John Lane

Somatic USP8 Gene Mutations Are a Common Cause of Pediatric Cushing Disease

Context: Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have been recently identified as the most common genetic alteration in patients with Cushing disease (CD). However, the frequency of these mutations in the pediatric population has not been extensively assessed. Objective: We investigated the status of the USP8 gene at the somatic level in a cohort of pediatric patients with corticotroph adenomas. Design and Methods: The USP8 gene was fully sequenced in both germline and tumor DNA samples from 42 pediatric patients with CD. Clinical, biochemical, and imaging data were compared between patients with and without somatic USP8 mutations. Results: Five different USP8 mutations (three missense, one frameshift, and one in-frame deletion) were identified in 13 patients (31%), all of them located in exon 14 at the previously described mutational hotspot, affecting the 14-3-3 binding motif of the protein. Patients with somatic mutations were older at disease presentation [mean 5.1 ± 2.1 standard deviation (SD) vs 13.1 ± 3.6 years, P = 0.03]. Levels of urinary free cortisol, midnight serum cortisol, and adrenocorticotropic hormone, as well as tumor size and frequency of invasion of the cavernous sinus, were not significantly different between the two groups. However, patients harboring somatic USP8 mutations had a higher likelihood of recurrence compared with patients without mutations (46.2% vs 10.3%, P = 0.009). Conclusion: Somatic USP8 gene mutations are a common cause of pediatric CD. Patients harboring a somatic mutation had a higher likelihood of tumor recurrence, highlighting the potential importance of this molecular defect for the disease prognosis and the development of targeted therapeutic options.

Corticotropinoma as a Component of Carney Complex

Known germline gene abnormalities cause one-fifth of the pituitary adenomas in children and adolescents, but, in contrast with other pituitary tumor types, the genetic causes of corticotropinomas are largely unknown. In this study, we report a case of Cushing disease (CD) due to a loss-of-function mutation in PRKAR1A, providing evidence for association of this gene with a corticotropinoma. A 15-year-old male presenting with hypercortisolemia was diagnosed with CD. Remission was achieved after surgical resection of a corticotropin (ACTH)-producing pituitary microadenoma, but recurrence 3 years later prompted reoperation and radiotherapy. Five years after the original diagnosis, the patient developed ACTH-independent Cushing syndrome, and a diagnosis of primary pigmented nodular adrenocortical disease was confirmed. A PRKAR1A mutation (c.671delG, p.G225Afs*16) was detected in a germline DNA sample from the patient, which displayed loss of heterozygosity in the corticotropinoma. No other germline or somatic mutations of interest were found. As corticotropinomas are not a known component of Carney complex (CNC), we performed loss of heterozygosity and messenger RNA stability studies in the patient’s tissues, and analyzed the effect of Prkar1a silencing on AtT-20/D16v-F2 mouse corticotropinoma cells. No PRKAR1A defects were found among 97 other pediatric CD patients studied. Our clinical case and experimental data support a role for PRKAR1A in the pathogenesis of a corticotroph cell tumor. This is a molecularly confirmed report of a corticotropinoma presenting in association with CNC. We conclude that germline PRKAR1A mutations are a novel, albeit apparently infrequent, cause of CD.

Loss-of-function mutations in the CABLES1 gene are a novel cause of Cushing’s disease

The CABLES1 cell cycle regulator participates in the adrenal–pituitary negative feedback, and its expression is reduced in corticotropinomas, pituitary tumors with a largely unexplained genetic basis. We investigated the presence of CABLES1 mutations/copy number variations (CNVs) and their associated clinical, histopathological and molecular features in patients with Cushing’s disease (CD). Samples from 146 pediatric (118 germline DNA only/28 germline and tumor DNA) and 35 adult (tumor DNA) CD patients were screened for CABLES1 mutations. CNVs were assessed in 116 pediatric CD patients (87 germline DNA only/29 germline and tumor DNA). Four potentially pathogenic missense variants in CABLES1 were identified, two in young adults (c.532G > A, p.E178K and c.718C > T, p.L240F) and two in children (c.935G > A, p.G312D and c.1388A > G, and p.D463G) with CD; no CNVs were found. The four variants affected residues within or close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein and impaired its ability to block cell growth in a mouse corticotropinoma cell line (AtT20/D16v-F2). The four patients had macroadenomas. We provide evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene. Its function might link two of the main molecular mechanisms altered in corticotropinomas: the cyclin-dependent kinase/cyclin group of cell cycle regulators and the epidermal growth factor receptor signaling pathway. Further studies are needed to assess the prevalence of CABLES1 mutations among patients with other types of pituitary adenomas and to elucidate the pituitary-specific functions of this gene.

