Mutations In TBX5 Research Articles

Novel Case of Isolated Peritoneal Carcinomatosis from Metastatic Prostate Cancer Carrying a Pathogenic BRCA Mutation Treated with Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy

We present the first case of isolated peritoneal carcinomatosis (PC) from prostate cancer carrying a previously unreported pathogenic BRCA2 mutation without other organ involvement. CT showed PC with no extra-peritoneal metastases. Treatment included neoadjuvant chemotherapy, cytoreductive surgery/hyperthermic intraperitoneal chemotherapy (CRS/HIPEC), and adjuvant chemotherapy. Patient is disease-free after 30-months. Germline genetic testing showed BRCA2 c.1813dupA (p.I605Nfs*11) mutation, reported in breast and ovarian cancers. Tumor genomic testing also revealed TMPRSS2, TBX3, and TP53 mutations. Given the comparable presentation and BRCA-mutation profile to ovarian cancer, BRCA2 mutations may contribute to disease phenotypes. CRS/HIPEC may provide long-term outcomes for this unique metastatic pattern.

TBX3 and TBX5 duplication: A family with an atypical overlapping Holt-Oram/ulnar-mammary syndrome phenotype

Holt-Oram syndrome (HOS) is a rare, autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. A wide spectrum of TBX5 mutations have been reported previously, most resulting in a null allele leading to haploinsufficiency. TBX5 gene duplications have been previously reported in association with typical and atypical HOS phenotypes. Ulnar-Mammary syndrome (UMS) is a distinct rare, autosomal dominant condition caused by mutations in the TBX3 gene. TBX5 and TBX3 are physically linked in cis on human chromosome 12 and contiguous chromosome 12q24 deletions comprising both TBX5 and TBX3 genes have been previously reported but to our knowledge, duplications have never been described. We report on a large German family with at least 17 affected individuals over 6 generations bearing a duplication at 12q24.21 identified on array-CGH comprising both TBX5 and TBX3 genes. Affected patients are presenting with HOS and UMS symptoms, consisting of variable limb anomalies involving the radial and the ulnar rays and cardiac findings such as congenital heart defects, persistent arterial duct or aortic stenosis, and non-classical symptoms, such as supernumerary nipples and cardiomyopathy. Fluorescence in situ hybridisation confirmed a tandem duplication at the 12q24.21 locus. This is the first report of a contiguous TBX3/TBX5 duplication associated with HOS/UMS phenotype.

Characterization of Stromal Tumor-infiltrating Lymphocytes and Genomic Alterations in Metastatic Lobular Breast Cancer.

Invasive lobular carcinoma (ILC) represents the second most common histologic breast cancer subtype after invasive ductal carcinoma (IDC). While primary ILC has been extensively studied, metastatic ILC has been poorly characterized at the genomic and immune level. We retrospectively assembled the multicentric EuroILC series of matched primary and metastatic samples from 94 patients with estrogen receptor (ER)-positive ILC. Stromal tumor-infiltrating lymphocytes (sTILs) were assessed by experienced pathologists. Targeted sequencing and low pass whole-genome sequencing were conducted to detect mutations and copy-number aberrations (CNAs). We compared the frequencies of the alterations in EuroILC with those from patients with ER-positive metastatic ILC (n = 135) and IDC (n = 563) from MSK-IMPACT. Low sTIL levels were observed in ILC metastases, with higher levels in the mixed nonclassic histology. Considering ILC metastases from EuroILC and MSK-IMPACT, we observed that >50% of tumors harbor genomic alterations that have previously been associated with endocrine resistance. A matched primary/metastasis comparison in EuroILC revealed mutations (AKT1, ARID1A, ESR1, ERBB2, or NF1) and CNAs (PTEN or NF1 deletion, CYP19A1 amplification) associated with endocrine resistance that were private to the metastasis in 22% (7/32) and 19% (4/21) of patients, respectively. An increase in CDH1, ERBB2, FOXA1, and TBX3 mutations, in CDH1 deletions and a decrease in TP53 mutations was observed in ILC as compared with IDC metastases. ILC metastases harbor genomic alterations that may potentially explain endocrine resistance in a large proportion of patients, and present genomic differences as compared with IDC metastases.

Novel mutations of TCTN3/LTBP2 with cellular function changes in congenital heart disease associated with polydactyly.

