basal cell carcinoma and rhabdomyosarcoma tumorigenesis in Ptch1+/- mice.

Basal cell carcinomas (BCCs) have relative genomic stability and relatively benign clinical behavior but whether these two are related causally is unknown. To investigate the effects of introducing genomic instability into murine BCCs, we have compared ionizing radiation-induced tumorigenesis in Ptch1(+/-) mice versus that in Ptch1(+/-) mice carrying mutant Blm alleles. We found that BCCs in Ptch1(+/-) Blm(tm3Brd/tm3Brd) mice had a trend toward greater genomic instability as measured by array comprehensive genomic hybridization and that these mice developed significantly more microscopic BCCs than did Ptch1(+/-) Blm(+/tm3Brd) or Ptch1(+/-) Blm(+/+) mice. The mutant Blm alleles also markedly enhanced the formation of rhabdomyosarcomas (RMSs), another cancer to which Ptch1(+/)(-) mice and PTCH1(+/)(-) (basal cell nevus syndrome) patients are susceptible. Highly recurrent but different copy number changes were associated with the two tumor types and included losses of chromosomes 4 and 10 in all BCCs and gain of chromosome 10 in 80% of RMSs. Loss of chromosome 11 and 13, including the Trp53 and Ptch1 loci, respectively, occurred frequently in BCCs, suggesting tissue-specific selection for genes or pathways that collaborate with Ptch deficiency in tumorigenesis. Despite the quantitative differences, there was no dramatic qualititative difference in the BCC or RMS tumors associated with the mutant Blm genotype.

The molecular mechanism underlying Roberts syndrome involves loss of ESCO2 acetyltransferase activity.

Roberts syndrome/SC phocomelia (RBS) is an autosomal recessive disorder with growth retardation, craniofacial abnormalities and limb reduction. Cellular alterations in RBS include lack of cohesion at the heterochromatic regions around centromeres and the long arm of the Y chromosome, reduced growth capacity, and hypersensitivity to DNA damaging agents. RBS is caused by mutations in ESCO2, which encodes a protein belonging to the highly conserved Eco1/Ctf7 family of acetyltransferases that is involved in regulating sister chromatid cohesion. We identified 10 new mutations expanding the number to 26 known ESCO2 mutations. We observed that these mutations result in complete or partial loss of the acetyltransferase domain except for the only missense mutation that occurs in this domain (c.1615T>G, W539G). To investigate the mechanism underlying RBS, we analyzed ESCO2 mutations for their effect on enzymatic activity and cellular phenotype. We found that ESCO2 W539G results in loss of autoacetyltransferase activity. The cellular phenotype produced by this mutation causes cohesion defects, proliferation capacity reduction and mitomycin C sensitivity equivalent to those produced by frameshift and nonsense mutations associated with decreased levels of mRNA and absence of protein. We found decreased proliferation capacity in RBS cell lines associated with cell death, but not with increased cell cycle duration, which could be a factor in the development of phocomelia and cleft palate in RBS. In summary, we provide the first evidence that loss of acetyltransferase activity contributes to the pathogenesis of RBS, underscoring the essential role of the enzymatic activity of the Eco1p family of proteins.

Copy number variations associated with autism spectrum disorders contribute to a spectrum of neurodevelopmental disorders.

PURPOSE: Autism spectrum disorders represent a range of neurodevelopmental disorders that have been shown to have a strong genetic etiological component. Microarray-based comparative genomic hybridization and other molecular cytogenetic techniques are discovering an increasing number of copy number variations in individuals with autism spectrum disorder. METHODS: We examined the yield of abnormal microarray-based comparative genomic hybridization findings in our laboratory for individuals referred for testing for autism spectrum disorder. We also examined the presence of autistic features among 151 additional individuals who were referred for microarray-based comparative genomic hybridization testing for indications other than autism spectrum disorder but had genomic alterations overlapping those found in cases referred for autism spectrum disorder. RESULTS: We identified 1461 individuals referred for testing for autism spectrum disorder, with likely significant abnormalities reported in approximately 11.6% of individuals analyzed with whole-genome arrays. These abnormalities include alterations that encompass novel candidate genes such as SNTG2, SOX5, HFE, and TRIP38. A minority of individuals with overlapping abnormalities (19%) had autistic features, and many of the copy number variations identified in our study are inherited (69% among those found in individuals with autism spectrum disorder). CONCLUSIONS: Our results suggest these copy number variations are one of multiple factors contributing to the development of an autism spectrum disorder phenotype. Additionally, the broad phenotypic spectrum of the patients with these copy number variations suggests that these copy number variations are not autism spectrum disorder-specific but likely more generally impair neurodevelopment.

Establishment and characterization of a cancer cell line derived from an aggressive childhood liver tumor.

BACKGROUND: Hepatoblastoma is a rare malignancy of childhood. The scarcity of adequate cell models has limited our understanding of this tumor. Here we describe and characterize a new human liver tumor cell line, Hep293TT, derived from an aggressive childhood hepatoblastoma. PROCEDURES: Hep293TT cells were established using primary tumor tissues from a 5-year-old Caucasian female child. This cell line has been maintained for more than 34 months and over 20 subcultures, and was characterized by histopathology, ELISA, genotype, cytogenetics, CGH array, immunohistochemistry, and molecular sequence analyses. RESULTS: Cells were confirmed to originate from parental tumor cells, secrete alpha-fetoprotein, and express hepatic markers and beta-catenin. Hep293TT cells were able to form colonies in soft agar. Tumorigenicity was demonstrated by induction of solid tumors after subrenal capsule injection in immunodeficient mice. Hep293TT cells demonstrated a highly aneuploid karyotype, and a whole genome CGH analysis revealed chromosomal imbalances in every chromosome. Allelotype analysis demonstrated loss of alleles at distal 11p15.5 as is typical of embryonal tumors. Both Hep293TT cells and the primary tumor contain a deletion of 351 nucleotides in beta-catenin, as has been seen in other hepatoblastoma tumors. The cell line expressed beta-catenin protein in both full-length and partially deleted forms, and expressed NOTCH2 protein characteristic of hepatoblasts. No mutation was detected in the APC, MYH, MLH1, or MSH2 genes. CONCLUSION: This cell line, Hep293TT, is a valuable resource for the study of childhood liver cancer and may potentially provide a tool in the development of new agents.