Evolution of germline TP53 variant classification in children with cancer.

Li-Fraumeni syndrome, caused by germline pathogenic variants in TP53, results in susceptibility to multiple cancers. Variants of uncertain significance (VUS) and reclassification of variants over time pose management concerns given improved survival with cancer surveillance for LFS patients. We describe the experience of TP53 variant reclassification at a pediatric cancer center. METHODS: We reviewed medical records (2010-2019) of 756 patients seen in Texas Children's Cancer Genetics Clinic. We noted initial TP53 classification and any reclassifications. We then classified TP53 variants following ClinGen TP53 variant curation expert panel recommendations using data from ClinVar, medical literature and IARC database. RESULTS: Of 234 patients tested for TP53, 27 (11.5%) reports contained pathogenic/likely pathogenic (P/LP) variants and 7 (3)% contained VUS. By January 2022, 4 of 6 unique VUS and 2 of 16 unique P/LP variants changed interpretations in ClinVar. Reinterpretation of these 4 VUS in ClinVar matched clinical decision at the time of initial report. Applying TP53 VCEP specifications classified 3 VUS to P/LP/benign, and one pathogenic variant to likely benign. CONCLUSIONS: Planned review of variant significance is essential, especially for patients with high probability of LFS.

Genomic profiling in Down syndrome acute lymphoblastic leukemia identifies histone gene deletions associated with altered methylation profiles.

Patients with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) have distinct clinical and biological features. Whereas most DS-ALL cases lack the sentinel cytogenetic lesions that guide risk assignment in childhood ALL, JAK2 mutations and CRLF2 overexpression are highly enriched. To further characterize the unique biology of DS-ALL, we performed genome-wide profiling of 58 DS-ALL and 68 non-DS (NDS) ALL cases by DNA copy number, loss of heterozygosity, gene expression and methylation analyses. We report a novel deletion within the 6p22 histone gene cluster as significantly more frequent in DS-ALL, occurring in 11 DS (22%) and only 2 NDS cases (3.1%) (Fisher's exact P=0.002). Homozygous deletions yielded significantly lower histone expression levels, and were associated with higher methylation levels, distinct spatial localization of methylated promoters and enrichment of highly methylated genes for specific pathways and transcription factor-binding motifs. Gene expression profiling demonstrated heterogeneity of DS-ALL cases overall, with supervised analysis defining a 45-transcript signature associated with CRLF2 overexpression. Further characterization of pathways associated with histone deletions may identify opportunities for novel targeted interventions.

Significant differences among physician specialties in management recommendations of BRCA1 mutation carriers.

The National Comprehensive Cancer Network (NCCN) has published guidelines for hereditary breast and ovarian cancer syndrome (HBOCS) management. Little data exist on compliance with these guidelines among different physician specialties. We performed an on-line case-based survey by randomly sampling physicians from five specialties, Family Medicine (FM), Obstetrics and Gynecology (OG), General Surgery (GS), Internal Medicine (IM), and Hematology and Oncology (HO). The physicians (n = 225) were asked to provide HBOCS management of healthy women ages 40-42 in the presence of a familial BRCA1 mutation. For women negative for the BRCA1 mutation, 59% of the physicians recommended appropriate surveillance although with significant differences among specialties; P = 0.01. Using an aggregate screening intensity score, physicians clearly recommended more intense screening for mutation positive than negative women (P < 0.0001), but only 16% of physicians followed NCCN guidelines for BRCA1-positive women. Seventy-six percent of all physicians recommended breast MRI with significant variation among specialties ranging from 62% of FM to 89% of OG (P = 0.0020). Similarly, 63% of physicians recommended prophylactic oophorectomy, with 76 and 78% of GS and OG compared to 38% of IM (P < 0.0001) and 57% recommended prophylactic mastectomy ranging from 84% of HO to 32% of FM (P < 0.0001). Independent of specialty, respondents with BRCA testing experience recommended more intense management than those without; P = 0.021. Management recommendations of BRCA1 mutation carriers are not consistent with NCCN guidelines and vary by medical specialty and genetic testing experience. Targeted education of physicians by specialty is needed, so that optimal management is offered to these high-risk women.

Revisiting the craniosynostosis-radial ray hypoplasia association: Baller-Gerold syndrome caused by mutations in the RECQL4 gene.