Congenital heart disease (CHD) associated with polydactyly involves various genes. We aimed to identify variations from genes related to complex CHD with polydactyly and to investigate the cellular functions related to the mutations. Blood was collected from a complex CHD case with polydactyly, and whole exome sequencing (WES) was performed. The CRISPR/Cas9 system was used to generate human pluripotent stem cell with mutations (hPSCs‐Mut) that were differentiated into cardiomyocytes (hPSC‐CMs‐Mut) and analysed by transcriptomics on day 0, 9 and 13. Two heterozygous mutations, LTBP2 (c.2206G>A, p.Asp736Asn, RefSeq NM_000428.2) and TCTN3 (c.1268G>A, p.Gly423Glu, RefSeq NM_015631.5), were identified via WES but no TBX5 mutations were found. The stable cell lines of hPSCs‐LTBP2mu/TCTN3mu were constructed and differentiated into hPSC‐CMs‐LTBP2mu/TCTN3mu. Compared to the wild type, LTBP2 mutation delayed the development of CMs. The TCTN3 mutation consistently presented lower rate and weaker force of the contraction of CMs. For gene expression pattern of persistent up‐regulation, pathways in cardiac development and congenital heart disease were enriched in hPSCs‐CM‐LTBP2mu, compared with hPSCs‐CM‐WT. Thus, the heterozygous mutations in TCTN3 and LTBP2 affect contractility (rate and force) of cardiac myocytes and may affect the development of the heart. These findings provide new insights into the pathogenesis of complex CHD with polydactyly.

Relationship Between Severity of T Cell Lymphopenia and Immune Dysregulation in Patients with DiGeorge Syndrome (22q11.2 Deletions and/or Related TBX1 Mutations): a USIDNET Study.

DiGeorge syndrome has substantial heterogeneity with variable immune deficiency and dysregulation. Implicated immunopathology includes reduced thymic output and increased peripheral homeostatic proliferation with Th2 skewing and expansion of self-reactive cells. We hypothesized that T cell lymphopenia severity will be associated with higher odds of autoimmunity and/or asthma. Using the US Immunodeficiency Network registry, we identified patients with 22q11.2 deletion (and/or TBX1). Initial absolute CD3+ T cell values were stratified: normal, 50-99% and below 50% of the lower limit of age-adjusted normal values. Patients with and without reported autoimmunity and asthma were compared using chi-square tests and multivariate logistic regression. Among 415 patients, autoimmunity was reported in 17 (4.1%), and asthma was reported in 28 (6.7%). Compared with those with no reported autoimmunity, patients with reported autoimmunity more frequently had low CD19+ B cells [3.3% (12/364) vs 28.6% (4/14); p = 0.002] and low IgG [6.2% (20/321) vs 29.4% (5/17); p = 0.005] levels. There were no statistically significant differences in other immune characteristics among those with and without reported asthma. Patients with absolute CD3 levels below 50% of age-adjusted normal values had higher odds of reported autoimmunity (n = 319, OR = 7.56, 95% CI = 1.58-36.17, p = 0.01) and reported asthma (n = 319, OR = 4.5, 95% CI = 1.06-18.93, p = 0.04) as compared with those with normal CD3 values, adjusted for age and low IgG. Absolute CD3+ T cell counts below 50% of age-adjusted normal values may be associated with higher odds of autoimmunity and/or asthma in patients with DiGeorge syndrome and be potentially useful to identify higher-risk patients.

Clinical and Genetic Findings in a Chinese Cohort of Patients with Digeorge Syndrome-Related Hypoparathyroidism

Objective: Patients with DiGeorge syndrome (DGS) are undiagnosed due to its diverse manifestations. We aimed to characterize the clinical manifestations in a group of Chinese patients of DGS with childhood-onset hypoparathyroidism (HP) as the primary referral, and to report a novel TBX1 mutation. Methods: In this single-center observational study, clinical features and biochemical indices were recorded in 26 patients with DGS and 114 patients with idiopathic HP (IHP). An in vitro functional experiment was launched to analyze the novel TBX1 missense mutation. Results: Compared with 114 patients of IHP (19.1 [13.5, 27.3] years old), 26 patients of DGS (14.9 [10.4, 20.3] years old) had the following differences: an earlier onset age of hypocalcemia; higher levels of serum parathyroid hormone, with a similar disease course; and lower doses of vitamin D preparation therapy. Among the 26 patients of DGS, only 3 of them were clinically diagnosed as this syndrome prior to genetic testing. A total of 25 patients of DGS were verified to have a TBX1 deletion and 1 case with a novel missense mutation of TBX1. The novel p.Y490C mutation in TBX1, located in the transactivation domain, was verified to decrease the transcriptional activity of the TBX1 protein. Conclusion: In this Chinese group of patients with DGS-related HP, a relatively earlier onset age and less severity of HP were found compared to that of patients with IHP. Less common extraparathyroid manifestations are clues for the diagnosis of DGS. Additionally, our discovery of a novel TBX1 missense mutation expands the mutation database of DGS. Abbreviations: DGS = DiGeorge syndrome; HP = hypoparathyroidism; IHP = idiopathic hypoparathyroidism; LCR = low copy repeat; PCR = polymerase chain reaction; PTH = parathyroid hormone; TAD = transactivation domain.

