Search

To develop an evidence-based clinical practice guideline for the use of exome and genome sequencing (ES/GS) in the care of pediatric patients with one or more congenital anomalies (CA) with onset prior to age 1 year or developmental delay (DD) or intellectual disability (ID) with onset prior to age 18 years. The Pediatric Exome/Genome Sequencing Evidence-Based Guideline Work Group (n = 10) used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) evidence to decision (EtD) framework based on the recent American College of Medical Genetics and Genomics (ACMG) systematic review, and an Ontario Health Technology Assessment to develop and present evidence summaries and health-care recommendations. The document underwent extensive internal and external peer review, and public comment, before approval by the ACMG Board of Directors. The literature supports the clinical utility and desirable effects of ES/GS on active and long-term clinical management of patients with CA/DD/ID, and on family-focused and reproductive outcomes with relatively few harms. Compared with standard genetic testing, ES/GS has a higher diagnostic yield and may be more cost-effective when ordered early in the diagnostic evaluation. We strongly recommend that ES/GS be considered as a first- or second-tier test for patients with CA/DD/ID.

Transient neonatal diabetes mellitus (TNDM) is a rare form of diabetes that presents in infancy and is characterized by intrauterine growth restriction and hyperglycemia without ketones on urinalysis. Patients are treated with insulin until remission, usually within the first year. Relapse to a permanent state may occur later in life, with a mean age of 14 years. The most common cause of TNDM is a chromosome 6q24 mutation that affects pancreatic β-cell function. Reports of relapse have been limited. We describe a case of an adolescent female with TNDM due to 6q24 hypomethylation who relapsed at 15 years of age with severe dental disease as the presenting sign.

A 3-year-old girl with developmental delays, hypotonia, and generalized seizures was diagnosed with a novel variant of a heterozygous mutation in the TBC1 domain containing kinase (TBCK) gene. Postmortem findings revealed severe hypotrophy of cerebral white matter, hypogenesis of the corpus callosum with reduced myelination and oligodendrocyte populations, and reactive gliosis, and venous angiomas of the cerebrum, brainstem, and cerebellum white matter. This report is the first to link a TBCK gene mutation to impaired white matter development with the targeting of central nervous system myelin and oligodendrocytes. The mechanism may involve inhibition of signaling through (mTORC1).

Background. Growth hormone (GH) treatment increases the adult height of short children born small for gestational age (SGA). Catch-up growth is associated with a younger age, shorter height, and prepubertal status at the onset of GH treatment. We report a 12 11/12-year-old girl born SGA who received GH for 5 years without catch-up growth and was diagnosed with Noonan Syndrome (NS). Results. A 5-year-and-9-month-old 46, XX girl born SGA was started on GH treatment at a dose of 0.32 mg/kg/week. Her midparental target height is 158.6 cm. Endocrine work up showed an IGF-1 level 69 ng/ml (Normal (N): 55–238 ng/ml), IGFBP3 2.6 mg/L (N: 1.9–5.2 mg/L), TSH 3.2 mIU/L (N: 0.35–5.5 mIU/L), and a normal skeletal survey. Height was 96 cm (0.1%; Ht SDS −2.9), weight 14 kgs (1%; Wt SDS −2.3), and Tanner 1 breast and pubic hair were observed. Due to the poor catch-up growth on GH treatment, she was referred to Genetics to elucidate genetic or syndromic causes of short stature. She was noted to have posteriorly rotated ears and slight down slanting of the palpebral fissures. Genetic findings showed a heterozygous pathogenic variant in PTPN11 (c.922A > G (p.Asn308Asp)) diagnostic for NS. This finding is de novo given negative parental testing. She was noted to have a heterozygous missense variant of unknown significance (VUS) in FGFR3: c.746C > A (p.Ser249Tyr). FGFR3 is associated with multiple skeletal dysplasias including thanatophoric dysplasia, achondroplasia, and Crouzon syndrome and hypochondroplasia. Clinical correlation is poor for these syndromes. Conclusion. Diminished catch-up growth and response to GH treatment in a child born SGA led to the diagnosis of NS. The concomitant diagnosis of SGA and NS may have affected the responsiveness of this child to the growth promoting effect of GH treatment.

