Characteristics Associated with Tumor Development in Individuals Diagnosed with Beckwith–Wiedemann Spectrum: Novel Tumor-(epi)Genotype-Phenotype Associations in the BWSp Population

Beckwith–Wiedemann Spectrum (BWSp) is the most common epigenetic childhood cancer predisposition disorder. BWSp is caused by (epi)genetic changes affecting the BWS critical region on chromosome 11p15. Clinically, BWSp represents complex molecular and phenotypic heterogeneity resulting in a range of presentations from Classic BWS to milder features. The previously reported tumor risk based on Classic BWS cohorts is 8–10% and routine tumor screening has been recommended. This work investigated the tumor risk and correlation with phenotype within a cohort of patients from Classic BWS to BWSp using a mixed-methods approach to explore phenotype and epigenotype profiles associated with tumor development through statistical analyses with post-hoc retrospective case series review. We demonstrated that tumor risk across BWSp differs from Classic BWS and that certain phenotypic features are associated with specific epigenetic causes; nephromegaly and/or hyperinsulinism appear associated with cancer in some patients. We also demonstrated that prenatal and perinatal factors that are not currently part of the BWSp classification may factor into tumor risk. Additionally, blood testing results are not necessarily synonymous with tissue testing results. Together, it appears that the current understanding from Classic BWS of (epi)genetics and phenotype correlations with tumors is not represented in the BWSp. Further study is needed in this complex population.

Prospective study of epigenetic alterations responsible for isolated hemihyperplasia/hemihypoplasia and their association with leg length discrepancy

Background Hemihyperplasia and hemihypoplasia result in leg length discrepancy (LLD) by causing skeletal asymmetry. Beckwith–Wiedemann syndrome (BWS) and Silver–Russell syndrome (SRS) are opposite growth-affecting disorders caused by opposite epigenetic alterations at the same chromosomal locus, 11p15, to induce hemihyperplasia and hemihypoplasia, respectively. Because of their somatic mosaicism, BWS and SRS show a wide spectrum of clinical phenotypes. We evaluated the underlying epigenetic alterations and potential epigenotype-phenotype correlations, focusing on LLD, in a group of individuals with isolated hemihyperplasia/hemihypoplasia. Results We prospectively collected paired blood-tissue samples from 30 patients with isolated hemihyperplasia/hemihypoplasia who underwent surgery for LLD. Methylation-specific multiplex-ligation-dependent probe amplification assay (MS-MLPA) and bisulfite pyrosequencing for differentially methylated regions 1 and 2 (DMR1 and DMR2) on chromosome 11p15 were performed using the patient samples. Samples from patients showing no abnormalities in MS-MLPA or bisulfite pyrosequencing were analyzed by single nucleotide polymorphism (SNP) microarray and CDKN1C Sanger sequencing. We introduced a metric named as the methylation difference, defined as the difference in DNA methylation levels between DMR1 and DMR2. The correlation between the methylation difference and the predicted LLD at skeletal maturity, calculated using a multiplier method, was evaluated. Predicted LLD was standardized for stature. Ten patients (33%) showed epigenetic alterations in MS-MLPA and bisulfite pyrosequencing. Of these, six and four patients had epigenetic alterations related to BWS and SRS, respectively. The clinical diagnosis of hemihyperplasia/hemihypoplasia was not compatible with the epigenetic alterations in four of these ten patients. No patients showed abnormalities in SNP array or their CDKN1C sequences. The standardized predicted LLD was moderately correlated with the methylation difference using fat tissue (r = 0.53; p = 0.002) and skin tissue (r = 0.50; p = 0.005) in all patients. Conclusions Isolated hemihyperplasia and hemihypoplasia can occur as a spectrum of BWS and SRS. Although the accurate differentiation between isolated hemihyperplasia and isolated hemihypoplasia is important in tumor surveillance planning, it is often difficult to clinically differentiate these two diseases without epigenetic tests. Epigenetic tests may play a role in the prediction of LLD, which would aid in treatment planning.

