Heterozygous Nonsense Mutation Research Articles

Sanger Sequencing Reveals Novel Variants in GLO-1, ACE, and CBR1 Genes in Patients of Early and Severe Diabetic Nephropathy.

Background and Objectives: Diabetes is a global health issue, with approximately 50% of patients developing diabetic nephropathy (DN) and 25% experiencing early and severe forms of the disease. The genetic factors contributing to rapid disease progression in a subset of these patients are unclear. This study investigates genetic variations in the GLO-1, CBR-1, and ACE genes associated with early and severe DN. Materials and Methods: Sanger DNA sequencing of the exons of CBR1, GLO1, and ACE genes was conducted in 113 patients with early and severe DN (defined as occurring within 10 years of the diagnosis of diabetes and with eGFR < 45 mL/min/1.73 m2) and 100 controls. The impact of identified genetic variations was analyzed using computational protein models created in silico with SWISS-Model and SWISS-Dock for ligand binding interactions. Results: In GLO1, two heterozygous missense mutations, c.102G>T and c.147C>G, and one heterozygous nonsense mutation, c.148G>T, were identified in patients. The SNP rs1049346 (G>A) at location 6:38703061 (GRCh38) was clinically significant. The c.147C>G mutation (C19S) was associated with ligand binding disruption in the GLO1 protein, while the nonsense mutation resulted in a truncated, non-functional protein. In CBR1, two heterozygous variations, one missense c.358G>A, and one silent mutation c.311G>C were observed, with the former (D120N) affecting the active site. No significant changes were noted in ACE gene variants concerning protein structure or function. Conclusions: The study identifies four novel and five recurrent mutations/polymorphisms in GLO1, ACE, and CBR1 genes associated with severe DN in Pakistani patients. Notably, a nonsense mutation in GLO1 led to a truncated, non-functional protein, while missense mutations in GLO1 and CBR1 potentially disrupt enzyme function, possibly accelerating DN progression.

A novel nonsense mutation c.747C>G in the NEUROD1 gene detected within a Chinese family affected by maturity-onset diabetes of the young type 6.

Highlights Maturity-onset diabetes of the young type 6 (MODY6) is a rare form of monogenic diabetes mellitus due to NEUROD1 gene mutation on chromosome 2q32. A 21-year-old woman exhibiting weight loss, polyuria, and hyperglycemia was initially misdiagnosed with type 1 diabetes mellitus. Considering the early-onset age, a three-generation family history of diabetes, and negative autoimmune antibodies, a MODY diagnosis was suspected. Genetic analysis revealed that she inherited a novel heterozygous nonsense NEUROD1 mutation c.747C>G (p.Tyr249*) from her father. Correct MODY6 diagnosis facilitates appropriate interventions.

Nonsense CD247 mutations show dominant-negative features in T-cell receptor expression and function

BackgroundThe invariant TCRζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its three immunoreceptor tyrosine‐based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects. ObjectiveTo analyze and model the potential impact on TCR expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity. MethodsJurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with wild-type CD247 or mutations ablating one (Q142X), two (Q101X), or three (Q70X) ITAMs. ResultsThree patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2+3, respectively. Both mutations were associated with low surface CD3 expression, normal intracellular CD247 levels using a transmembrane-specific antibody but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO could not restore normal surface CD3 expression (only 60%, 22% and 10%, respectively), whereas in WT they reduced it (to 39%, 19% and 9% of normal levels), and both effects were ITAM number dependent. All six transfectants showed reduced CD69 induction (25-50%), indicating that they were unable to signal downstream properly neither isolated nor associated with wild-type CD247. ConclusionOur results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.

Application of whole exome sequencing technology in the diagnosis of hereditary eye diseases