OR33-06 Potential Role of Mutations in TBX3 in Human Weight Regulation

Introduction: Tbx3 has been shown to play a role in the terminal specification of hypothalamic melanocortin neurons during neonatal development & in maintaining the plasticity of their peptidergic role in adulthood in animal experiments (1). The absence of humans with biallelic mutations in TBX3 & the conservation of the critical domains across species emphasizes its essential role in life. Heterozygous mutations in humans have been associated with ulnar mammary syndrome (UMS) with a spectrum of phenotype including obesity. Based on these observations, we hypothesized that heterozygous mutations in the conserved regions of TBX3 may play an important role in the weight regulation pathway in humans. Methods: The Genetics of Early Childhood Obesity (GECO) study enrolls children with severe (BMI > 120% of 95th %tile of CDC reference) early onset (< 6 years) obesity. Whole exome sequencing (WES) was performed in a subset of proband-parent trios. Peripheral mononuclear cells (PBMCs) from selected families were collected to generate induced pluripotent stem cells using non-integrating Sendai virus. Differentiation into disease-relevant hypothalamic neurons was performed using the published protocol (2). Loss-of-function models & isogenic controls were created using CRISPR/Cas9 & their cellular/molecular phenotypes were obtained at several time points during the course of differentiation. Results: We have identified a family with heterozygous mutation in TBX3 (p.His205Tyr, c.613 C>T, g.115118728 G>A). The proband is an 11-year old boy with morbid obesity (BMI 43.9 kg/m2, BMIz + 3.25), advanced bone age, precocious puberty & type 2 diabetes. Trio analysis ruled out recessive & compound heterozygote causative variants, & none of the identified de novo variants were considered pathogenic. His mother suffers from severe obesity (BMI 38 kg/m2 post-bariatric surgery) supporting an autosomal dominant inheritance of the phenotype. The putative causal variant in TBX3 segregates in the proband, mother & maternal grandmother. Located in the DNA binding domain of T-box, the variant is predicted to be deleterious by 4 in silico algorithms & rare in population-based databases (mean allele frequency 0.006% in gNOMAD, absent in ClinVar). Consistent with the variable penetrance of the phenotype in UMS, neither mother nor child have the classic features, but, the mother has uterine anomalies causing 6 spontaneous abortions & was unable to breast feed due to inverted nipples. Ongoing functional studies in human hypothalamic neurons suggest that a decrease in melanocortin signaling possibly explaining the phenotype in this family. Conclusions: Mutations in TBX3 in humans may have a role as a monogenic cause of obesity and disease-relevant hypothalamic stem cells can serve as models to study them. Ref: 1) Quarta et al. Nat Metab 2019, 1(2), 222-35; 2) Wang et al. JCI, 125(2): 796-808

Phenotype and outcome of pulmonary arterial hypertension patients carrying aTBX4mutation

TBX4 mutation causes small patella syndrome (SPS) and/or pulmonary arterial hypertension (PAH). The characteristics and outcomes of PAH associated with TBX4 mutations are largely unknown. We report the clinical, functional, radiologic, histologic and haemodynamic characteristics and outcomes of heritable PAH patients carrying a TBX4 mutation from the French pulmonary hypertension (PH) network. 20 patients were identified in 17 families. They were characterised by a median age at diagnosis of 29 years (0-76 years) and a female to male ratio of three. Most of the patients (70%) were in New York Heart Association (NYHA) functional class III or IV with a severe haemodynamic impairment (median pulmonary vascular resistance (PVR) of 13.6 (6.2-41.8) Wood units). Skeletal signs of SPS were present in 80% of cases. Half of the patients had mild restrictive or obstructive limitation and diffusing capacity of the lung for carbon monoxide (D LCO) was decreased in all patients. High-resolution computed tomography (HRCT) showed bronchial abnormalities, peri-bronchial cysts, mosaic distribution and mediastinal lymphadenopathies. PAH therapy was associated with significant clinical improvement. At follow-up (median 76 months), two patients had died and two had undergone lung transplantation. One-year, three-year and five-year event-free survival rates were 100%, 94% and 83%, respectively. Histologic examination of explanted lungs revealed alveolar growth abnormalities, major pulmonary vascular remodelling similar to that observed in idiopathic pulmonary arterial hypertension (IPAH) and accumulation of cholesterol crystals within the lung parenchyma. PAH due to TBX4 mutations may occur with or without skeletal abnormalities across a broad age range from birth to late adulthood. PAH is usually severe and associated with bronchial and parenchymal abnormalities.