Baller-Gerold syndrome (BGS) is a rare autosomal recessive condition with radial aplasia/hypoplasia and craniosynostosis (OMIM 218600). Of >20 cases reported so far, a few appear atypical and have been reassigned to other nosologic entities, including Fanconi anaemia, Roberts SC phocomelia, and Pfeiffer syndromes after demonstration of corresponding cytogenetic or molecular abnormalities. Clinical overlap between BGS, Rothmund-Thomson syndrome (RTS), and RAPADILINO syndrome is noticeable. Because patients with RAPADILINO syndrome and a subset of patients with RTS have RECQL4 mutations, we reassessed two previously reported BGS families and found causal mutations in RECQL4 in both. In the first family, four affected offspring had craniosynostosis and radial defect and one of them developed poikiloderma. In this family, compound heterozygosity for a R1021W missense mutation and a g.2886delT frameshift mutation of exon 9 was found. In the second family, the affected male had craniosynostosis, radial ray defect, poikiloderma, and short stature. He had a homozygous splice site mutation (IVS17-2A>C). In both families, the affected offspring had craniosynostosis, radial defects, and growth retardation, and two developed poikiloderma. Our results confirm that BGS in a subgroup of patients is due to RECQL4 mutations and could be integrated into a clinical spectrum that encompasses RTS and RAPADILINO syndrome.

Outcomes of systematic screening for optic pathway tumors in children with Neurofibromatosis Type 1.

Optic pathway tumors (OPT) occur in about 15% of individuals with Neurofibromatosis Type 1 (NF1) and may effect substantial visual loss. Because their growth is not predictable at the time of discovery, neuroimaging for OPT in asymptomatic NF1 patients remains controversial. We evaluated the outcomes of systematic screening by both MRI and ophthalmic examinations for OPT in young children with NF1 seen at multi-disciplinary clinics for Neurofibromatosis and Genetics at one institution between 1996 and 2001. We report on 84 children who presented with NF1 under age 6 years, of whom 13 children presented with either known OPT or abnormal MRI findings and 11 children had OPTs identified by neuroimaging, including two children with abnormal eye examinations at presentation (one with strabismus and one with optic atrophy). Nine OPTs were detected in asymptomatic subjects with normal ophthalmic examinations. Three children with chiasmal lesions enlarging on subsequent MRI were treated with carboplatin and vincristine. After treatment, the vision in each involved eye was intact. In contrast, the 13 children with OPT diagnosed outside of screening guidelines included five children with substantial visual loss. Our observations suggest that early recognition of NF1 promotes appropriate surveillance and allows early intervention to reduce complications of OPT. This analysis supports prospective studies to compare the outcomes of systematic screening with neuroimaging to screening with ophthalmic examinations alone in children with NF1.

Cell cycle-dependent expression and nucleolar localization of hCAP-H.

Condensin is a conserved 13S heteropentamer composed of two nonidentical structural maintenance of chromosome (SMC) family proteins, in Xenopus XCAP-C and XCAP-E, and three regulatory subunits, XCAP-D2, XCAP-G, and XCAP-H. Both biochemical and genetic analyses have demonstrated an essential role for the 13S condensin complex in mitotic chromosome condensation. Further, a potential requirement for condensin in completion of chromatid arm separation in early anaphase is demonstrated by the mutational phenotypes of the Drosophila homologues of XCAP-H, barren and XCAP-C, DmSMC4. In this study we have investigated the expression and subcellular distribution of hCAP-H, the human homolog of XCAP-H, in order to better understand its cellular functions. Transcription of hCAP-H was restricted to proliferating cells with highest expression during the G(2) phase of the cell cycle. In contrast, cellular hCAP-H protein levels were constant throughout the cell cycle. hCAP-H was found to be associated with mitotic chromosomes exhibiting a nonuniform but symmetric distribution along sister chromatids. The symmetry of hCAP-H association with sister chromatids suggests that there are sequence-dependent domains of condensin aggregation. During interphase hCAP-H, -C, and -E, have distinct punctate nucleolar localization, suggesting that condensin may associate with and modulate the conformation and function of rDNA. hCAP-H association with condensed chromatin was not observed in the early phase of chromosome condensation when histone H3 phosphorylation has already taken place. This finding is consistent with the hypothesis that histone H3 phosphorylation precedes condensin-mediated condensation.

Expression and localization of the CDC34 ubiquitin-conjugating enzyme in pediatric acute lymphoblastic leukemia.