Objective: To evaluate the application of whole exome sequencing (WES) in the diagnosis of hereditary eye diseases. Methods: A total of 24 patients who came to the Obstetrics and Gynecology Hospital of Fudan University for reproductive genetic counseling from December 2020 to December 2023 with the main complaint of congenital eye disorders were included in this study. All cases had no known infections or exposure to known teratogenic drugs, karyotype and chromosome microarray analysis (CMA) abnormalities. Genomic DNA was extracted from the peripheral blood of the probands and their family members and tested for WES. Among them, three individual WES and 21 Trio WES were performed. Potential pathogenic sites were screened and analyzed by Sanger sequencing. For RPGRIP1:c.1611+26G>A site, minigene vector was constructed and RT-qPCR was performed to detect the effect of mRNA splicing. Results: A total of 24 families were collected in this study, of which 20 yielded positive results, achieving a diagnosis rate of 83.3% (20/24). The results involved 21 genes and identified 30 distinct variants, 19 of which were new variants reported. Prenatal diagnostic analysis of family 3 revealed that the fetus carried a c.6970G>T heterozygous nonsense mutation in the PRPF8 gene. The results of RT-PCR with the minigene vector at the non-classical splice site in family 24 indicated that the transcription product of the mutant plasmid was partially retained 104 bp in intron 12, resulting in a p.Glu538Valfs*12 alteration of the protein. Conclusions: The high detection rate of WES in the diagnosis of hereditary eye diseases further supports the advantages of its application as an important molecular detection tool for determining the etiology of hereditary eye diseases.

Characteristic craniofacial defects associated with a novel USP9X truncation mutation

Germline loss-of-function mutations in USP9X have been reported to cause a wide spectrum of congenital anomalies. Here, we report a Japanese girl with a novel heterozygous nonsense mutation in USP9X who exhibited intellectual disability with characteristic craniofacial abnormalities, including hypotelorism, brachycephaly, hypodontia, micrognathia, severe dental crowding, and an isolated submucous cleft palate. Our findings provide further evidence that disruptions in USP9X contribute to a broad range of congenital craniofacial abnormalities.

Generation of human induced pluripotent stem cell lines derived from four Rett syndrome patients with MECP2 mutations

Rett syndrome is characterized by severe global developmental impairments with autistic features and loss of purposeful hand skills. Here we show that human induced pluripotent stem cell (hiPSC) lines derived from four Japanese female patients with Rett syndrome are generated from peripheral blood mononuclear cells using Sendai virus vectors. The generated hiPSC lines showed self-renewal and pluripotency and carried heterozygous frameshift, missense, or nonsense mutations in the MECP2 gene. Since the molecular pathogenesis caused by MECP2 dysfunction remains unclear, these cell resources are useful tools to establish disease models and develop new therapies for Rett syndrome.

CRISPR/Cas9-mediated editing of ACTB in induced pluripotent stem cells: A model for investigating human ACTB loss-of-function and genetic adaptive responses

Heterozygous beta-actin (ACTB) indel and nonsense mutations are linked to developmental disorders. We generated two CRISPR/Cas9 human induced pluripotent stem cell (iPSC) lines, WTSIi018-B-19 and WTSIi018-B-20, carrying heterozygous and homozygous indel mutations in ACTB exon 4. Both iPSCs exhibited normal cell morphology, expression of pluripotency markers, and the ability to differentiate into the three primary germ layers. While iPSCs with a heterozygous ACTB mutation maintain genome integrity, homozygous mutants showed a loss of heterozygosity in chromosome three. These mutants provide a powerful model to study the onset, progression, and complex interplay of genetic compensation and phenotypic variation of ACTB-related diseases.

Molecular mechanism analysis of a family with hereditary coagulation F Ⅺ deficiency caused by compound heterozygous mutations

A 34 year old female patient was scheduled to undergo surgical resection due to a "breast nodule". Preoperative examination revealed an activated partial thromboplastin time (APTT) of 66.2 seconds, coagulation factor Ⅺ activity (FⅪ: C) of 2%, and FⅪ antigen (FⅪ: Ag) of 40.3%. The patient and family members showed no abnormal bleeding symptoms. Diagnosed as hereditary coagulation factor Ⅺ deficiency. Genetic testing revealed that the F11 gene had a heterozygous nonsense mutation in exon 10, c.1107C>A (p.Tyr351stop), and a heterozygous missense mutation in exon 13, c.1562A>G (p.Tyr503Cys). The father and son were p Heterozygous carriers of Tyr351stop mutation, while the mother and daughter are p Heterozygous carriers of Tyr503Cys mutations. The in vitro expression results showed that p The Tyr351stop mutation resulted in a significant decrease in the transcription level of F11 gene, while p The Tyr503Cys mutation has no effect on the transcription level and protein expression level of F11 gene, but it leads to a significant decrease in the level of FⅪ:C in the cell culture supernatant.

Autosomal Dominant Retinitis Pigmentosa Secondary to TOPORS Mutations: A Report of a Novel Mutation and Clinical Findings.