Abstract PD8-06: The genomic landscape of metastatic special histologic types of breast cancer

Abstract Introduction: Breast cancer (BC) is heterogeneous and comprises diverse entities with distinctive phenotypes, biology and clinical presentations. There are 21 special histologic types, accounting for approximately 20% of invasive BCs. Large-scale sequencing studies have focused preferentially on common histologic forms, and data on the genomic landscape of special histologic types, particularly in the metastatic setting, are scarce. Here, we sought to define the repertoire of somatic genetic alterations of metastatic special histologic types of BC. Materials and methods: We conducted a reanalysis of MSK-IMPACT targeted sequencing data of 346 special histologic types of BC from Razavi et al (Cancer Cell 2018), including invasive lobular (ILC; primary, n=129; metastasis, n=150), mucinous (primary n=11, metastasis n=5), micropapillary (primary n=8, metastasis n=13) and metaplastic carcinomas (primary, n=6; metastasis, n=11). Somatic single nucleotide variants (SNVs) and copy number alterations (CNAs) were inferred using a validated bioinformatics pipeline, and mutational signatures were assessed with SigMA. Genetic alterations of metastatic special histologic type BCs were compared to those of primary tumors and metastatic invasive ductal carcinomas of no special type (IDC-NSTs) matched by age, menopausal status and estrogen receptor (ER)/HER2 status at a 1:2 ratio for ILCs and at a 1:3 ratio for the remaining BCs. Results: The most frequently mutated genes in metastatic ILCs were CDH1 (71%), PIK3CA (49%), ESR1 (20%) and KMT2C (19%). ESR1 mutations were more frequent in metastatic than in primary ILCs (20% vs 3%; P&amp;lt;0.01). Metastatic ILCs, as compared to matched metastatic IDC-NSTs, had a higher frequency of mutations in CDH1 (71% vs 2%; P&amp;lt;0.001), KMT2C (19% vs 10%; P&amp;lt;0.05), TBX3 (16% vs 5%; P&amp;lt;0.001), ERBB2 (12% vs 3%; P&amp;lt;0.01) and FOXA1 (10% vs 2%; P&amp;lt;0.05), and a lower frequency of mutations in TP53 (16% vs 36%; P&amp;lt;0.001) and GATA3 (6% vs 16%; P&amp;lt;0.01). GATA3, NCOR1 and ESR1 (each, 40%) were the most frequently mutated genes in metastatic mucinous BCs. ESR1 and NCOR were numerically more frequent in metastatic than in primary mucinous BCs (40% vs 0%; P&amp;gt;0.05). Metastatic mucinous BCs had a numerically higher frequency of mutations in ESR1 (40% vs 6%; P&amp;gt;0.05) and GATA3 (40% vs 0%; P&amp;gt;0.05), and lower frequency of TP53 mutations (20% vs 53%; P&amp;gt;0.05) than matched metastatic IDC-NSTs. PIK3CA and TP53 were the most recurrently mutated genes in metastatic micropapillary BCs and were numerically more frequent in metastatic than in primary tumors (PIK3CA, 61% vs 25%; TP53, 46% vs 25%; P&amp;gt;0.05). PIK3CA was more frequently mutated in metastatic micropapillary BCs than IDC-NSTs (61% vs 23%; P&amp;lt;0.05). Metastatic metaplastic BCs most frequently harbored mutations in TP53 (72%) and PIK3CA (45%), with comparable frequencies in primary metaplastic BCs. ATM (27% vs 3%; P&amp;lt;0.05), MAP3K13 and YAP1 (27% vs 0%; P&amp;lt;0.05) were more frequently mutated in metastatic metaplastic BCs than in matched IDC-NSTs. No differences in the frequency of CNAs were seen between metastatic BCs of special histologic type and matched primary tumors or metastatic IDC-NSTs. Akin to metastatic IDC-NSTs, aging or APOBEC were the predominant mutational SNV signatures in metastatic ILCs, micropapillary and metaplastic BCs. Conclusions: The repertoire of somatic genetic alterations of primary and metastatic special histologic type BCs markedly overlaps. Nonetheless, there is an enrichment of mutations in ESR1 in metastatic BCs of typically ER-positive special histologic type. Metastatic BCs of special histologic type show an enrichment of similar genetic alterations as primary tumors, such as a higher frequency of mutations in CDH1, FOXA1 and TBX3 in ILCs, a lower frequency of PIK3CA and TP53 mutations in mucinous BCs and a higher frequency of PIK3CA and TP53 mutations in micropapillary and metaplastic BCs. Citation Format: Fresia Pareja, Simon SK Lee, Francisco Beca, Lorenzo Ferrando, Pier Selenica, David N Brown, Hannah Y Wen, Hong Zhang, Edi Brogi, Pedram Razavi, Sarat Chandarlapaty, Britta Weigelt, Jorge S Reis-Filho. The genomic landscape of metastatic special histologic types of breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD8-06.