Genetic characterization of short stature patients with overlapping features of growth hormone insensitivity syndromes

Context and objective Growth hormone insensitivity (GHI) in children is characterized by short stature, functional IGF-I deficiency and normal or elevated serum GH concentrations. The clinical and genetic etiology of GHI is expanding. We undertook genetic characterization of short stature patients referred with suspected GHI and features which overlapped with known GH-IGF-I axis defects. Design and methods Between 2008 and 2020, our center received 149 GHI referrals for genetic testing. Genetic analysis utilized a combination of candidate gene sequencing (CGS), whole exome sequencing (WES), array comparative genomic hybridization (aCGH) and a targeted whole genome short stature gene panel. Results Genetic diagnoses were identified in 80/149 subjects (54%) with 45/80 (56%) having known GH-IGF-I axis defects (GHR n=40, IGFALS n=4, IGFIR n=1). The remaining 35/80 (44%) had diagnoses of 3M syndrome (n=10) (OBSL1 n=7, CUL7 n=2 and CCDC8 n=1), Noonan syndrome (n=4) (PTPN11 n=2, SOS1 n=1 and SOS2 n=1), Silver-Russell syndrome (n=2) (Loss of methylation on chromosome 11p15 and uniparental disomy for chromosome 7), Class 3-5 copy number variations (n=10) and disorders not previously associated with GHI (n=9) (Barth syndrome, Autoimmune lymphoproliferative syndrome, Microcephalic osteodysplastic primordial dwarfism Type II, Achondroplasia, Glycogen storage disease Type IXb, Lysinuric protein intolerance, Multiminicore Disease, MACS syndrome and Bloom syndrome). Conclusion We report the wide range of diagnoses in 149 patients referred with suspected GHI, which emphasizes the need to recognize GHI as a spectrum of clinical entities in undiagnosed short stature patients. Detailed clinical and genetic assessment may identify a diagnosis and inform clinical management.

Prospective Study of Epigenetic Alterations Responsible for Isolated Hemihyperplasia/Hemihypoplasia and Their Association With Leg Length Discrepancy

Background: Hemihyperplasia and hemihypoplasia result in leg length discrepancy (LLD) by causing skeletal asymmetry. Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) are opposite growth-affecting disorders caused by opposite epigenetic alterations at the same chromosomal locus, 11p15, to induce hemihyperplasia and hemihypoplasia, respectively. Because of their somatic mosaicism, BWS and SRS show a wide spectrum of clinical phenotypes. We evaluated the underlying epigenetic alterations and potential epigenotype-phenotype correlations, focusing on LLD, in a group of individuals with pure isolated hemihyperplasia/hemihypoplasia.Results: We prospectively collected paired blood-tissue samples from 30 patients with pure isolated hemihyperplasia/hemihypoplasia who underwent surgery for LLD. Methylation-specific multiplex-ligation-dependent probe amplification assay (MS-MLPA) and bisulfite pyrosequencing for differentially methylated regions 1 and 2 (DMR1 and DMR2) on chromosome 11p15 were performed using the patient samples. Samples from patients showing no abnormalities in MS-MLPA or bisulfite pyrosequencing were analyzed by single nucleotide polymorphism (SNP) microarray and CDKN1C Sanger sequencing. We introduced a metric named as the methylation difference, defined as the difference in DNA methylation levels between DMR1 and DMR2. The correlation between the methylation difference and the predicted LLD at skeletal maturity, calculated using a multiplier method, was evaluated. Predicted LLD was standardized for stature. Ten patients (33%) showed epigenetic alterations in MS-MLPA and bisulfite pyrosequencing. Of these, six and four patients had epigenetic alterations related to BWS and SRS, respectively. The clinical diagnosis of hemihyperplasia/hemihypoplasia was not compatible with the epigenetic alterations in four of these ten patients. No patients showed abnormalities in SNP array or their CDKN1C sequences. The methylation difference was strongly correlated with the standardized predicted LLD in patients with epigenetic alterations (r = 0.76; p = 0.01).Conclusions: Pure isolated hemihyperplasia and hemihypoplasia can occur as a spectrum of BWS and SRS. Although the accurate differentiation between isolated hemihyperplasia and isolated hemihypoplasia is important in tumor surveillance planning, it is often difficult to clinically differentiate these two diseases without epigenetic tests. Epigenetic tests may play a role in the prediction of leg length discrepancy, which would aid in treatment planning.

