BMPR1A Mutations Research Articles

Juvenile polyposis syndrome in children: The impact of SMAD4 and BMPR1A mutations on clinical phenotype and polyp burden.

A constitutional disease-causing variant (DCV) in the SMAD4 or BMPR1A genes is present in 40%-60% of patients with juvenile polyposis syndrome (JPS). The aim of this study was to characterize the clinical course and polyp burden in children with DCV-positive JPS compared to DCV-negative JPS. Demographic, clinical, genetic, and endoscopic data of children with JPS were compiled from eight international centers in the ESPHGAN/NASPGHAN polyposis working group. A total of 124 children with JPS were included: 69 (56%) DCV-negative and 55 (44%) DCV-positive (53% SMAD4 and 47% BMPR1A) with a median (interquartile range) follow-up of 4 (2.8-6.4) years. DCV-positive children were diagnosed at an older age compared to DCV-negative children [12 (8-15.7) years vs. 5 (4-7) years, respectively, p < 0.001], had a higher frequency of family history of polyposis syndromes (50.9% vs. 1.4%, p < 0.001), experienced a greater frequency of extraintestinal manifestations (27.3% vs. 5.8%, p < 0.001), and underwent more gastrointestinal surgeries (16.4% vs. 1.4%, p = 0.002). The incidence rate ratio for the development of new colonic polyps was 6.15 (95% confidence interval 3.93-9.63, p < 0.001) in the DCV-positive group compared to the DCV-negative group, with an average of 12.2 versus 2 new polyps for every year of follow-up. There was no difference in the burden of polyps between patients with SMAD4 and BMPR1A mutations. This largest international cohort of pediatric JPS revealed that DCV-positive and DCV-negative children exhibit distinct clinical phenotype. These findings suggest a potential need of differentiated surveillance strategies based upon mutation status.

Molecular and immune landscape by cyclin dependent kinase (CDK) 4/6 expression and TP53 mutational status in mismatch repair deficient/microsatellite instability-high (dMMR/MSI-H) colorectal cancer (CRC).

190 Background: TP53 mutation (mut) has been reported to be associated with lower anti-tumor immunity and worse response to immune checkpoint inhibition (ICI) in CRC. Recent data suggest that CDK4/6 inhibition may enhance the efficacy of ICI in CRC by modulation of TP53 pathway mediators. We sought to characterize the impact of CDK4/6 expression on mutational profile, tumor microenvironment (TME), and clinical outcomes in patients with dMMR/MSI-H CRC stratified by TP53 mut status. Methods: Tumor profiling was performed for 13,942 samples by NextGen Sequencing on DNA (592-gene panel or WES) and RNA (WTS) at Caris Life Sciences (Phoenix, AZ). Of these, 932 specimens were dMMR/MSI-H. Cohorts were created based on top (Q4) and bottom (Q1) quartiles of CDK4/6 RNA expression (transcripts per million) and further divided based on TP53 mut. Chi-square, Fishers-exact, and Mann Whitney tests were used to determine statistical significance and adjusted for multiple hypothesis testing by Benjamini-Hochberg ( q &lt; 0.05). Cell infiltration in the TME was estimated by quanTIseq. Insurance claims data was obtained to calculate ICI-survival using Kaplan-Meier estimates from the initiation of treatment to last contact. Results: CDK4 &amp; 6 tumor expression levels positively correlated in our dataset ( ρ = 0.53, P &lt; 0.05) and were not significantly different in primary vs. metastatic sites. CDK6-Q4 tumors were enriched for mutations in APC, HNF1A, KRAS and DNMT3A, while CDK6-Q1 was associated with BRAF mutations and PDL1+ tumors ( P &lt; 0.05). Mutations in ARID1A, CDH1, KMT2A, FANCL, NF2 and BMPR1A showed opposite trends in CDK6-Q4 compared to Q1 depending on TP53 mut. CDK6-Q4 tumors were associated with higher expression of immune checkpoint genes including CTLA4, CD274, PDCD1LG12, LAG3, CD80, CD86, PDCD1, HAVCR2, IFNG (fold change [FC] Q4 vs Q1: 1.65-3.23, q &lt; 0.05); alongside a TME characterized by higher infiltration rates of B cells, M2 macrophages, and NK cells (FC: 1.19-1.61, q &lt; 0.05), independent of TP53 mut. The T-cell inflamed signature score was also higher in CDK6-Q4 vs Q1 ( q &lt; 0.05). Similar results were observed for CDK4. In patients with TP53 mut tumors, CDK6 expression greater than the median was associated with longer ICI survival (HR = 0.46, 95% CI 0.22-0.95, P = 0.032), whereas no difference was observed in patients not stratified by TP53 mut. Conclusions: Our comprehensive analysis is the first to show distinct mutational profiles according to TP53 mut status, and differential expression of immune-related genes and TME cell infiltration independent of TP53 mut in CDK4/6 high vs low dMMR/MSI-H CRC. In our series, CDK6 expression correlated with ICI treatment benefit in TP53 mut tumors, warranting further studies to explore the potential of targeting the CDK4/6 axis to enhance ICI efficacy in CRC.