Mutation screening of genes associated with congenital talipes equinovarus in pakistani families

Objectives: Congenital talipes equinovarus (CTEV) is characterized by midfoot cavus, forefoot adducts, hindfoot varus, and equinus. The condition leads to difficulty in walking. CTEV is one of the most common congenital birth defects and an incidence of 1–2/1000 newborns has been reported. The etiology of the CTEV is not completely understood. Multiple etiological factors, both environmental and genetic, have been implicated in the pathogenesis of CTEV. Genetic factors associated with the development of CTEV include mutations in genes related to limb patterning and development of muscle and blood vessels. The present study aims to screen four genes to detect the mutation underlying CTEV in families from Pakistan. Methods: Peripheral blood samples were collected from ten individuals from two Pakistani families with CTEV segregating in an autosomal recessive manner. Genomic DNA was isolated, followed by primer designing and Sanger sequencing of four known genes associated with the isolated form of CTEV (TBX4, PITX1, HOXD12, and HOXD9). Results: Clinical diagnoses of the affected individuals were made by consultant orthopedics. Sequencing analyses revealed that the four candidate genes screened here are not responsible for the CTEV phenotype in both families. Conclusion: Failure to detect sequence variant in four known genes associated with the isolated form of CTEV lead us to the conclusion that TBX4, PITX1, HOXD12, and HOXD9 mutations are not responsible for CTEV in both families. Therefore, genome-wide studies, including whole-genome single nucleotide polymorphism genotyping and whole-exome sequencing, are required to identify the underlying genetic defects in these families.

A novel TBX5 mutation predisposes to familial cardiac septal defects and atrial fibrillation as well as bicuspid aortic valve.

TBX5 has been linked to Holt-Oram syndrome, with congenital heart defect (CHD) and atrial fibrillation (AF) being two major cardiac phenotypes. However, the prevalence of a TBX5 variation in patients with CHD and AF remains obscure. In this research, by sequencing analysis of TBX5 in 178 index patients with both CHD and AF, a novel heterozygous variation, NM_000192.3: c.577G>T; p.(Gly193*), was identified in one index patient with CHD and AF as well as bicuspid aortic valve (BAV), with an allele frequency of approximately 0.28%. Genetic analysis of the proband’s pedigree showed that the variation co-segregated with the diseases. The pathogenic variation was not detected in 292 unrelated healthy subjects. Functional analysis by using a dual-luciferase reporter assay system showed that the Gly193*-mutant TBX5 protein failed to transcriptionally activate its target genes MYH6 and NPPA. Moreover, the mutation nullified the synergistic transactivation between TBX5 and GATA4 as well as NKX2-5. Additionally, whole-exome sequencing analysis showed no other genes contributing to the diseases. This investigation firstly links a pathogenic variant in the TBX5 gene to familial CHD and AF as well as BAV, suggesting that CHD and AF as well as BAV share a common developmental basis in a subset of patients.