Novel Variant in PLAG1 in a Familial Case with Silver–Russell Syndrome Suspicion

Silver–Russell syndrome (SRS) is a rare growth-related genetic disorder that is mainly associated with prenatal and postnatal growth retardation. Molecular causes are not clear in all cases, the most common ones being loss of methylation on chromosome 11p15 (≈50%) and maternal uniparental disomy for chromosome 7 (upd(7)mat) (≈10%). However, pathogenic variants in genes such as CDKN1C, HMGA2, IGF2, or PLAG1 have also been described. Previously, two families and one sporadic case have been reported with PLAG1 alterations. Here, we present a case of a female with clinical suspicion of SRS (i.e., intrauterine and postnatal growth retardation, triangular face, psychomotor delay, speech delay, feeding difficulties). No alterations in methylation or copy number were detected at chromosomes 11p15 and 7 using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). The custom panel study by next-generation sequencing (NGS) revealed a frameshift variant in the PLAG1 gene (NM_002655.3:c.551delA; p.(Lys184Serfs *45)). Familial studies confirmed that the variant was inherited from the mother and it was also present in other family members. New evidence of pathogenic alterations in the HMGA2-PLAG1-IGF2 pathway suggest the importance of studying and taking into account these genes as alternative molecular causes of Silver–Russell syndrome.

CRISPRi-mediated functional analysis of lung disease-associated loci at non-coding regions

Genome-wide association studies have identified lung disease-associated loci; however, the functions of such loci are not well understood in part because the majority of such loci are located at non-coding regions. Hi-C, ChIP-seq and eQTL data predict potential roles (e.g. enhancer) of such loci; however, they do not elucidate the molecular function. To determine whether these loci function as gene-regulatory regions, CRISPR interference (CRISPRi; CRISPR/dCas9-KRAB) has been recently used. Here, we applied CRISPRi along with Hi-C, ChIP-seq and eQTL to determine the functional roles of loci established as highly associated with asthma, cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Notably, Hi-C, ChIP-seq and eQTL predicted that non-coding regions located at chromosome 19q13 or chromosome 17q21 harboring single-nucleotide polymorphisms (SNPs) linked to asthma/CF/COPD and chromosome 11p15 harboring an SNP linked to IPF interact with nearby genes and function as enhancers; however, CRISPRi indicated that the regions with rs1800469, rs2241712, rs12603332 and rs35705950, but not others, regulate the expression of nearby genes (single or multiple genes). These data indicate that CRISPRi is useful to precisely determine the roles of non-coding regions harboring lung disease-associated loci as to whether they function as gene-regulatory regions at a genomic level.

SUN-172 A False Positive Result in Newborn Screening for Congenital Adrenal Hyperplasia (CAH) in a Girl with Beckwith Wiedemann Syndrome