Human fetuses with trisomy 21 (T21) have atypical brain development that is apparent sonographically in the second trimester. Prenatal diagnosis provides a potential opportunity to begin treatment in utero. We hypothesize that by analyzing and integrating dysregulated gene expression and pathways common to humans with DS and mouse models we can discover novel targets for therapy. Here, we tested the safety and efficacy of apigenin (4’, 5, 7-trihydroxyflavone), identified using this approach, in both human amniocytes from fetuses with T21 and in the Ts1Cje mouse model. The experiments compared treated to untreated results in T21 and euploid cells, as well as in Ts1Cje mice and their wild-type littermate controls. T21 cells cultured with apigenin (2µM) had significantly reduced oxidative stress and improved antioxidant defense response in vitro. Apigenin (333-400 mg/kg/day), mixed with chow, was initiated prenatally to the dams and fed to the pups over their lifetimes. There was no significant increase in birth defects or pup deaths resulting from prenatal apigenin treatment. Apigenin significantly improved several developmental milestones and spatial olfactory memory in Ts1Cje neonates. In addition, we noted sex-specific effects on exploratory behavior and long-term hippocampal memory in adult mice, with males showing significantly more improvement than females. Global gene expression analyses demonstrated that apigenin targets similar signaling pathways through common upstream regulators both in vitro and in vivo. These studies provide proof-of-principle that apigenin has therapeutic effects in preclinical models of Down syndrome.ONE SENTENCE SUMMARYAs a candidate prenatal treatment for Down syndrome, apigenin improved oxidative stress/antioxidant capacity imbalance and reduced pathways associated with inflammation in human cells while improving aspects of behavior in the Ts1Cje mouse model.

Background DICER1 syndrome is an autosomal dominant condition due to mutations in the DICER1 gene, located on chromosome 14q32.13. Patients are at increased risk for malignant and benign tumors, including pleuropulmonary blastoma (PPB), cystic nephroma, ovarian Sertoli-Leydig cell tumors, multinodular goiter (MNG) and differentiated thyroid cancer (DTC). MNG is very common in patients with DICER1 Syndrome but data on incidence is lacking. MNG is more common in females than males. Case presentation: 22 year old man who originally presented with pleuropulmonary blastoma, Type 3 at 3 years of age. His treatment included pulmonectomy, radiation of 46.6 Gy to thorax, and alkylating agents including Cisplatin, Ifosphamide and Cytoxan. He developed frontal lobe metastasis over the course of 3 years and was treated with focal cranial radiation. His father and maternal uncle had history of lung cancer. He was evaluated at the Endocrine clinic at 12 years 10 months for short stature. His height was 132.5 cm (SDS -2.96), weight 29.7 kg (SDS -2.40), and BMI 16.99kg/m² (SDS -0.61). On examination he had normal thyroid exam and Tanner 2 bilateral 4 cc testicles. He was treated with Levothyroxine for subclinical hypothyroidism (TSH: 5.96 uIU/ml (0.35–5.5) and Free T 4: 0.99 ng/dl (0.8–1.80) and growth hormone (GH) for growth hormone deficiency (peak GH was 7.7 ng/ml after Arginine and Clonidine GH stimulation test). At 14 years 10 month he developed respiratory distress. CT scan of chest showed right lower pole nodule 1.6 x 1.5 x 1.4 cm. Ultrasound of thyroid showed right thyroid solid mid pole isoechoic nodule 1.4 x 1.7 x 1.3 cm with multiple enlarged bilateral cervical nodes, largest left supraclavicular region > 1 cm. Biopsy of the right nodule was negative for malignancy. Over the course of 2 years he developed new right thyroid isoechoic nodule in the lower pole 2.1 x 2.5 x 1.9 cm and new left thyroid isoechoic nodule in the upper pole 1.0 x 0.5 x 0.5 cm. Biopsy was negative for malignancy. Due to his PPB, MNG and family history of lung cancer he was evaluated at our genetic cancer clinic and tested positive for germline DICER1 pathogenic variant c.4605_4606del (p.Cys1535Trpfs*3) Conclusion: Our 22 year old male presented with pleuropulmonary blastoma and over the course of few years developed MNG. Genetic testing was positive for germline DICER1 pathogenic variant c.4605_4606del (p.Cys1535Trpfs*3). Our case illustrates the importance of consideration of: 1) Testing children with PPB for DICER1 Syndrome as there are screening recommendations including regular thyroid ultrasound and examinations to look for MNG or other features concerning for thyroid gland neoplasia. 2) MNG is uncommon in children and detection of this should raise suspicion for consideration of testing for DICER1 Syndrome.