Ubiquitin-dependent protein degradation impacts many cellular processes.However, the regulation of ubiquitin-conjugating enzymes (UBCs) in cancer is unknown. We find that the human CDC34 UBC protein is expressed at a 3-4 fold higher level (P < 0.001) in pediatric T cell than in pre-B-cell acute lymphoblastic leukemia (ALL) before treatment in two independent patient sets. The level of CDC34 mRNA was similar in both types of leukemia. CDC34 expression levels in normal resting T cells, B cells and activated T lymphocytes was comparable with pre-B-cell ALL. CDC34 protein (but not mRNA) was also increased in T-cell ALL compared with pre-B-cell ALL cell lines. The difference in expression was not attributable to mutation or associated with altered CDC34 stability. Immunohistochemistry and cellular fractionation reveals a heterogeneous CDC34 expression pattern including cells containing primarily cytoplasmic or nuclear protein. Thus, a feature of pediatric T-cell ALL is posttranscriptional up-regulation and heterogeneous localization of the human CDC34 UBC.

Clinical manifestations in a cohort of 41 Rothmund-Thomson syndrome patients.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive genodermatosis characterized by a poikilodermatous rash starting in infancy, small stature, skeletal abnormalities, juvenile cataracts, and predisposition to specific cancers. We have identified a contemporary cohort of 41 patients to better define the clinical profile, diagnostic criteria, and management of patients with RTS. Patients with the diagnosis of RTS were ascertained by referrals from dermatology, ophthalmology, genetics, and oncology or from direct contact with the patient's family. Medical information was obtained from interviews with physicians, patients, and their parents and a review of medical records. The age range at ascertainment was 9 months to 42 years (28 males and 13 females; M:F, 2:1). All subjects displayed a characteristic rash. Thirteen subjects had osteosarcoma (OS) (32%), eight had radial defects (20%), seven had gastrointestinal findings (17%), two had cataracts (6%), and one had skin cancer (2%). Twenty-two of 28 patients without OS were less than 15 years old and thus remain at significant risk for this tumor. This case-series study reveals a clinical profile of RTS that includes a higher prevalence of OS and fewer cataracts, compared with historical reports. These differences may reflect either allelic or genetic heterogeneity. This study documents the frequency of clinical anomalies in a contemporary cohort of RTS patients and revises guidelines for diagnosis and management of RTS.

FBN1 exon 2 splicing error in a patient with Marfan syndrome.

Mutations in FBN1 cause the autosomal dominant condition, Marfan syndrome. A single-base mutation that results in a skipping of exon 2 of FBN1 was found in a Marfan patient. By sequencing this proband's entire FBN1 gene and comparing the mutated DNA sequence with proband's unaffected family numbers, we confirmed this alteration was the causative mutation. The skipping of exon 2 creates a frameshift and premature termination codon, and forms a truncated fibrillin-1 composed only of 55 amino acids of N-terminus plus 45 nonsense amino acids. The mRNA transcription levels of the mutated FBN1 allele and the deposition of fibrillin-1 into extracellular matrix in fibroblast cells culture were assessed.

Neoplasms in neurofibromatosis 1 are related to gender but not to family history of cancer.

The risk of malignancies among persons with neurofibromatosis 1 (NF1) is higher than in the general population, but the excess risk has not been precisely estimated. The effects of gender and inheritance pattern on cancer risk are unclear. Therefore, we conducted a historical cohort study to determine cancer risk factors by contacting 138 Caucasian NF1 patients originally seen at Baylor College of Medicine (BCM) in Houston between 1978 and 1984. A total of 304 patients of all ethnic groups were evaluated at BCM during this period. We successfully located 173 patients, 138 of who were Caucasian. We computed standardized incidence ratios (SIRs) with the age-, gender-, and time period-specific rates from the Connecticut Tumor Registry for 2,094 person-years of observation (median follow-up = 16 years). Eleven incident tumors were reported. Females were at much higher risk of cancer than males (SIR = 5.6, 95% confidence interval (CI) 2.7-10.3 and SIR = 0.6; 95% CI, 0.0-3.0, respectively). We found no elevated cancer risk in unaffected first-degree relatives, regardless of whether the proband had cancer or not (SIR = 1.1 95% CI, 0.6-1.8 and SIR = 1.0, 95% CI, 0.6-1.5, respectively). Our results suggest that malignancy in the proband is not the result of a modifying gene that has a significant impact on general cancer risk.

Mammography behavior after receiving a negative BRCA1 mutation test result in the Ashkenazim: a community-based study.