Re-recognition of BMPR1A-related polyposis: beyond juvenile polyposis and hereditary mixed polyposis syndrome.

Bone morphogenetic protein receptor type 1A (BMPR1A) is responsible for two individual Mendelian diseases: juvenile polyposis syndrome and hereditary mixed polyposis syndrome 2, which have overlapping phenotypes. This study aimed to elucidate whether these two syndromes are just two subtypes of a single syndrome rather than two isolated syndromes. We sequenced the BMPR1A gene in 186 patients with polyposis and colorectal cancer, and evaluated the clinicopathological features and phenotypes of the probands and their available relatives with BMPR1A mutations. BMPR1A germline mutations were found in six probands and their three available relatives. The numbers of frameshift, nonsense, splice-site, and missense mutations were one, one, two, and two, respectively; two of the six mutations were novel. Typical juvenile polyps were found in only three patients. Two patients had colorectal cancer rather than any polyps. Diseases in BMPR1A germline mutation carriers vary from mixed polyposis to sole colorectal cancer, and typical juvenile polyps do not always occur in these carriers. The variety of phenotypes reflected the features of BMPR1A-mutation carriers, which should be recognized as a spectrum of one syndrome. Genetic testing may be a good approach to identifying BMPR1A-related syndromes.

Abstract 5898: Bone morphogenic protein receptor 2 (BMPR2) as a potential germline driver in Juvenile Polyposis Syndrome (JPS)

Abstract Summary: Juvenile Polyposis Syndrome is a pediatric cancer predisposition syndrome for which approximately half of patients do not have a known germline driver; we propose BMPR2 as a possible germline driver of disease in mutation-negative JPS. Background: Juvenile Polyposis Syndrome (JPS) is a cancer predisposition syndrome characterized in some cases by inactivating germline mutations in BMPR1A or SMAD4. However, in 40-60% of patients the germline driver is unknown. Methods: Through whole exome sequencing, an individual with high polyp burden and negative genetic testing for variants in SMAD4 and BMPR1A was found to have a potentially inactivating germline variant in BMPR2. Under an IRB-approved protocol, patient-derived, three-dimensional organoids were established from adjacent colon and polyp tissue of this individual, as well as an individual with a BMPR1A deletion and age- and sex-matched controls. Proliferation, metabolic activity, and protein expression of each line were measured by Western blot, Ki67 staining, EdU labelling, cell titer glo (CTG) assay. Results: Distinct phenotypic differences are evident between colonoids from an individual with a BMPR1A deletion and both the colon and polyp BMPR2 variant colonoids, such as increased crypt budding and Ki67 staining. CTG assay data indicate higher metabolic activity in BMPR2 variant colonoids in comparison to normal controls (p= 0.0104.) Through Western blot analysis, both BMPR2 colon and polyp colonoids showed decreased SMAD4 expression and phosphorylation. Conclusions: These data demonstrate that clear phenotypic differences exist between colonoid lines established from normal controls, individuals with known JPS predisposition genes, and a novel candidate driver of disease, BMPR2. Proliferation and protein expression data suggest that both known and candidate genotypes are consistent with a hyperproliferative phenotype. In vitro data support BMPR2 as a candidate germline driver of the JPS phenotype, and additional study is ongoing regarding downstream pathway regulation in colonoids with a BMPR2 variant. Citation Format: Suzanne P. MacFarland, Bridgid Greed, Michael H. Xie, Garrett M. Brodeur, Zvi Cramer, Maiah H. Dent, Melani M. Duvall, Kathryn E. Hamilton, James Howe, Tatiana Karakasheva, Petar Mamula, Christopher Lengner. Bone morphogenic protein receptor 2 (BMPR2) as a potential germline driver in Juvenile Polyposis Syndrome (JPS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5898.