The Development of Proportional Reasoning in Qatar

Abstract The development of proportional reasoning in the Arabian Gulf State of Qatar and the United States was examined. The sample consisted of 554 children from Qatar and the U.S. from the fifth, sixth, and seventh grades. The children were administered two paper and pencil tests: the Orange Juice Test developed by Noelting (1980), which measured proportional reasoning, and the Ravens Standard Progressive Matrices Test, which was used as a nonverbal measure of general intelligence. Significant age group, sex, and country main effects and a significant Sex × Country interaction for proportional reasoning were determined even after controlling for nonverbal intelligence.

Association of mitochondrial DNA copy number with cardiovascular disease

Importance Mitochondrial dysfunction is a core component of the aging process and may play a key role in atherosclerotic cardiovascular disease. Mitochondrial DNA copy number (mtDNA-CN), which represents the number of mitochondria per cell and number of mitochondrial genomes per mitochondrion, is an indirect biomarker of mitochondrial function. Objective To determine whether mtDNA-CN, measured in an easily accessible tissue (buffy coat/circulating leukocytes), can improve risk classification for cardiovascular disease (CVD) and help guide initiation of statin therapy for primary prevention of CVD. Design, Setting, and Participants Prospective, population-based cohort analysis including 21 870 participants (20 163 free from CVD at baseline) from 3 studies: Cardiovascular Health Study (CHS), Atherosclerosis Risk in Communities Study (ARIC), and Multiethnic Study of Atherosclerosis (MESA). The mean follow-up was 13.5 years. The study included 11 153 participants from ARIC, 4830 from CHS, and 5887 from MESA. Analysis of the data was conducted from March 10, 2014, to January 29, 2017. Exposures Mitochondrial DNA-CN measured from buffy coat/circulating leukocytes. Main Outcomes and Measures Incident CVD, which combines coronary heart disease, defined as the first incident myocardial infarction or death owing to coronary heart disease, and stroke, defined as the first nonfatal stroke or death owing to stroke. Results Of the 21 870 participants, the mean age was 62.4 years (ARIC, 57.9 years; MESA, 62.4 years; and CHS, 72.5 years), and 54.7% of participants were women. The hazard ratios for incident coronary heart disease, stroke, and CVD associated with a 1-SD decrease in mtDNA-CN were 1.29 (95% CI, 1.24-1.33), 1.11 (95% CI, 1.06-1.16), and 1.23 (95% CI, 1.19-1.26). The associations persisted after adjustment for traditional CVD risk factors. Addition of mtDNA-CN to the 2013 American College of Cardiology/American Heart Association Pooled Cohorts Equations for estimating 10-year hard atherosclerosis CVD risk was associated with improved risk classification (continuous net reclassification index, 0.194; 95% CI, 0.130-0.258; P < .001). Mitochondrial DNA-CN further improved sensitivity and specificity for the 2013 American College of Cardiology/American Heart Association recommendations on initiating statin therapy for primary prevention of ASCVD (net 221 individuals appropriately downclassified and net 15 individuals appropriately upclassified). Conclusions and Relevance Mitochondrial DNA-CN was independently associated with incident CVD in 3 large prospective studies and may have potential clinical utility in improving CVD risk classification.

Association between mitochondrial DNA copy number and sudden cardiac death: Findings from the Atherosclerosis Risk in Communities study (ARIC)

Aims Sudden cardiac death (SCD) is a major public health burden. Mitochondrial dysfunction has been implicated in a wide range of cardiovascular diseases including cardiomyopathy, heart failure, and arrhythmias, but it is unknown if it also contributes to SCD risk. We sought to examine the prospective association between mtDNA copy number (mtDNA-CN), a surrogate marker of mitochondrial function, and SCD risk. Methods and results We measured baseline mtDNA-CN in 11 093 participants from the Atherosclerosis Risk in Communities (ARIC) study. mtDNA copy number was calculated from probe intensities of mitochondrial single nucleotide polymorphisms (SNP) on the Affymetrix Genome-Wide Human SNP Array 6.0. Sudden cardiac death was defined as a sudden pulseless condition presumed due to a ventricular tachyarrhythmia in a previously stable individual without evidence of a non-cardiac cause of cardiac arrest. Sudden cardiac death cases were reviewed and adjudicated by an expert committee. During a median follow-up of 20.4 years, we observed 361 SCD cases. After adjusting for age, race, sex, and centre, the hazard ratio for SCD comparing the 1st to the 5th quintiles of mtDNA-CN was 2.24 (95% confidence interval 1.58-3.19; P-trend <0.001). When further adjusting for traditional cardiovascular disease risk factors, prevalent coronary heart disease, heart rate, QT interval, and QRS duration, the association remained statistically significant. Spline regression models showed that the association was approximately linear over the range of mtDNA-CN values. No apparent interaction by race or by sex was detected. Conclusion In this community-based prospective study, mtDNA-CN in peripheral blood was inversely associated with the risk of SCD.