PURPOSE: To define the impact of a negative BRCA1 test result on subsequent breast cancer screening behavior in women. METHODS: Longitudinal study of a community-based sample of Ashkenazi Jews offered testing for the 185delAG BRCA1 mutation in 1996. Of 309 participants, 118 women were mutation negative, of average risk (based on family history of cancer), unaffected with breast cancer, and provided complete data at baseline, and Year 1 and Year 2 follow-up questionnaires. RESULTS: Women age 50 and older had 91.7% compliance with mammography for the year prior to entry (baseline), 88.3% during Year 1, 91.7% during Year 2 (no significant change; P = 0.775). Women under age 50 demonstrated an increase in mammography (49.2% at baseline, 62.7% Year 1, and 67.1% Year 2; P = 0.035). Both groups demonstrated significant decreases in breast cancer worry and perceived risk. Logistic regression analysis on having a mammogram at Year 2 showed that age, physician recommendation, worry, and perceived risk were all significant. CONCLUSION: Receipt of negative BRCA1 test results in a cohort of Ashkenazi Jewish women did not have a negative impact on mammography behavior 2 years after genetic testing.

Psychological and behavioral factors associated with colorectal cancer screening among Ashkenazim.

BACKGROUND: Psychological and behavioral factors related to annual colorectal cancer (CRC) screening were examined in a sample of Ashkenazi Jewish individuals. Identification of factors related to regular CRC screening in this population is important because of the possibility of a heightened incidence of CRC. METHODS: Eligible participants were 171 Ashkenazi Jewish adults 40 years or older attending an educational program about breast cancer genetics. Compliance with recommended guidelines for digital rectal examination and fecal occult blood test in the past year were dependent measures. Demographic variables, family history of CRC, perceived risk, physician recommendation, and worry about cancer were independent measures. RESULTS: Digital rectal examinations and fecal occult blood tests had been obtained in the past year by 46 and 31% of the participants, respectively. A logistic regression showed that physician recommendation was related significantly to obtaining digital rectal examinations. Physician recommendation and education were related significantly to obtaining fecal occult blood tests. Although participants with family histories of CRC perceived themselves as being at increased risk of developing CRC, and were more worried about developing colon cancer, they were no more likely to adhere to CRC screening guidelines than those without such histories. CONCLUSIONS: Overall, compliance with recommended CRC screening was low even among high-risk individuals. Physicians play a key role in motivating people to comply with CRC screening. Physicians need to en courage all asymptomatic patients 50 years and older to be screened for CRC.

Human Cdc34 and Rad6B ubiquitin-conjugating enzymes target repressors of cyclic AMP-induced transcription for proteolysis.

Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes. However, few in vivo targets of the mammalian Cdc34 and Rad6 ubiquitin-conjugating enzymes are known. A yeast-based genetic assay to identify proteins that interact with human Cdc34 resulted in three cDNAs encoding bZIP DNA binding motifs. Two of these interactants are repressors of cyclic AMP (cAMP)-induced transcription: hICERIIgamma, a product of the CREM gene, and hATF5, a novel ATF homolog. Transfection assays with mammalian cells demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteolysis of hICERIIgamma and hATF5. This degradation requires an active ubiquitin-conjugating enzyme and results in abrogation of ICERIIgamma- and ATF5-mediated repression of cAMP-induced transcription. Consistent with these results, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B-/-) or transfected with dominant negative and antisense constructs of human CDC34. Based on the requirement for CREM/ICER and Rad6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1-Cullin-F-box ubiquitin protein ligase subunits Cul-1 and Cul-2, which are associated with Cdc34 activity during murine testicular development. Cdc34, Rad6B, and the Cullin proteins are expressed in a developmentally regulated manner, with distinctly different patterns for Cdc34 and the Cullin proteins in germ cells. The Cdc34 and Rad6B proteins are significantly elevated in meiotic and postmeiotic haploid germ cells when chromatin modifications occur. Thus, the stability of specific mammalian transcription factors is the result of complex targeting by multiple ubiquitin-conjugating enzymes and may have an impact on cAMP-inducible gene regulation during both meiotic and mitotic cell cycles.

Growth deficiency and malnutrition in Bloom syndrome.