Sequencing for novel mutation screening in juvenile polyposis syndrome

Juvenile polyposis syndrome (JPS), a rare polyposis syndrome in childhood, is characterized by multiple diffuse hamartomatous polyps in the gastrointestinal tract (GI) with an estimated incidence of one in 50,000 to 100,000 live births. Although a juvenile polyp is a non-cancerous lesion, affected individuals have an increased risk of GI malignancy. JPS is a disease related to loss-of-function mutations in BMPR1A and SMAD4 involved in the biological maintenance of cellular growth. Pathogenic mutations in those genes have been identified in approximately 50–60% of JPS cases. Our report describes two JPS cases with loss-of-function mutations of BMPR1A identified by a high-throughput nucleotide sequencing. Both cases had a similar clinical manifestation, symptomatic lower GI bleeding. Both patients underwent a total colectomy after histological confirmation. Consequently, Family members who had had genetic confirmation of these mutations were invited to be screened by enteroscopy. JPS is a hereditary disease caused by dominantly inherited loss-of-function of the BMPR1A and SMAD4 genes . Identifying loss-of-function mutations in particular genes allows early screening, diagnosis, and prevention of malignancy among the index cases and their families. High-throughput genome sequencing is a current molecular approach for improving the precision of mutation detection and minimizing time to diagnosis.

Comprehensive Analysis of R-Spondin Fusions and RNF43 Mutations Implicate Novel Therapeutic Options in Colorectal Cancer

Gene fusions involving R-spondin (RSPOfp) and RNF43 mutations have been shown to drive Wnt-dependent tumor initiation in colorectal cancer. Herein, we aimed to characterize the molecular features of RSPOfp/RNF43 mutated (mut) compared with wild-type (WT) colorectal cancers to gain insights into potential rationales for therapeutic strategies. A discovery cohort was classified for RSPOfp/RNF43 status using DNA/RNA sequencing and IHC. An independent cohort was used to validate our findings. The discovery cohort consisted of 7,245 colorectal cancer samples. RSPOfp and RNF43 mutations were detected in 1.3% (n = 94) and 6.1% (n = 443) of cases. We found 5 RSPO fusion events that had not previously been reported (e.g., IFNGR1-RSPO3). RNF43-mut tumors were associated with right-sided primary tumors. No RSPOfp tumors had RNF43 mutations. In comparison with WT colorectal cancers, RSPOfp tumors were characterized by a higher frequency of BRAF, BMPR1A, and SMAD4 mutations. APC mutations were observed in only a minority of RSPOfp-positive compared with WT cases (4.4% vs. 81.4%). Regarding RNF43 mutations, a higher rate of KMT2D and BRAF mutations were detectable compared with WT samples. Although RNF43 mutations were associated with a microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) phenotype (64.3%), and a tumor mutation burden ≥10 mt/Mb (65.8%), RSPOfp was not associated with MSI-H/dMMR. The validation cohort replicated our genetic findings. This is the largest series of RSPOfp/RNF43-mut colorectal cancers reported to date. Comprehensive molecular analyses asserted the unique molecular landscape associated with RSPO/RNF43 and suggested potential alternative strategies to overcome the low clinical impact of Wnt-targeted agents and immunotherapy.

Phenotypic diversity among juvenile polyposis syndrome patients from different ethnic background

Juvenile polyposis syndrome (JPS), has diverse phenotypes. Aim: To assess mutation rate, clinical features and genotype-phenotype correlation among Israeli JPS kindreds from different ethnicities.MethodsPatients’ data were extracted retrospectively from 5 centers.ResultsThirty five kindreds (49 patients) were included. Thirty one (89%) Jewish [10 (32%) Ashkenazi; 9 (29%) Sephardi; 11 (35%) non-Russia former Soviet-Union countries (NRFSU), one (3%) unknown]. 40/49 individuals from 27 families underwent genetic testing. Among them 34, from 21 families (85, 78%, respectively) had a pathogenic mutation: BMPR1A n = 15 (71%), SMAD4 n = 6 families (29%). While no SMAD4 mutation was described among Jewish families from NRFSU, 7 NRFSU families carried a founder mutation comprising a large genomic deletion of BMPR1A. GI involvement was reported in 42 patients (86%): colonic polyps (n = 40, 95%, > 50 polyps n = 14, 35%) and 12 underwent colonic resection. Fourteen patients (34%) had gastric or small bowel involvement (n = 5) and 4\\14 underwent gastrectomy due to polyp burden. Families from NRFSU had more gastric involvement (66.7% vs. 22.2%- Sephardic and 20%- Ashkenazi Jews; p = 0.038), with more gastric polyps (p = 0.017).ConclusionsWe demonstrated a high rate of mutation detection in the heterogeneous population of Israel. Patients from NRFSU with BMPR1A mutation had high rate of gastric involvement.