Homozygous Null TBX4 Mutations Lead to Posterior Amelia with Pelvic and Pulmonary Hypoplasia

The development of hindlimbs in tetrapod species relies specifically on the transcription factor TBX4. In humans, heterozygous loss-of-function TBX4 mutations cause dominant small patella syndrome (SPS) due to haploinsufficiency. Here, we characterize a striking clinical entity in four fetuses with complete posterior amelia with pelvis and pulmonary hypoplasia (PAPPA). Through exome sequencing, we find that PAPPA syndrome is caused by homozygous TBX4 inactivating mutations during embryogenesis in humans. In two consanguineous couples, we uncover distinct germline TBX4 coding mutations, p.Tyr113∗ and p.Tyr127Asn, that segregated with SPS in heterozygous parents and with posterior amelia with pelvis and pulmonary hypoplasia syndrome (PAPPAS) in one available homozygous fetus. A complete absence of TBX4 transcripts in this proband with biallelic p.Tyr113∗ stop-gain mutations revealed nonsense-mediated decay of the endogenous mRNA. CRISPR/Cas9-mediated TBX4 deletion in Xenopus embryos confirmed its restricted role during leg development. We conclude that SPS and PAPPAS are allelic diseases of TBX4 deficiency and that TBX4 is an essential transcription factor for organogenesis of the lungs, pelvis, and hindlimbs in humans.

P1801Multiple members with an isolated atrial septal defect phenomenon presented in a family with Holt-Oram syndrome

Abstract Background Holt-Oram syndrome (HOS), characterized by upper limb malformations, congenital heart diseases (CHD) and/or cardiac conduction abnormalities, is an autosomal dominant disease. Almost all the HOS patients suffer from cardiac and limb abnormalities at the same time. Among them, atrial septal defect (ASD) is the most common cardiac anomaly. The unusual phenotype in a pedigree including multiple members with isolated ASD and a few with isolated limb deformity attracted attention. Aim To detect mutant genes of ASD in this pedigree and make a definitive diagnosis. To investigate the mutant type of the gene and illustrate the possible mechanism of heterogeneous phenotype. Methods Echocardiography, electrocardiography and physical examination were given to a four-generation Chinese HOS family. Of all 11 patients in this pedigree, eight patients had isolated ASD and one patient had isolated finger deformity. In addition, two patients suffered from both diseases (Figure. 1). Whole Exome Sequencing was performed on the proband and his relatives including three with ASD only, one with finger deformity only and one normal person. Sanger sequencing was performed on biological relatives in this pedigree to valid rare variants. Single nucleotide polymorphisms and insertions/deletions were identified using the GATK program. Pathogenicity was predicted in software like SIFT, Ployphen-2, MutationTaster and CADD_phred. Results An exon site mutation (c.100dupG:p.Ala34fs) of TBX5 was detected in all the patients of this pedigree. This mutation site is located in front of T-box and most probably leads to haploinsufficiency of TBX5 protein. In addition, only 42 variants (1 splicing single nucleotide variants (SNV), 15 insertions/deletions, 24 nonsynonymous SNVs, 1 stopgain SNVs and 1 unknown) were found in 29 genes, which are related to cardiac disease. Figure 1 Conclusions Our study revealed a mutation (c.100dupG:p.Ala34fs) of TBX5 and further demonstrated the possibility of HOS-related TBX5 mutation in patients with congenital ASD only. This study clarified the diagnosis of this pedigree, provided genetic counseling and promoted the prevention and treatment of HOS. Further research is needed on mechanisms that may lead to phenotypic variation between and within families, such as random monoallelic expression. Acknowledgement/Funding None

TBX1 is required for normal stria vascularis and semicircular canal development

Little is known about the role of TBX1 in post-otocyst stages of inner ear development. Here, we report on mice with a missense mutation of Tbx1 that are viable with fully developed but abnormally formed inner ears. Mutant mice are deaf due to an undeveloped stria vascularis and show vestibular dysfunction associated with abnormal semicircular canal formation. We show that TBX1 is expressed in endolymph-producing strial marginal cells and vestibular dark cells of the inner ear and is an upstream regulator of Esrrb, which previously was shown to control the developmental fate of these cells. We also show that TBX1 is expressed in sensory cells of the crista ampullaris, which may relate to the semicircular canal abnormalities observed in mutant mice. Inner ears of mutant embryos have a non-resorbed fusion plate in the posterior semicircular canal and a single ampulla connecting anterior and lateral canals. We hypothesize that the TBX1 missense mutation prevents binding with specific co-regulatory proteins. These findings reveal previously unknown functions of TBX1 during later stages of inner ear development.