Purpose: Mutations in Topoisomerase I-binding RS protein (TOPORS) have been previously documented and have been described to result in pathological autosomal dominant retinitis pigmentosa (adRP). In our study, we describe the various genotypes and clinical/phenotypic manifestations of TOPORS-related mutations of our unique patient population in Rural Appalachia. Methods: The medical records of 416 patients with inherited retinal disease at the West Virginia University Eye Institute who had undergone genetic testing between the years of 2015-2022 were reviewed. Patients found to have pathologic RP and mutations related to TOPORS were then analyzed. Results: In total, 7 patients (ages 12-70) were identified amongst three unique families. All patients were female in our study. The average follow-up period was 7.7 years. A mother (70 yr) and daughter (51 yr) had a novel heterozygous nonsense point mutation in TOPORS c.2431C > T, p.Gln811X (Exon 3) that led to premature termination of the desired protein resulting in early onset vision loss, cataract formation, and visual field restriction. The mother developed a full-thickness macular hole which was successfully repaired. Five other patients were found to have previously described TOPORS mutations. Visual field loss was progressive with age in both cohorts. Conclusions: Seven patients at our institution were identified to have mutations in TOPORS resulting in autosomal dominant retinitis pigmentosa. Two patients were found to have novel truncating mutations in the TOPORS gene resulting in profound night blindness and visual field loss, recurrent macular edema, and in one individual, epiretinal membrane formation leading to a macular hole which was able to be successfully repaired.

Generation of the human induced pluripotent stem cell line (IBKMOLi003-A) from PBMCs of a vascular Ehlers-Danlos syndrome (vEDS) patient carrying the heterozygous nonsense mutation c.430C > T (p.Q105*) in the COL3A1 gene

Ehlers-Danlos syndrome (EDS) belongs to a spectrum of rare heritable connective tissue disorders and is characterised by hyperextensibility, joint hypermobility and tissue fragility. Peripheral blood mononuclear cells (PBMCs) from a vascular EDS (vEDS) patient, known as the rarest EDS subtype, carrying a heterozygous nonsense mutation c.430C > T (p.Q105*) in the COL3A1 gene, which is essential for type III collagen synthesis, were reprogrammed into induced pluripotent stem cells (iPSCs). The generated iPSCs exhibit high expression of pluripotency-associated markers, possess trilineage differentiation capacity and reveal a normal karyotype. This novel patient-specific cell line enables in-depth pathophysiological studies of vEDS.

A Chinese patient with Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder that has been reported in all ethnicities, with several identifiable pathogenic variants. There have been reported cases indicating that RTS may lead to low birth weight in fetuses, but specific data on the fetal period are lacking. Genetic testing for RTS II is currently carried out by identifying pathogenic variants in RECQL4. In order to determine the cause, we performed whole-genome sequencing (WGS) analysis on the patient and his parents. Variants detected by WGS were confirmed by Sanger sequencing and examined in family members. After analyzing the WGS data, we found a heterozygous nonsense mutation c.2752G>T (p.Glu918Ter) and a novel frameshift insertion mutation c.1547dupC (p.Leu517AlafsTer23) of RECQL4, which is a known pathogenic/disease-causing variant of RTS. Further validation indicated these were compound heterozygous mutations from parents. Our study expands the mutational spectrum of the RECQL4 gene and enriches the phenotype spectrum of Chinese RTS patients. Our information can assist the patient's parents in making informed decisions regarding their future pregnancies. This case offers a new perspective for clinicians to consider whether to perform prenatal diagnosis.

Phenotype and genotype analyses of two pedigrees with inherited fibrinogen deficiency