BMPR1A and SMAD4 mutations in juvenile polyposis syndrome: clinicopathological and genetic data from two congolese patients

BackgroundJuvenile polyposis syndrome (JPS) is a rare genetic disorder due to germline mutations in BMPR1A or in SMAD4 that are both tumor suppressor genes. The main clinical manifestation is the presence of multiple hamartomatous polyps preferentially located in gastrointestinal tract, mostly in colorectal site. The polyps can degenerate and lead to inherited malignant tumors. Materials and MethodsAmong 3157 patients with malignant and benign tumors diagnosed in Brazzaville teaching hospital, 43 persons with polyposis in colorectal tube were recruited. The positive diagnosis was based on endoscopic examination, World Health Organization and JASS guidelines clinical criteria, histopathological analysis and next-generationsequencing of DNAs extracted from peripheral blood of the patients. ResultsAmong 43 patients having polyposis, two were male patients identified with JPS representing 4.65% of polyposis and 0.06% of all tumors. Histological examination revealed typical JPS features. In those two males, whole genome comparative microarray did not identify relevant copy number imbalances. DNA sequencing of a comprehensive gene cancer genes panel identified specific mutations in the BMPR1A and SMAD4 genes. ConclusionWe have shown that BMPR1A and SMAD4 gene disorders are responsible for JPS in congolese patients as well and a novel mutation has been identified in one individual. As the condition predisposes highly to hereditary colorectal cancer, a comprehensive assessment of the JPS including genetic testing should be implemented in order to facilitate early diagnosis in Congo Brazzaville.

Juvenile polyposis syndrome might be misdiagnosed as familial adenomatous polyposis: a case report and literature review

BackgroundJuvenile polyposis syndrome (JPS) is a rare disorder characterized by the presence of multiple juvenile polyps in the gastrointestinal tract, and germline mutations in SMAD4 or BMPR1A. Due to its rarity and complex clinical manifestation, misdiagnosis often occurs in clinical practice.Case presentationA 42-year-old man with multiple pedunculated colorectal polyps and concomitant rectal adenocarcinoma was admitted to our hospital. His mother had died of colon cancer. He was diagnosed with familial adenomatous polyposis (FAP) and underwent total proctocolectomy and ileal pouch anal anastomosis. Two polyps were selected for pathological examination. One polyp had cystically dilated glands with slight dysplasia. The other polyp displayed severe dysplasia and was diagnosed as adenoma. Three years later, his 21-year-old son underwent a colonoscopy that revealed more than 50 pedunculated colorectal juvenile polyps. Both patients harbored a germline pathogenic mutation in BMPR1A. Endoscopic resection of all polyps was attempted but failed. Finally, the son received endoscopic resection of polyps in the rectum and sigmoid colon, and laparoscopic subtotal colectomy. Ten polyps were selected for pathological examination. All were revealed to be typical juvenile polyps, with cystically dilated glands filled with mucus. Thus, the diagnosis of JPS was confirmed in the son. A review of the literatures revealed that patients with JPS can sometimes have adenomatous change. Most polyps in patients with JPS are benign hamartomatous polyps with no dysplasia. A review of 767 colorectal JPS polyps demonstrated that 8.5% of the polyps contained mild to moderate dysplasia, and only 0.3% had severe dysplasia or cancer. It is difficult to differentiate juvenile polyps with dysplasia from adenoma, which could explain why juvenile polyps have been reported to have adenomatous changes in patients with JPS. Therefore, patients with JPS, especially those with concomitant dysplasia and adenocarcinoma, might be easily diagnosed as FAP in clinical practice.ConclusionsJuvenile polyp with dysplasia is often diagnosed as adenoma, which might lead to the misdiagnosis of JPS as FAP. The differential diagnosis of JPS versus FAP, should be based on comprehensive evaluation of clinical presentation, endoscopic appearance and genetic investigations; not on the presence or absence of adenoma.

Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles

ObjectiveSporadic early-onset colorectal cancer (EOCRC) has bad prognosis, yet is poorly represented by cell line models. We examine the key mutational and transcriptomic alterations in an organoid biobank enriched in...

Clinical and Histologic Overlap and Distinction Among Various Hamartomatous Polyposis Syndromes.