Abstract 602: Mir-182-5p is a Conserved Downstream Effector of Tbx5 Involved in Heart Development and Arrhythmia in Zebrafish

Background: TBX5 mutations cause Holt-Oram syndrome (HOS) characterized by upper limb and cardiac malformations, but can also contribute to early-onset of atrial fibrillation. Focusing on miRNAs involved in TBX5 regulatory circuits with a cardiac relevant role, we identified miR-182-5p, belonging to miR-183 cluster, found upregulated in Tbx5-depleted hearts of mouse and zebrafish embryos. Methods: To functionally analyse the miR-182-5p role in developing heart, miR-182-5p was dysregulated in zebrafish zygotes of Tg(Myl7:EGFP) and Tg(myl7:gCaMP) transgenic lines. To stably deregulate miR-182-5p in zebrafish heart we exploited the Gal4/UAS system to restrict the miR-182 expression into cardiac context. For physiological analyses we performed the mechanogram of cardiac contraction and electrocardiogram recording. To understand miR-182-5p downstream regulation, in silico analyses, followed by ddPCR/real-time quantifications on dissected zebrafish hearts and rescue experiments both in transient and stable miR-182-5p overexpressing zebrafish embryos were performed. Results: Depletion of Tbx5 from cardiomyocytes increased the expression of miR-182 cluster family that is controlled by Kruppel-like factor 4 (KLF4), a transcription factor repressed by Tbx5. Both transient and stable upregulation of miR-182 in zebrafish affect heart morphology, calcium handling and the onset of arrhythmia while its cardiac-specific downregulation decreases cardiac defects in zebrafish HOS hearts. Expression analyses on selected miR-182-5p putative targets revealed that several calcium channel proteins resulted downregulated in miR-182-5p overexpressing hearts. Transgenic zebrafish line stably overexpressing miR-182-5p in the heart manifested arrhythmia overtime with or without cardiac structural defects. Conclusion: We identified miR-182-5p as a potential suitable target to interfere in the circuit between upstream genetic abnormalities and downstream effectors leading to arrhythmia occurrence.

Tbx5 inhibits hedgehog signaling in determination of digit identity.

Dominant TBX5 mutation causes Holt-Oram syndrome (HOS), which is characterized by limb defects in humans, but the underlying mechanistic basis is unclear. We used a mouse model with Tbx5 conditional knockdown in Hh-receiving cells (marked by Gli1+) during E8 to E10.5, a previously established model to study atrial septum defects, which displayed polydactyly or hypodactyly. The results suggested that Tbx5 is required for digit identity in a subset of limb mesenchymal cells. Specifically, Tbx5 deletion in this cell population decreased cell apoptosis and increased the proliferation of handplate mesenchymal cells. Furthermore, Tbx5 was found to negatively regulate the Hh-signaling activity through transcriptional regulation of Ptch1, a known Hh-signaling repressor. Repression of Hh-signaling through Smo co-mutation in Tbx5 heterozygotes rescued the limb defects, thus placing Tbx5 upstream of Hh-signaling in limb defects. This work reveals an important missing component necessary for understanding not only limb development but also the molecular and genetic mechanisms underlying HOS.

DNA‐binding specificity of TBX5 and its coding variants associated with Holt‐Oram Syndrome

Transcription factors (TFs) are important sequence‐specific DNA binding proteins that regulate gene expression and control cellular state. Evaluation of the DNA‐binding properties of transcription factors is fundamental for understanding their possible roles in controlling gene regulatory networks. TBX5 is a transcription factor that has been identified as an important regulator in organ development. TBX5 mutations have been associated to Holt‐Oram Syndrome, a genetic disorder that affects upper limb formation and often the heart. We aim to determine the specificity of DNA‐binding of TBX5 and its T‐Box DNA‐binding domain and how it changes when the protein mutates. We have cloned TBX5 and its T‐Box domain that contains a His‐ and Strep‐Tag affinity tags that we plan to use for purifying after we overexpress it in E. coli cells. We will then analyze the DNA‐binding specificity of TBX5 and the changes it conveys when it mutates using Cognate Site Identification by High‐Throughput Systematic Evolution of Ligands by Exponential enrichment (HT‐SELEX), as this will allow us to better understand the impact of TBX5 mutations on its function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A de novo TBX3 mutation presenting as dorsalization of the little fingers: A forme fruste phenotype of ulnar-mammary syndrome

Ulnar-mammary syndrome (UMS) is a rare syndromic limb malformation caused by heterozygous mutations in TBX3. The name highlights the two commonly involved body parts i.e. mammary gland and ulnar ray of the upper limbs, although a more extensive systemic involvement is also known to occur. Here, we report the surprising finding of a patient with a de novo mutation in TBX3 whose clinical presentation is limited to dorsalization of both little fingers and slightly deep 4th web spaces. We review the literature to confirm that this should be considered as a forme fruste phenotype of UMS.