OBJECTIVES: To describe the growth and nutritional status of a pediatric population with Bloom syndrome. STUDY DESIGN: Longitudinal growth data from 148 patients in the Bloom's Syndrome Registry (85 male, 63 female) were compiled retrospectively from physician and parent records to develop graphed statistics of weight-for-age, height-for-age, fronto-occipital circumference-for-age, and weight-for-height for both sexes with comparisons with the normal population. RESULTS: Term birth measurements confirm that the growth deficiency of Bloom syndrome has prenatal onset. Stunting persists throughout life, and an adolescent growth spurt is not apparent from the smoothed data. Growth continues by at least 1 cm/yr until age 21 years for both sexes. More than half of children with Bloom syndrome are significantly wasted until age 8 years, which is not related to early death or underlying malignancy. The mean body mass index for adults with Bloom syndrome after age 25 years is low normal (n = 22, mean = 20.2 kg/m2). CONCLUSIONS: Children with Bloom syndrome have significant growth retardation and wasting.

Psychological impact of receiving negative BRCA1 mutation test results in Ashkenazim.

PURPOSE: Most DNA test results for breast/ovarian cancer susceptibility are negative. Because negative test results might be interpreted incorrectly and may have serious psychological and behavioral implications, determining the psychological impact of such results is important. METHODS: A community-based sample of 289 Ashkenazim was tested for 185delAG. The 199 mutation-negatives provided data at baseline and follow-up. Increased risk participants included those who received negative test results but remained at increased risk because positive family and/or personal histories of breast or ovarian cancer made the results uninformative. Average risk meant those who tested negative and had negative family and personal histories of breast or ovarian cancer. Using a logistic regression analysis, both groups' psychological distress levels were compared at baseline and at 1 and 6 months after notification of DNA test results. RESULTS: A logistic regression analysis showed significant but small differences in cancer-specific distress after 6 months between increased and average risk participants (P < 0.006). Increased risk participants reported more distress than average risk. General distress declined among all participants after 1 month. Although baseline and follow-up differences in cancer-specific distress obtained by the increased and average risk participants were statistically significant, none of the absolute levels observed reflected especially high degrees of stress. CONCLUSIONS: Receipt of negative DNA test results does not have a deleterious psychological impact, whether results are informative or uninformative.

Screening and clinical implications for BRCA1 and BRCA2 mutation carriers.

In this article, we review the history of testing for mutations in breast cancer susceptibility genes and discuss the current state of testing for mutations in BRCA1 and BRCA2 in different clinical settings including at-risk individuals and cancer patients. The risk of breast cancer. other associated malignancies and prognosis in carriers of these mutations are reviewed. A final section includes discussion of current recommendations for surveillance and the need for further research to identify environmental and genetic factors which modify the risk of developing cancer in mutation carriers.

Reconstitution of a MEC1-independent checkpoint in yeast by expression of a novel human fork head cDNA.

A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae. CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S. cerevisiae, including mec1, rad9, rad24, dun1, and rad53. CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains. Presence of CHES1 but not a control vector resulted in G2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain. CHES1 can also suppress the lethality, UV sensitivity, and G2 checkpoint defect of a mec1 null mutation. In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain. Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors. Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site. Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast.

Screening for 185delAG in the Ashkenazim.

A study was initiated to assess interest, educational effectiveness, and implications of genetic testing for the common BRCA1 mutation, 185delAG, in the Ashkenazim. Of 333 individuals who attended group sessions, 309 (92%) participated in the study. Participants were categorized as having negative family history (67%), positive family history (defined, by a relaxed criterion, as one first-degree relative or two second-degree relatives with breast [premenopausal] or ovarian cancer) (22%), positive personal history (7%), and both positive personal history and positive family history (4%). Group education was effective, as shown by the improvement in participant scores from pre- to posteducation tests. For the 289 individuals (94%) who requested testing, the major reasons included concern for their own risk, concern for the risk of their children, and desire to learn about surveillance options. The most common reason given by participants who declined testing was concern about health insurance. Six participants found to be heterozygous for the 185delAG mutation received results and were offered genetic counseling. Participants had consented for additional testing without receiving results and were screened for the 6174delT mutation in BRCA2, and seven were found to be positive. All identified carriers reported at least one first- or second-degree relative with a history of breast or ovarian cancer, although they did not all meet our study criteria for positive family history. Given these outcomes, we conclude that screening for breast and ovarian cancer susceptibility is most appropriate for individuals with a positive personal or positive family cancer history. We propose a guideline for future studies designed to identify individuals who may benefit from genetic testing for inherited breast and ovarian cancer.