INTRODUCTION:Hamartomatous polyposis syndromes (HPS) are rare autosomal-dominant inherited disorders associated with gastrointestinal (GI) tract and other cancers. HPS include Peutz-Jeghers syndrome (PJS), juvenile polyposis syndrome (JPS), and phosphatase and tensin homolog hamartomatous tumor syndromes (PHTS). Diagnosis, management, and outcome prediction of HPS pose a clinical challenge. To characterize genotype, phenotype, histology and outcomes of individuals with HPS.METHODS:A retrospective cohort study (2004–2017) of consecutive patients that were clinically diagnosed with HPS that visited a specialized GI oncology clinic. Demographic, clinicopathological, and genetic data were obtained from medical records.RESULTS:Fifty-two individuals from 34 families were included. Common clinical manifestations were GI bleeding (40% JPS, 23% PJS, and 25% PHTS) and bowel obstruction (46.15% PJS and 11.4% JPS). Twenty patients (38.4%) underwent surgery, 5 of whom required multiple procedures. Higher polyp burden was associated with the need for surgery (P = 0.007). Polyp histology varied widely with 69.2% of patients exhibiting histology different from the syndrome hallmark. GI cancer history was positive in 65%, 40%, and 50% of JPS, PJS, and PHTS families, respectively. Five (9.6%) patients developed cancers (one patient each had small bowel-1, colon-1, and thyroid-1, one patient had both small bowel adenocarcinoma and breast cancer, and one had both breast cancer and liposarcoma). Twenty (38.4%) patients tested positive for STK11, PTEN, SMAD4, BMPR1A, or AKT1 mutations: Sanger sequencing and multi-gene next generation sequencing panels detected mutations in 40.9% and 100% of tested cases, respectively.DISCUSSION:HPS patients present versatile phenotypes with overlapping clinical and histological characteristics. Polyp burden is associated with the need for surgery. Next-generation sequencing increases mutation detection.

MON-235 Bone Morphogenic Protein Receptor Variants: A New Cause of Primary Ovarian Insufficiency

Primary ovarian insufficiency (POI) is a major cause of infertility. This disease is characterized by amenorrhea with an increase in gonadotropin levels and affects 1% of women before the age of 40. POI may be the result of a broad spectrum of disorders caused by two main mechanisms: abnormal follicular development and follicle depletion due to a defect in their formation or an aberrantly rapid depletion of the stock. Although the majority of cases are idiopathic, POI can be trigged by autoimmune disease, infectious agents, iatrogenic effects, or genetic causes. It may also be part of syndromic diseases such as Turner, Fragile-X or Blepharophimosis Ptosis Epicanthus Inversus syndrome. However, in the majority of cases, the cause of POI is unknown suggesting that new causative genes are yet to be discovered. We conducted a Whole Exome Sequencing on 69 Caucasian women with sporadic POI and performed a bioinformatics analysis on a specific subset of 420 coherent candidate genes1. We identified variations in about fifty genes potentially linked to POI, including new genes never described to be associated with the diseases aetiology, such as AuTophagy related Genes (ATG)2, KHDRBS1 gene encoding an RNA-binding protein3, NOTCH2 gene4... We also found variants in genes encoding Bone Morphogenetic Protein Receptors (BMPR1A and BMPR1B) in three patients. These receptors bind ligands of TGFβ superfamily. Mutations of members of this family (e.g. BMP15, GDF9) are known to be involved in the etiology of POI. BMPR encode transmembrane receptors with serine-threonine kinase activity expressed in the granulosa cells of growing follicles. Missense variations, located in the kinase domain of both receptors 1A and 1B, are predicted to be deleterious in silico. We studied the signaling pathway of these receptors: SMAD proteins phosphorylation, measurement of their transcriptional activity and expression of specific target genes and showed that they are deleterious. Bmpr1a -/- and Bmpr1b-/- mouse models develop infertility due to a reduced spontaneous ovulation and compromised cumulus expansion respectively, indicating that both genes play a crucial role in fertility. Altogether, our study describes the first BMPR1A and 1B mutations associated with POI and increases the number of genes formally implicated as being responsible for this condition. 1Patiño LC, Beau I, Carlosama C, Buitrago JC, González R, Suárez CF, Patarroyo MA, Delemer B, Young J, Binart N, Laissue P. Hum Reprod. 2017; 32(7):1512-1520. 2Delcour C, Amazit L, Patino L, Magnin F, Fagart J, Delemer B, Young J, Laissue P, Binart N, Beau I. Genet Med. 2018 (in press) 3Carlosama C, Patiño L, Beau I, Morel A, Delemer B, Young J, Binart N, Laissue P. Clin Endocrinol (Oxf). 2018 (in press) 4Patiño LC, Beau I, Morel A, Delemer B, Young J, Binart N, Laissue P. Hum Mutat. 2018 (in press)