Objective: To analyze the phenotype and genotype of two pedigrees with inherited fibrinogen (Fg) deficiency caused by two heterozygous mutations. We also preliminarily probed the molecular pathogenesis. Methods: The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and plasma fibrinogen activity (Fg∶C) of all family members (nine people across three generations and three people across two generations) were measured by the clotting method. Fibrinogen antigen (Fg:Ag) was measured by immunoturbidimetry. Direct DNA sequencing was performed to analyze all exons, flanking sequences, and mutated sites of FGA, FGB, and FGG for all members. Thrombin-catalyzed fibrinogen polymerization was performed. ClustalX 2.1 software was used to analyze the conservatism of the mutated sites. MutationTaster, PolyPhen-2, PROVEAN, SIFT, and LRT online bioinformatics software were applied to predict pathogenicity. Swiss PDB Viewer 4.0.1 was used to analyze the changes in protein spatial structure and molecular forces before and after mutation. Results: The Fg∶C of two probands decreased (1.28 g/L and 0.98 g/L, respectively). The Fg∶Ag of proband 1 was in the normal range of 2.20 g/L, while it was decreased to 1.01 g/L in proband 2. Through genetic analysis, we identified a heterozygous missense mutation (c.293C>A; p.BβAla98Asp) in exon 2 of proband 1 and a heterozygous nonsense mutation (c.1418C>G; p.BβSer473*) in exon 8 of proband 2. The conservatism analysis revealed that Ala98 and Ser473 presented different conservative states among homologous species. Online bioinformatics software predicted that p.BβAla98Asp and p.BβSer473* were pathogenic. Protein models demonstrated that the p.BβAla98Asp mutation influenced hydrogen bonds between amino acids, and the p.BβSer473* mutation resulted in protein truncation. Conclusion: The dysfibrinogenemia of proband 1 and the hypofibrinogenemia of proband 2 appeared to be related to the p.BβAla98Asp heterozygous missense mutation and the p.BβSer473* heterozygous nonsense mutation, respectively. This is the first ever report of these mutations.

FRI018 A Novel Truncating Variant Of EBF2 Disrupts Human Adipocyte Differentiation In Lipodystrophy Syndromes: An Example Of A Discovery From A Clinical Translational Pipeline

Abstract Disclosure: M.C. Foss de Freitas: Advisory Board Member; Self; PTC Therapeutics. N. Wys: None. M. Udler: None. L. Pais: None. A. Monteiro da Rocha: None. O.A. MacDougald: None. E.A. Oral: Consulting Fee; Self; Regeneron Pharmaceuticals, Aegerion Pharmaceuticals, Ionis Pharmaceuticals Inc., Third Rock Ventures, Rejuvenate Inc. Grant Recipient; Self; Regeneron Pharmaceuticals, Aegerion Pharmaceuticals, Ionis Pharmaceuticals Inc., Novo Nordisk, Fractyl Laboratories, Rhythm pharmaceuticals, GID Dynamics. Other; Self; Aegerion Pharmaceuticals. T. Chun: None. Objective/Goals: Partial Lipodystrophy is characterized by the gradual loss of adipose tissues, which progresses after birth. The genetic and molecular underpinning of many cases of partial lipodystrophy have not been fully understood. Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy, we initiated deep sequencing for our patients with partial lipodystrophy syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel truncating EBF2 variant. Methods: We performed loss-of-function and gene rescue experiments using 3T3-L1 and human primary subcutaneous preadipocytes. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen). After lentivirus transfection (Full-length EBF2 or truncated variant EBF2), cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone, insulin and isobutyl methylxanthine. Total RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was synthesized using IScript (Bio-Rad). Real-time qPCR was performed using TaqMan probes for Pparg and Fabp4, two key adipogenesis markers. Results: The patient was found to carry a heterozygous nonsense mutation in exon 6 of 16 (NM_022659.4) of EBF2, causing the premature termination of the protein at amino acid position 165 (EBF2 8:26033143 C&amp;gt;A (hg38), c.493G&amp;gt;T, p.Glu165Ter). Adipogenesis was significantly suppressed in 3T3-L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed a marked reduction of intracellular lipid content and Pparg and Fabp4 expression (&amp;gt;80% reduction). With lentiviral gene transfer, EBF2 fully rescued adipogenic potential, whereas the truncated variant EBF2 p.Glu165Ter did not. Of note, 3T3-L1 cells transfected with the EBF2 variant impaired adipogenesis, suggesting an inhibitory effect of the EBF2 variant on adipogenesis. We next confirmed the inhibition in adipogenesis with the EBF2 variant in primary human preadipocytes from subcutaneous adipose tissues. Using lentiviral gene transfer in human preadipocytes, we observed that the full-length EBF2 increased the gene expression of PPARg and FABP4 while the truncated EBF2 variant did not. Conclusion: Our data suggest that EBF2 is indispensable for adipogenesis of mouse and human preadipocytes. The truncating variant of EBF2 p.Glu165Ter likely plays a pathogenic role in the partial lipodystrophy presentation of this patient. Whole genome (or exome) sequencing undertaken in patients with partial lipodystrophy and subsequent biological functional analysis may lead to identification of novel mechanisms driving novel discoveries. Presentation: Friday, June 16, 2023