Abstract A25: Prevalence of Klinefelter syndrome among males aged 0-19 years diagnosed with germ cell tumors: A report from the Children’s Oncology Group

Previous studies have suggested that males with Klinefelter syndrome (KS; 47, XXY) may be more likely to develop germ cell tumors (GCTs), particularly mediastinal GCTs. Due to the rarity of pediatric GCTs, there are no reports characterizing the prevalence of KS among males diagnosed with GCTs. We used data from a Children’s Oncology Group epidemiology study (2008-2015) to evaluate the prevalence of KS in males (n=433) diagnosed with GCTs (aged 0-19 years). These 433 cases provided saliva samples and had one parent who was willing to participate in the study and complete a questionnaire including questions about health history, demographics, and environmental exposures. Tumor characteristics (location and histology) were abstracted from pathology reports provided by the treating institution. GCT cases were classified as having KS if they had evidence of an extra copy of the X chromosome based on evaluation of array data from Illumina HumanCoreExome-12 genotyping chips. Genvisis was used to identify samples with sex aneuploidy and to determine the parent-of-origin for the nondisjunction. Using chi-square tests, we examined differences in age at diagnosis, race/ethnicity, tumor location and histology, and a number of birth characteristics between KS-GCT cases and GCT cases without chromosomal abnormalities (n=415). Using publically available data, we estimated the 1-year risk and corresponding Risk Ratio (RR) and 95% Confidence Interval (95% CI) of a male with KS developing a GCT. Based on analysis of array genotyping data, 3% (n=13) of male GCT cases had KS. Among these 13 cases, the extra X chromosome was of maternal origin in 7 cases and of paternal origin in 6 cases. Of the 13 KS cases with genotyping data, 5/9 (56%) KS-GCT cases with parental questionnaire data reported receiving a diagnosis of KS. The average age at GCT diagnosis for cases with genotyping-detected KS (n=13) was 13.8 years (standard deviation [SD], 4.4 years) compared with 12.5 years (SD, 6.2 years) for GCT cases without chromosomal abnormalities (n=415; p=0.45). We did not observe significant differences in patient or birth characteristics including race, birth weight and length, maternal age, paternal age, and the use of fertility drugs between the two groups. We confirmed that KS-GCT cases were significantly more likely to be diagnosed with extragonadal tumors (GCT [n=69/411; 17%]; KS-GCT [n=11/13; 85%]; p In our large case series of males diagnosed with GCTs, we observed that 3% of GCT cases (n=13/433) were also carriers of an extra X chromosome based on array genotyping data and were thus classified as having KS. Males aged 0-19 years with KS experience a large increase in risk of developing a GCT compared with males from the general population. Collectively, these findings suggest that young males with KS should be monitored for the development of a GCT. Similarly, the possibility of KS should be considered in males diagnosed with a mediastinal GCT during childhood or adolescence and these patients should be tested for the presence of KS. Citation Format: Lindsay A. Williams, Nathan Pankratz, John Lane, Michelle Roesler, Michaela Richardson, A. Lindsay Frazier, James Amatruda, Jenny N. Poynter. Prevalence of Klinefelter syndrome among males aged 0-19 years diagnosed with germ cell tumors: A report from the Children’s Oncology Group [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A25.

Klinefelter syndrome in males with germ cell tumors: A report from the children’s oncology group: A report from the children\x92s oncology group

Males with Klinefelter syndrome (KS) (47,XXY) may be more likely to develop germ cell tumors (GCTs), particularly mediastinal GCTs. To date, there are no reports characterizing the prevalence of KS among male GCT cases.

Abstract A37: De novo and transmitted germline TP53 variation in pediatric osteosarcoma: A report from the Children's Oncology Group

Pathogenic germline variation in TP53, usually accompanied by familial Li-Fraumeni syndrome (LFS), is known to underly a small proportion of pediatric osteosarcoma (OS). However, the extent to which de novo mutations and rare but mildly deleterious variation in TP53 are found in the genomes of OS patients is only now coming into focus with next-generation sequencing. We recruited 285 patients with OS diagnosed at Citation Format: Logan Spector, Ian Lock, John Lane, Aaron Sarver, Mark Krailo, Rajaram Nagarajan, Nathan Pankratz. De novo and transmitted germline TP53 variation in pediatric osteosarcoma: A report from the Children9s Oncology Group. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A37.