3323 Juvenile Polyposis Syndrome Patients Without a Mutation in SMAD4 or BMPR1A: Clinical Presentation and Novel Drivers of Disease

OBJECTIVES/SPECIFIC AIMS: Juvenile Polyposis Syndrome (JPS) is an inherited cancer predisposition syndrome sometimes attributed to a germline mutation in SMAD4 or BMPR1A. However, many patients meet clinical criteria for JPS without having a pathogenic alteration in either gene. Herein, we perform a cross-sectional analysis of JPS patients at a pediatric and adult tertiary referral center to understand potential differences in the clinical presentation and outcomes of patients with or without a known causative gene mutation. Additionally, we conduct whole exome sequencing (WES) on a subset of the pediatric patients to evaluate for novel genomic drivers of disease. METHODS/STUDY POPULATION: Data were abstracted from medical charts using IRB-approved protocols at the Children’s Hospital of Philadelphia (CHOP) and the University of Pennsylvania (Penn). Records were reviewed for patients with a clinical diagnosis of JPS and genetic testing result and seen at either institution in the last 10 years (2008-2018). Patients recruited for sequencing were consented for blood draw through the CHOP IRB protocol, and had whole exome sequencing completed at 70X depth, with data analyzed through institutional pipeline. RESULTS/ANTICIPATED RESULTS: Records were reviewed for 41 patients at CHOP and 19 patients at Penn, for a total of 60 JPS patients. Mean age of CHOP cohort was 11 years: 58.5% male, mean length of follow up 3.9 years. Mean age Penn cohort was 33 years: 47.4% male, mean length of follow up 9.3 years. In the pediatric cohort, 7 patients (17%) had a mutation in BMPR1A (n=6) or SMAD4 (n=1); in the adult cohort, 15 patients (79%) had a mutation in BMPR1A (n=3) or SMAD4 (n=12). The average number of polyps in the pediatric cohort was not significantly higher in patients with a SMAD4 or BMPR1A mutation (9.3 polyps/year of surveillance with a SMAD4 or BMPR1A mutation, vs 5.7 polyps/year; p=0.19). In combined cohort review, all individuals that required gastrectomy and/or colectomy (n=8) as well as all those who developed gastrointestinal cancer (n=3) had a mutation in SMAD4 or BMPR1A. Of the patients who underwent whole exome sequencing (n=13), potential causative germline mutations were identified in four patients (30.8%); all potential drivers identified were within the TNF/BMP pathway. DISCUSSION/SIGNIFICANCE OF IMPACT: This data from a dual-institution review demonstrates that the rate of SMAD4/BMPR1A mutation in JPS is lower in a pediatric cohort compared to an adult cohort. Furthermore, although individuals with JPS may have similar clinical presentations in childhood regardless of whether or not a causative mutation is present, the presence of a mutation in SMAD4 or BMPR1A is associated with a more severe course of disease in adulthood. Further study and a larger cohort will be required to fully validate these findings. Approximately 30% of patients who underwent germline WES had a potential novel driver identified, with further validation underway.

Somatic clones heterozygous for recessive disease alleles of BMPR1A exhibit unexpected phenotypes in Drosophila.

The majority of mutations studied in animal models are designated as recessive based on the absence of visible phenotypes in germline heterozygotes. Accordingly, genetic studies primarily rely on homozygous loss-of-function to determine gene requirements, and a conceptually-related 'two-hit model' remains the central paradigm in cancer genetics. Here we investigate pathogenesis due to somatic mutation in epithelial tissues, a process that predominantly generates heterozygous cell clones. To study somatic mutation in Drosophila, we generated inducible alleles that mimic human Juvenile polyposis-associated BMPR1A mutations. Unexpectedly, four of these mutations had no phenotype in heterozygous carriers but exhibited clear tissue-level effects when present in somatic clones of heterozygous cells. We conclude that these alleles are indeed recessive when present in the germline, but nevertheless deleterious when present in heterozygous clones. This unforeseen effect, deleterious heteromosaicism, suggests a 'one-hit' mechanism for disease initiation that may explain some instances of pathogenesis associated with spontaneous mutation.

Screening of BMPR1a for pathogenic mutations in familial colorectal cancer type X families from Newfoundland.