THU236 Mild Asymptomatic Hypercalcemia Secondary To A Novel Calcium-sensing Receptor Mutation

Abstract Disclosure: J. Tarkoff: None. Background: Familial hypocalciuric hypercalcemia (FHH) is a rare cause of mild hypercalcemia that is usually secondary to heterozygous mutations of the calcium-sensing receptor (CaSR) with autosomal dominant inheritance. The distinction between this entity and primary hyperparathyroidism can be difficult. Clinical Case: A 15-year-old female presented to the endocrinology clinic for an incidentally found elevated total calcium of 10.9 mg/dl (8.9-10.4) and elevated ionized calcium 5.5 mg/dl (4.8-5.3 mg/dl). Her labs at that time also showed a PTH of 29 pg/mL (12-71) and phosphorus 3.5 mg/dl (2.5-4.5 mg/dl). She denied any symptoms of hypercalcemia and there is no reported family history of mineral disorders or fractures. She does not take any medications. Her physical exam was unremarkable. Our evaluation showed calcium 10.5 mg/dl, phosphorus 4 mg/dl, magnesium 2.2 mg/dl (1.5-2.5 mg/dl) 25-hydroxyvitamin D 32 ng/ml (30-100 ng/ml), fractional excretion of calcium (FeCa) based upon spot urine sample = 0.0104, TSH 1.29 mIU/L (0.5-4.3 mIU/L), alkaline phosphatase 79 U/L (45-150 U/L), and normal renal function. Given her spot FeCa being &amp;lt;0.02, sequencing of the CASR gene was offered to the family because of potential overlap of her FeCa with primary hyperparathyroidism for which the treatment would be surgical (1). Commercial genetic testing was notable for a previously unreported heterozygous nonsense mutation, c.3001C&amp;gt;T (p.Gln1001X). It is expected to disrupt the final 78 amino acids of the protein in its intracellular domain and may play a role in preventing downstream signaling. At follow-up without treatment, her calcium has ranged between 10-10.9 mg/dl and she remains asymptomatic. Clinical lesson/conclusion: Hypocalciuria in the setting of hypercalcemia should raise the suspicion of FHH even without family history. Genetic testing of the CaSR is a crucial step in facilitating the diagnosis of FHH, especially given the importance of distinguishing it from primary hyperparathyroidism. Reference: (1) Christensen, S.E., Nissen, P.H., Vestergaard, P., Heickendorff, L., Brixen, K. and Mosekilde, L. (2008), Discriminative power of three indices of renal calcium excretion for the distinction between familial hypocalciuric hypercalcemia and primary hyperparathyroidism: a follow-up study on methods. Clinical Endocrinology, 69: 713-720. Presentation: Thursday, June 15, 2023

Tooth ultrastructure changes induced by a nonsense mutation in the FAM83H gene: insights into the diversity of amelogenesis imperfecta.

The current research on single-nucleotide polymorphism (SNP) mutation sites at different positions of the FAM83H gene and their phenotypic changes leading to amelogenesis imperfecta (AI) is inconsistent. We identified a previously reported heterozygous nonsense mutation c.1192C>T (p.Q398*) in the FAM83H gene and conducted a comprehensive analysis of the dental ultrastructure and chemical composition changes induced by this mutation. Additionally, we predicted the protein feature affected by this mutation site. The aim was to further deepen our understanding of the diversity of AI caused by different mutation sites in the FAM83H gene. Whole-exome sequencing (WES) and Sanger sequencing were used to confirm the mutation sites. Physical features of the patient's teeth were investigated using various methods including cone beam computer tomography (CBCT), scanning electron microscopy (SEM), contact profilometry (roughness measurement), and a nanomechanical tester (nanoindentation measurement). The protein features of wild-type and mutant FAM83H were predicted using bioinformatics methods. One previously discovered FAM83H heterozygous nonsense mutation c.1192C>T (p.Q398*) was detected in the patient. SEM revealed inconsistent dentinal tubules, and EDS showed that calcium and phosphorus were lower in the patient's dentin but higher in the enamel compared to the control tooth. Roughness measurements showed that AI patients' teeth had rougher occlusal surfaces than those of the control tooth. Nanoindentation measurements showed that the enamel and dentin hardness values of the AI patients' teeth were both significantly reduced compared to those of the control tooth. Compared to the wild-type FAM83H protein, the mutant FAM83H protein shows alterations in stability, hydrophobicity, secondary structure, and tertiary structure. These changes could underlie functional differences and AI phenotype variations caused by this mutation site. This study expands the understanding of the effects of FAM83H mutations on tooth structure. Our study enhances our understanding of the genetic basis of AI and may contribute to improved diagnostics and personalized treatment strategies for patients with FAM83H-related AI.