Background: Congenital Adrenal Hyperplasia (CAH) comprises a spectrum of autosomal recessive diseases, resulting in enzymatic defects in the cortisol secretion. CAH newborn screening can avoid neonatal mortality in children with the salt-wasting form and prevent incorrect gender assignments in females. The occurrence of false-positive results creates diagnostic difficulties presenting therapeutic implications. Beckwith Wiedemann Syndrome (BWS) is a congenital disease characterized by somatic overgrowth, increased risk of neonatal hypoglycemia, and development of embryonic tumors. BWS is due to (epi)genetic changes involving growth-regulating genes with good genotype-phenotype correlation. The adrenal gland is frequently involved and may present diffuse cytomegaly of the adrenal cortex1. We reported a BWS newborn girl with a false-positive diagnosis of CAH in the screening. Case report: The patient was born at 39 weeks from an uneventful cesarean section, 5.6kg (>p97) and 52cm (>p97), referred to the Endocrinology service due to abnormal neonatal tests (neonatal 17-OHP: 96ng/mL) collected at 6 days old. At 14 days old, she was 6.3 kg (Z:+5.59), and 58cm (Z:+2.47), BMI: 18.7 kg/m2 (Z:+4.45), and with typical female external genitalia, ruling out the diagnosis of classic CAH. She presented some syndromic characteristics as macroglossia, ogival palate, orbital hypertelorism, hepatomegaly, and umbilical hernia. At 1 month and 14 days old, serum 17OHP was 7.4ng/mL, androstenedione: 6.1 ng/mL, total testosterone: 279ng/dL, 11-deoxycortisol: 2.11ng/mL, cortisol: 5.0ug/dL, and ACTH: 54pg/mL. At five months old she evolved with normalization of serum 17OHP, androstenedione and testosterone levels (1.36ng/mL, <0.50ng/mL, and 37ng/dL, respectively), but still with high DHEAS levels: 2913ng/mL. At 11 months old, DHEAS also normalized, confirming that it was transient hyperactivity of the zona reticulata. A molecular test was performed in a blood sample by MLPA, showing a gain of methylation in the imprinting control region 1 (ICR1) of chromosome 11p15, which controls two imprinted genes, H19 and IGF-2, confirming the clinical diagnosis of BWS. The hypermethylation of ICR1 is largely related to the Wilms tumor. The patient was diagnosed with bilateral Wilms tumor at 11 months old and undergone chemotherapy without adequate response requiring left nephrectomy at 1 year and 5 days old. Conclusion: We presented the first description of false-positive diagnosis of CAH in the newborn screening of a girl with Beckwith Wiedemann syndrome, probably due to a transient overactivation of the zona reticulata. References: 1.Brioude F, Kalish JM, Mussa A, Foster AC, Bliek J, Ferrero GB, et al. Expert consensus document: clinical and molecular diagnosis, screening and management of Beckwith-Wiedemann syndrome: an international consensus statement. Nat Rev Endocrinol. 2018;14(4):229-49.

SAT-231 A Case of Metastatic Pheochromocytoma Associated with Beckwith-Wiedemann Syndrome

Introduction: Beckwith-Wiedemann Syndrome (BWS) is an autosomal dominant disorder of chromosome 11p15 that results in increased IGF-2 and CDK1NC. This leads to excessive cell proliferation and tumor formation. The following highlights a case of metastatic pheochromocytoma in a patient with BWS. Clinical Case: A 30-year-old male presented with sudden onset blurry vision without any associated complaints. His past medical history was significant for BWS. His family history was negative for uncontrolled hypertension, sudden death, thyroid cancer or hyperparathyroidism. Physical examination was notable for an elevated systolic blood pressure of 200/160 mm of hg and fundoscopy revealed features of hypertensive emergency. Laboratory investigations revealed an elevated plasma normetanephrine [10445 pg/ml (normal: <148)], metanephrine [93 pg/ml (normal: <57)], total metanephrine [10538 pg/ml (normal: <205)], epinephrine [134 pg/ml (normal:<50)], norepinephrine [23526 pg/ml (normal: 112-658), total catecholamine level [23660 (normal: 123-671pg/ml)] and dopamine [403 pg/ml (normal<30)] levels. His PTH, corrected serum calcium, gastrin, insulin, carcinoembryonic antigen, calcitonin levels and basal metabolic panel were all normal. MRI of the abdomen demonstrated bilateral adrenal nodules with a large mass encasing the celiac axis along with evidence of hepatic lesions. I-123 MIBG scan showed mild radioactive tracer uptake in the adrenal nodules and mass near the celiac axis but not in the hepatic lesions. PET scan confirmed MRI findings and was negative for any evidence of malignancy in the chest, pelvis and skeleton. MRI of the brain was negative for metastasis as well as pituitary abnormalities. Ultrasound-guided liver biopsy was positive for malignant cells that stained positive for chromogranin and synaptophysin confirming the diagnosis of metastatic pheochromocytoma. He was treated with phenoxybenzamine, diltiazem and lisinopril. He underwent cycles of cyclophosphamide, vincristine and dacarbazine. Genetic testing revealed a variant in SDHD gene which was of uncertain significance. Repeat biochemical testing on follow up after a year and a half showed a decreased plasma normetanephrine [487pg/ml] and metanephrine levels [110 pg/ml] in comparison to his levels on presentation. Repeat imaging revealed a decrease in tumor burden including bilateral adrenal nodules, celiac axis mass and hepatic metastases. Conclusion: This is an unusual case of malignant pheochromocytoma in the absence of SDHB mutation in a patient with BWS. Genetic causes in these patients are yet to be determined. However, genes H19 and KCNQ1OT1 have been implicated in addition to IGF-2 and CDK1NC