The Canadian province of Newfoundland and Labrador (NL) reports one of the highest incidence rates of familial colorectal cancer (CRC) worldwide. The NL population is an invaluable resource for studying genetic disorders because of a unique ancestry, and a willingness to participate in research studies. Familial colorectal cancer type X (FCCTX) describes a cluster of families with strong predisposition for CRC, of unknown etiology. A putative link between FCCTX and BMPR1a mutations has been identified in the Finnish population; however these findings have not been independently replicated. To investigate a potential connection between BMPR1a and FCCTX, we screened a cohort of 22 probands from unrelated NL FCCTX families using Sanger sequencing. This analysis did not independently replicate findings seen in Finland; as no candidate pathogenic BMPR1a mutations were uncovered. Our findings highlight that BMPR1a mutations are not a major contributor of FCCTX incidence in NL. Further investigation of additional FCCTX populations may assist in delineating a role for BMPR1a, if any, in FCCTX globally.

Association of autoimmune thyroiditis and celiac disease with Juvenile Polyposis due to 10q23.1q23.31 deletion: Potential role of PI3K/Akt pathway dysregulation

Juvenile Polyposis (JP) is a rare hereditary condition characterized by diffuse hamartomatous gastrointestinal polyposis, associated with a significantly increased risk of neoplastic transformation. Most of the cases are caused by SMAD and BMPR1A mutations, while 10q23 microdeletions, encompassing both PTEN and BMPR1A oncogenes, are extremely rare, typically associated with more aggressive JP, and extraintestinal features overlapping with PTEN Hamartoma Tumor Syndrome. We present the first case of a young female with multiple autoimmune disorders (i.e. thyroiditis and celiac disease), associated with JP, cardiac defects and epilepsy, who carries a de novo heterozygous 10q23.1q23.31 deletion. The dysregulation of the PI3K/Akt pathway is advanced as the putative mechanism connecting autoimmune, malformative and neoplastic disorders. A literature review of clinical manifestation, gene alterations and the treatment of patients with 10q23 deletion is also provided, highlighting the importance of comprehensive, long-term, multi-disciplinary management, aimed at early identification and treatment of both intestinal and extraintestinal disorders.

Germline mutations in seemingly sporadic pancreatic cancer.

312 Background: Genetic predisposition underlies approximately 10% of pancreatic cancer (PC). Germline mutations in BRCA1, BRCA2, PALB2, ATM, TP53, MLH1, MSH2, MSH6, PMS2, PRSS1, APC, BMPR1A, STK11, CDKN2A, and SMAD4 are associated with PC susceptibility. Since young onset is not a hallmark of hereditary PC, we have hypothesized that seemingly sporadic cases may carry germline mutations in these genes. Methods: To investigate the prevalence of germline mutations in these 15 genes among sporadic PC cases, we analyzed lymphocyte DNA next generation sequencing data from 296 PC cases ascertained over 1 year from two PC referral centers in Toronto and Montreal. Results: We identified 21 mutations in 5 of the 15 genes (10 in BRCA2, 2 in BRCA1, 5 in ATM, 1 in p16, 3 in PRSS1) in 19 individuals. One individual carried two BRCA2 mutations that are usually observed together, and another individual carried both a PRSS1 and ATM mutation, however, in the absence of pancreatitis, PRSS1 mutations are not known to increase risk of PC. Incidences of 1.7% and 4.1% in ATM and BRCA-1 or -2 were observed. Approximately half of these mutations (4/9 for BRCA2, 1/2 for BRCA1, and 1/1 for p16) were known founder mutations in the French Canadian, Ashkenazi Jewish, Dutch or Asian populations. Since the role of ATMin PC predisposition remains under investigation, we obtained further evidence by testing for loss of the wild-type allele in cases with available tumor tissue. We observed loss of the wild-type allele in the 2 cases tested. Interestingly, the final pathology for the resected specimen from one of these two cases favored an ampullary cancer rather than a PC diagnosis. Conclusions: Since there are screening implications for the relatives of carriers and potentially treatment considerations for patients using therapies targeting DNA repair defects, these findings suggest a role for reflex testing of incident cases for at least population-specific recurrent mutations. Our findings also suggest that ATM germline mutations may give rise to ampullary cancer, underlie 1.4% of incident PC cases and imply broader testing considerations. For patients, testing may direct therapy choices if these tumors are targetable using agents that exploit DNA repair defects.