10523 Background: Breast cancer is a heterogenous disease and management is complex. Advances in next generation sequencing has allowed genetic testing to be more accessible. However, conveying results to patients and care team can be challenging due to various variant classifications. Diagnostic results have the potential to guide management. Nondiagnostic results can be misinterpreted. The extent to which preoperative genetic testing affects management of newly diagnosed breast cancer is unknown. Methods: Newly diagnosed breast cancer patients were identified via review of breast tumor board between May 2019 and March 2019 followed by chart review to collect detailed information. Results: 408 newly diagnosed breast cancer cases were queried. Genetic evaluation was recommended and completed in 68%, not recommended (did not meet NCCN criteria) in 30% and declined in 2.7%. The genetic evaluation recommended cohort was associated with a higher mastectomy rate in comparison with when not recommended (31% vs. 9%, p=0.0001). Of those who completed genetic testing: 12% harbored a pathogenic/likely pathogenic variant (PV/LPV), 26% had a nondiagnostic variant of uncertain significance (VUS) and 61% had negative testing. Comparison between nondiagnostic test results (negative and VUS) and diagnostic test results revealed significantly increased number of women in the diagnostic group who chose mastectomy over breast conservation therapy (BCT, nondiagnostic 20% vs diagnostic 39%, p=0.018). When negative, VUS, and PV/LPV were each independently analyzed, diagnostic test results again revealed a significantly increased number of mastectomies over BCT (p0.5). Conclusions: Germline genetic testing in patients with newly diagnosed breast cancer impacts clinical management. Those harboring a diagnostic result were more likely to choose mastectomy over BCT. Not surprisingly, mastectomy rate was higher among those where genetic evaluation was recommended, possibly due to concerning personal or family history. The mastectomy rate was higher among those with a diagnostic result, indicating an understanding of the genetic testing significance by patients and the care team. More importantly, those who harbored nondiagnostic VUS did not make significantly different surgical choices compared with negative genetic testing, highlighting the critical role of proper genetic counseling and being part of the care team. We conclude consideration of genetic evaluation is clinically useful and feasible without affecting the surgical timeline.[Table: see text]

Turner syndrome is a sex chromosome aneuploidy with characteristic malformations. Amniotic fluid, a complex biological material, could contribute to the understanding of Turner syndrome pathogenesis. In this pilot study, global gene expression analysis of cell-free RNA in amniotic fluid supernatant was utilized to identify specific genes/organ systems that may play a role in Turner syndrome pathophysiology. Cell-free RNA from amniotic fluid of five mid-trimester Turner syndrome fetuses and five euploid female fetuses matched for gestational age was extracted, amplified, and hybridized onto Affymetrix(®) U133 Plus 2.0 arrays. Significantly differentially regulated genes were identified using paired t tests. Biological interpretation was performed using Ingenuity Pathway Analysis and BioGPS gene expression atlas. There were 470 statistically significantly differentially expressed genes identified. They were widely distributed across the genome. XIST was significantly down-regulated (p < 0.0001); SHOX was not differentially expressed. One of the most highly represented organ systems was the hematologic/immune system, distinguishing the Turner syndrome transcriptome from other aneuploidies we previously studied. Manual curation of the differentially expressed gene list identified genes of possible pathologic significance, including NFATC3, IGFBP5, and LDLR. Transcriptomic differences in the amniotic fluid of Turner syndrome fetuses are due to genome-wide dysregulation. The hematologic/immune system differences may play a role in early-onset autoimmune dysfunction. Other genes identified with possible pathologic significance are associated with cardiac and skeletal systems, which are known to be affected in females with Turner syndrome. The discovery-driven approach described here may be useful in elucidating novel mechanisms of disease in Turner syndrome.

Anatomical and functional brain abnormalities begin during fetal life in Down syndrome (DS). We hypothesize that novel prenatal treatments can be identified by targeting signaling pathways that are consistently perturbed in cell types/tissues obtained from human fetuses with DS and mouse embryos. We analyzed transcriptome data from fetuses with trisomy 21, age and sex-matched euploid controls and embryonic day 15.5 forebrains from Ts1Cje, Ts65Dn and Dp16 mice. The new datasets were compared to other publicly available datasets from humans with DS. We used the human Connectivity Map (CMap) database and created a murine adaptation to identify FDA-approved drugs that can rescue affected pathways. USP16 and TTC3 were dysregulated in all affected human cells and two mouse models. DS-associated pathway abnormalities were either the result of gene dosage specific effects or the consequence of a global cell stress response with activation of compensatory mechanisms. CMap analyses identified 56 molecules with high predictive scores to rescue abnormal gene expression in both species. Our novel integrated human/murine systems biology approach identified commonly dysregulated genes and pathways. This can help to prioritize therapeutic molecules on which to further test safety and efficacy. Additional studies in human cells are ongoing prior to pre-clinical prenatal treatment in mice.