A transcriptomics analysis of the Tbx5 paralogues in zebrafish.

TBX5 is essential for limb and heart development. Mutations in TBX5 are associated with Holt-Oram syndrome in humans. Due to the teleost specific genome duplication, zebrafish have two copies of TBX5: tbx5a and tbx5b. Both of these genes are expressed in regions of the lateral plate mesoderm and retina. In this study, we perform comparative RNA sequencing analysis on zebrafish embryos during the stages of lateral plate mesoderm migration. This work shows that knockdown of the Tbx5 paralogues results in altered gene expression in many tissues outside of the lateral plate mesoderm, especially in the somitic mesoderm and the intermediate mesoderm. Specifically, knockdown of tbx5b results in changes in somite size, in the differentiation of vasculature progenitors and in later patterning of trunk blood vessels.

Engineering Regenerative Thymic Tissues to Restore Long-Term T Cell Lymphopoiesis

The thymus is a primary lymphoid organ that plays a critical role in the development of adaptive T cell immunity and central tolerance. Bone marrow-derived lymphoid progenitor cells migrate into the thymus and interact with thymic epithelial cells (TECs) through sequential positive and negative selection to mature. Thymus-educated mature T cells express a diverse, MHC-restricted and self-tolerant T cell receptor (TCR) repertoire that protects against infection and prevents autoimmunity. Patients born with congenital thymic aplasia, due to 22q11 Deletion Syndrome, or mutations in TBX1, FOXN1 or CHD7, present with complete absence of T cells and a severe combined immunodeficiency (SCID)-like phenotype. Bone marrow transplantation does not cure the thymic defect in these patients and severe infections occur within the first year of life if left untreated. Allogenic thymus transplantation has provided proof of principle that HLA-unmatched pediatric donor thymic tissues can lead to successful immune reconstitution with the emergence of a diverse TCR V-beta repertoire. However, post-transplant organ-specific autoimmunity remains a major concern. Currently allogeneic thymus transplantation is no longer available in the US leaving a deadly therapeutic void for patients born without thymus. Patient-specific or histocompatible thymic tissues derived from pluripotent stem cells could address the critically unmet need, and also a broader range of clinical applications including immune reconstitution post hematopoietic stem cell transplantation (HSCT) and tolerance induction for solid donor organs.The thymus contains two major non-hematological components: the thymic stromal cells and the extracellular matrix (ECM). The thymic stromal layer is composed of thymic epithelial cells and mesenchymal cells. The thymic ECM forms a three-dimensional (3D) network to provide physical support and nutrition to thymic stromal cells.Methods: To address the need for histocompatible regenerative thymic tissues, we aim to differentiate fully functional thymic epithelial progenitor cells (TEPCs) from human pluripotent stem cells (hPSCs) and further generate 3D transplantable organoids using engineered matrix proteins that mimic the native thymic microenvironment.Results: We have developed a novel platform to generate hPSC-derived TEPCs by dissecting the key signaling pathways that govern human thymic ontogeny. These hPSC-derived TEPCs express the defining markers of TEPC-fate, such as FoxN1, Cytokeratin 8, Cytokeratin 5, Delta-like Canonical Notch Ligand 4 (DLL4) and MHC class II. Previous studies have shown FoxN1 to be the master regulator controlling thymic development, however, little is known about its regulatory network. Elucidating and validating the factors that initiate and maintain FoxN1 expression is the key to successfully engineer sustainable thymic tissues. We have identified a combination of morphogens that can maintain the expression of FoxN1, DLL4 and AIRE of primary TECs in culture. To gain insight into the composition of primary thymic ECM proteins and adapt their characteristics beyond the features of commercially available 3D hydrogels, we analyzed a series of human fetal thymic tissues using whole transcriptome analysis. Our current work focuses on adapting our 2D culture protocol to sustain hPSC-TEPCs in 3D matrix-based organoids. Ongoing studies test the capacity of hPSC-TECPs to promote T cell maturation and the development of a diverse TCR repertoire in an athymic xenograft mouse model (NSG-FoxN1null).Conclusions: hPSC can be differentiated in vitro into TEPC-fate and developed into thymic organoids using custom-designed protein matrices. Studies to test sustainability and functionality of the engineered thymic organoids in vivo are currently under way. DisclosuresNo relevant conflicts of interest to declare.