Expanding the genotype\u2013phenotype spectrum in hereditary colorectal cancer by gene panel testing

Hereditary syndromes causing colorectal cancer include both polyposis and non-polyposis syndromes. Overlapping phenotypes between the syndromes have been recognized and this make targeted molecular testing for single genes less favorable, instead there is a gaining interest for multi-gene panel-based approaches detecting both SNVs, indels and CNVs in the same assay. We applied a panel including 19 CRC susceptibility genes to 91 individuals of six phenotypic subgroups. Targeted NGS-based sequencing of the whole gene regions including introns of the 19 genes was used. The individuals had a family history of CRC or had a phenotype consistent with a known CRC syndrome. The purpose of the study was to demonstrate the diagnostic difficulties linked to genotype-phenotype diversity and the benefits of using a gene panel. Pathogenicity classification was carried out on 46 detected variants. In total we detected sixteen pathogenic or likely pathogenic variants and 30 variants of unknown clinical significance. Four of the pathogenic or likely pathogenic variants were found in BMPR1A in patients with unexplained familial adenomatous polyposis or atypical adenomatous polyposis, which extends the genotype-phenotype spectrum for this gene. Nine patients had more than one variant remaining after the filtration, including three with truncating mutations in BMPR1A, PMS2 and AXIN2. CNVs were found in three patients, in upstream regions of SMAD4, MSH3 and CTNNB1, and one additional individual harbored a 24.2 kb duplication in CDH1 intron1.

Mutation analysis of 13 driver genes of colorectal cancer-related pathways in Taiwanese patients.

To investigate the driver gene mutations associated with colorectal cancer (CRC) in the Taiwanese population. In this study, 103 patients with CRC were evaluated. The samples consisted of 66 men and 37 women with a median age of 59 years and an age range of 26-86 years. We used high-resolution melting analysis (HRM) and direct DNA sequencing to characterize the mutations in 13 driver genes of CRC-related pathways. The HRM assays were conducted using the LightCycler® 480 Instrument provided with the software LightCycler® 480 Gene Scanning Software Version 1.5. We also compared the clinicopathological data of CRC patients with the driver gene mutation status. Of the 103 patients evaluated, 73.79% had mutations in one of the 13 driver genes. We discovered 18 novel mutations in APC, MLH1, MSH2, PMS2, SMAD4 and TP53 that have not been previously reported. Additionally, we found 16 de novo mutations in APC, BMPR1A, MLH1, MSH2, MSH6, MUTYH and PMS2 in cancerous tissues previously reported in the dbSNP database; however, these mutations could not be detected in peripheral blood cells. The APC mutation correlates with lymph node metastasis (34.69% vs 12.96%, P = 0.009) and cancer stage (34.78% vs 14.04%, P = 0.013). No association was observed between other driver gene mutations and clinicopathological features. Furthermore, having two or more driver gene mutations correlates with the degree of lymph node metastasis (42.86% vs 24.07%, P = 0.043). Our findings confirm the importance of 13 CRC-related pathway driver genes in the development of CRC in Taiwanese patients.

Somatic alterations in juvenile polyps from BMPR1A and SMAD4 mutation carriers.

Juvenile polyposis syndrome (JPS) is a rare autosomal dominant disorder predisposing to gastrointestinal hamartomatous polyps and cancer with a pathogenic SMAD4 or BMPR1A germline mutation (1st-hit) being identified in about 40-50% of patients. Little is known, however, about the occurrence and nature of somatic alterations (2nd-hit) in SMAD4-/BMPR1A-related juvenile polyps. In this study, we screened 25 polyps from three patients carrying either a pathogenic SMAD4 (c.1244-1247delACAG) or BMPR1A (c.583C>T; p.Gln195*) germline mutation for somatic alterations. The SMAD4-related polyps were also analyzed for SMAD4 protein expression by immunohistochemistry. Despite comprehensive screening for loss of heterozygosity (LOH), mutations in the coding sequence, chromosomal rearrangements, and promoter methylation, no somatic alterations could be identified in 14 SMAD4-related polyps. SMAD4 protein expression, however, was lost in 8 (57%) of 14 juvenile polyps with 6 showing concomitant loss in both, the epithelial and stromal, compartments. In the BMPR1A-related polyps, five out of nine (56%) displayed LOH. Further analysis of selected polyps revealed that LOH was gene copy number neutral and had occurred in the epithelial compartment. The heterogeneity of genetic mutations and protein expression levels indicates that different modes of gene inactivation can be operational in SMAD4- and BMPR1A-related polyp formation. Our observation, that about half of BMPR1A-related polyps displayed LOH, predominantly in the epithelial compartment, is compatible with BMPR1A acting as a tumour suppressor gene. Still, it remains to be determined whether juvenile polyp development generally requires loss of BMPR1A expression or, as observed in some SMAD4-related polyps, can occur despite normal protein expression.