We sought to establish a blood-neural barrier (BNB) model of astrocyte contact with endothelial cells (EC) to test the hypothesis that transforming growth factor beta (TGF beta) promotes an EC barrier-phenotype. Astrocyte-EC contact induced BNB properties in EC. Transendothelial resistance was augmented by direct contact between astrocytes-EC, but not by astrocyte-conditioned medium or astrocyte-EC coculture conditioned medium. Coculture of EC and astrocytes led to significant increase in endothelial occludin levels and junctional localization. EC gamma-glutamyl-transferase (GGT) activity was increased by direct contact with astrocytes, by conditioned medium from cocultures or by TGF beta1. Coculture inhibited EC proliferation with no effect on astrocyte proliferation. A neutralizing antibody to TGF beta decreased GGT activity in cocultures and increased cell number. Whereas total TGF beta was not significantly altered by coculture, activated TGF beta increased in astrocyte-EC cocultures. In summary, astrocyte-EC contact induces BNB characteristics in EC and locally activated TGF beta is responsible for part of the induction.

Pericytes have been suggested to play a role in regulation of vessel stability; one mechanism for this stabilization may be via pericyte-derived vascular endothelial growth factor (VEGF). To test the hypothesis that differentiation of mesenchymal cells to pericytes/smooth muscle cells (SMC) is accompanied by VEGF expression, we used endothelial cell (EC) and mesenchymal cell cocultures to model cell–cell interactions that occur during vessel development. Coculture of EC and 10T1/2 cells, multipotent mesenchymal cells, led to induction of VEGF expression by 10T1/2 cells. Increased VEGF expression was dependent on contact between EC-10T1/2 and was mediated by transforming growth factorβ (TGFβ). A majority of VEGF produced in coculture was cell- and/or matrix-associated. Treatment of cells with high salt, protamine, heparin, or suramin released significant VEGF, suggesting that heparan sulfate proteoglycan might be sequestering some of the VEGF. Inhibition of VEGF in cocultures led to a 75% increase in EC apoptosis, indicating that EC survival in cocultures is dependent on 10T1/2-derived VEGF. VEGF gene expression in developing retinal vasculature was observed in pericytes contacting newly formed microvessels. Our observations indicate that differentiated pericytes produce VEGF that may act in a juxtacrine/paracrine manner as a survival and/or stabilizing factor for EC in microvessels.