Germline Variants in Phosphodiesterase Genes and Genetic Predisposition to Pediatric Adrenocortical Tumors

Phosphodiesterases (PDEs) form a superfamily of enzymes that catalyze the hydrolysis of cyclic nucleotides adenosine 3′5′-cyclic monophosphate (cAMP) and guanosine 3′5′-cyclic monophosphate (cGMP) to their inactive 5′ monophosphates. cAMP plays a critical role as a second messenger in endocrine tissues, and activation of cAMP signaling has been reported in endocrine tumors. Germline variants in PDEs have been associated with benign cortisol-secreting adrenocortical adenomas and testicular germ cell cancer but not adrenocortical carcinoma. We performed whole genome sequencing (WGS) and whole exome sequencing (WES) of paired blood and tumor samples from 37 pediatric adrenocortical tumors (ACTs). Germline inactivating variants in PDEs were observed in 9 of 37 (24%) patients. Tumor DNA analysis revealed loss of heterozygosity, with maintenance of the mutated allele in all cases. Our results suggest that germline variants in PDEs and other regulators of the cAMP-signaling pathway may contribute to pediatric adrenocortical tumorigenesis, perhaps by cooperating with germline hypomorphic mutant TP53 alleles and uniparental disomy of chromosome 11p15 (Beckwith–Wiedemann syndrome).

Cognitive Profiles and Brain Volume Are Affected in Patients with Silver–Russell Syndrome

Context There is little information on cognitive function in Silver–Russell syndrome (SRS), and no neuroimaging studies are available so far. Objective To assess cognitive function and brain volumes in patients with SRS. Design/Setting Wechsler Intelligence Scale and brain magnetic resonance on a 3-Tesla scanner with Voxel-based morphometry analysis were performed between 2016 and 2018 in a single tertiary university center. Partecipants 38 white subjects with clinical diagnosis of SRS confirmed by molecular analysis: 30 of these patients (mean age 12.6 ± 10 years) were enrolled for cognitive assessment; 23 of the 30 performed neuroimaging sequences. A control group of 33 school-aged children performed cognitive assessment while 65 age and sex-matched volunteers were included for the neuroradiological assessment. Main Outcomes Intelligence quotient, Verbal Comprehension Index (VCI), Perceptual Reasoning Index (PRI), Working Memory Index (WMI), Processing Speed Index, and brain volume. Results The mean overall IQ score was 87.2 ± 17, and it was significantly lower in the maternal uniparental disomy of chromosome 7 (mUPD7) group at the age of 6 to 16 years compared to loss of methylation on chromosome 11p15 (11p15 LOM) group and to controls. VCI, PRI, and WMI were significantly higher in 11p15 LOM group and in control group than in mUPD7 group at the age of 6 to 16 years. There were no significant differences in cognitive scores between 11p15 LOM school-aged patients and the control group. SRS patients showed lower brain volume compared to controls at the frontal/temporal poles and globi pallidi. Conclusions Patients with mUPD7 had an impaired cognitive profile. The brain volume at the frontal/temporal lobes and at the globi pallidi was reduced in patients with SRS.