Fetal cell-free mRNA in amniotic fluid (AF) is a valuable source of information regarding the developing fetus. Nucleic acids isolated from AF provide novel information compared to other sources of cell-free nucleic acids, such as maternal plasma (Zhong et al., 2006). Genomic and transcriptomic information from AF is of special interest because it derives solely from the fetus, and may therefore offer unique perspectives. The discovery-driven approach of using transcriptomic analysis has contributed novel information about the pathogenesis of several genetic disorders, such as Down syndrome and polyhydramnios in the setting of twin–twin transfusion syndrome (Larrabee et al., 2005; Slonim et al., 2009). The successful extraction of fetal RNA from cell-free AF supernatant and its subsequent hybridization to oligonucleotide microarrays has already demonstrated the wealth of new information this nucleic acid source has to offer (Hui and Bianchi, 2010). A possible impediment to the expanded utilization of AF is the cost and labor associated with extraction of DNA and RNA. A method using the combination of Trizol and the RNeasy Maxi Kit (QIAGEN, Hilden, Germany) has been used in our laboratory to successfully extract RNA from AF supernatant. However, this method does not allow the simultaneous processing of large numbers of samples and includes several possible sources of error, such as the division of samples into smaller aliquots and a precipitation step. Recently, a new kit was made commercially available to extract nucleic acids from human plasma or serum, the QIAamp Circulating Nucleic Acid Kit (QIAGEN). This kit utilizes a vacuum extraction method that allows throughput of large volumes on one mini column and eliminates the need for precipitation of the final sample. The purpose of this study was to compare RNA extraction from AF using this new method to our laboratory’s previously reported standard methods (Larrabee et al., 2005; Slonim et al., 2009). This study was approved by the Tufts Medical Center Institutional Review Board. Five residual samples of AF supernatant that were obtained for routine clinical indications at Tufts Medical Center were used [46,XY (n = 2) and 46,XX (n = 3)]. The gestational ages ranged from 15 4/7 weeks to 17 3/7 weeks. After the initial collection of AF, samples were processed and frozen at −80 °C within 12 h. The samples had been stored at −80 °C for up to 9 months before RNA extraction was performed. The samples of ≥ 10 mL were each split into two 5 mL aliquots for the two different extraction methods. For the standard approach, total RNA was extracted from one 5 mL aliquot of each AF sample using 15 mL of Trizol LS Reagent (Invitrogen) and 4 mL of chloroform per sample. Subsequent reactions were divided into smaller fractions, consisting of 750 μL of Trizol, 250 μL of AF supernatant sample, and 200 μL of chloroform per reaction in order to utilize the microcentrifuge necessary for the protocol. This method resulted in approximately 20 different reactions per 5 mL sample. Extracted RNA was then purified using the RNeasy Maxi Kit (QIA-GEN) as per the manufacturer’s protocol. RNA was precipitated from the original volume of approximately 650 μL and then rehydrated in 20 μL of RNase-free water. Briefly, 3 M NaOAc and 100% ethanol were added to the sample, the sample incubated at −20 °C for 4 h, the RNA was pelleted by centrifugation and the precipitated RNA was washed by adding 80% ethanol, followed by centrifugation and rehydration of the RNA. For the new protocol, total RNA was extracted from the second 5 mL aliquot from all samples using the QIAamp Circulating Nucleic Acid Kit (QIAGEN), according to the ‘Isolation of Circulating Nucleic Acids from Plasma or Serum’ protocol as recommended by the manufacturer. Briefly, the sample was lysed and then applied to a column via a vacuum pump. The RNA (bound to the spin column) was then washed three times and eluted with 20 μL of RNase-free water. On-column DNase digestion (RNase-Free DNase Set, QIAGEN) was performed during extraction in both methods. All extracted RNA samples were then stored at −80 °C for approximately one week prior to real-time quantitative reverse transcriptase (qRT) PCR amplification. Real time qRT-PCR was performed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with primer and probe sequences as previously reported (Maron et al., 2008), using an ABI 7900 Sequence Detection System with TaqMan One-Step RT-PCR Master Mix reagents kit (Applied Biosystems, Foster City, CA, USA). All qRT-PCR experiments were performed in duplicate with negative controls on each plate. The quality of total RNA in extracted RNA samples was assessed by an Agilent Bioanalyzer 2100 (Foster City, CA, USA). All statistical analyses were performed with SPSS 11.5 software (SPSS Inc., Somers, NY, USA). Independent t-tests were performed between the mean cycle of amplification of GAPDH between the samples extracted using the two different methods to determine if there were statistically significant expression differences. Statistical significance was set at a threshold level of p < 0.05. Our results demonstrated that RNA extracted using the new method amplified at a significantly earlier cycle threshold than RNA extracted using the standard method (p = 0.001, Figure 1). This demonstrates significantly improved quantity of extracted RNA using the newer method. In order to gather qualitative data, the bioanalyzer results for both extraction methods were analyzed. These results were inconclusive, with similar quality RNA obtained from both methods. We speculate this is due to the overall low concentration and degraded nature of the RNA obtained from both methods. These findings demonstrate that neither method has a significant effect on the quality of isolated RNA. This impeded qualitative comparisons but still allowed qRT-PCR amplification. The new extraction method also lowered the risk of pipetting error by eliminating the step of splitting the samples into 1.5 mL tubes and a precipitation step. The protocol changes also significantly decreased the time to process the samples, with the new method requiring only 2.5 h, whereas the standard method requires 8.5 h. The new method is also more cost effective, with the total cost of the standard method being $53.33/sample compared to $21.11/sample for the new method ($USD). Figure 1 Comparison of the mean Ct values of each extraction method. RNA extracted using the QIAamp Circulating Nucleic Acid Kit (CNA Kit) amplified significantly earlier than RNA extracted using Trizol and the Maxi Kit (p = 0.001). Bars represent standard deviation ... In summary, the new method of isolating cell-free RNA from AF samples using the QIAamp Circulating Nucleic Acid Kit represents overall improvement in many ways. It is both more cost effective and less labor intensive than the standard method of RNA extraction from AF. By increasing the quantity of extracted RNA this new method makes downstream applications more accessible and robust, thereby offering an improved workflow to further understand both normal fetal development and the pathogenesis of many genetic disorders.