PLPPR4 haploinsufficiency causes neurodevelopmental disorders by disrupting synaptic plasticity via mTOR signalling.

Phospholipid phosphatase related 4 (PLPPR4), a neuron-specific membrane protein located at the postsynaptic density of glutamatergic synapses, is a putative regulator of neuronal plasticity. However, PLPPR4 dysfunction has not been linked to genetic disorders. In this study, we report three unrelated patients with intellectual disability (ID) or autism spectrum disorder (ASD) who harbour a de novo heterozygous copy number loss of PLPPR4 in 1p21.2p21.3, a heterozygous nonsense mutation in PLPPR4 (NM_014839, c.4C > T, p.Gln2*) and a homozygous splice mutation in PLPPR4 (NM_014839: c.408 + 2 T > C), respectively. Bionano single-molecule optical mapping confirmed PLPPR4 deletion contains no additional pathogenic genes. Our results suggested that the loss of function of PLPPR4 is associated with neurodevelopmental disorders. To test the pathogenesis of PLPPR4, peripheral blood mononuclear cells obtained from the patient with heterozygous deletion of PLPPR4 were induced to specific iPSCs (CHWi001-A) and then differentiated into neurons. The neurons carrying the deletion of PLPPR4 displayed the reduced density of dendritic protrusions, shorter neurites and reduced axon length, suggesting the causal role of PLPPR4 in neurodevelopmental disorders. As the mTOR signalling pathway was essential for regulating the axon maturation and function, we found that mTOR signalling was inhibited with a higher level of p-AKT, p-mTOR and p-ERK1/2, decreased p-PI3K in PLPPR4-iPSCs neurons. Additionally, we found silencing PLPPR4 disturbed the mTOR signalling pathway. Our results suggested PLPPR4 modulates neurodevelopment by affecting the plasticity of neurons via the mTOR signalling pathway.

Infant Alstrom syndrome diagnosed by a new gene mutation: a case report.

Alstrom syndrome is a rare autosomal recessive disorder resulting from an ALMS1 gene mutation. Here, we present the clinical data of a case of an infant diagnosed with Alstrom syndrome through whole-exome sequencing. A 2-month-old male infant was admitted to Sichuan Provincial Maternity and Child Health Care Hospital on 30 May 2019 after "coughing for half a day and dyspnea for 2 hours". He was diagnosed with severe pneumonia, acute congestive heart failure, Grade III cardiac function, acute respiratory failure, and myocarditis. After treatment, he was discharged with a prescription for oral medication. After a 4-month follow-up, the patient's left ventricle exhibited spherical enlargement and a decrease in left ventricular function. The infant's whole-exome sequencing results revealed compound heterozygous mutations in the ALMS1 gene: c.2179dup (p. Y727Lfs*12), a frameshift mutation, that was heterozygous and originated from the mother, while c.11140C>T (p. Q3714*) was a heterozygous nonsense mutation that originated from the father. Both mutations are classified as "category 1-pathogenic mutations" according to the American College of Medical Genetics and Genomics (ACMG) assessment. A novel ALMS1 mutation was identified in this case report, highlighting the importance of genetic testing for the early diagnosis of Alstrom syndrome.

Establishment of human induced pluripotent stem cell lines, KMUGMCi006, from a patient with Tuberous sclerosis complex (TSC) bearing mosaic nonsense mutations in the Tuberous sclerosis complex 2 (TSC2) gene

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by neuropsychiatric symptoms and multiple dysplastic organ lesions, caused by loss of function mutations in either TSC1 or TSC2. The peripheral blood mononuclear cells (PBMCs) from a patient carrying mosaic nonsense mutation of TSC2 gene were reprogrammed using the CytoTune-iPS2.0 Sendai Reprogramming Kit. The human induced pluripotent cell (hiPSC) lines with the mutation and without the mutation were established. The heterozygous nonsense mutation in TSC2 will cause the truncated protein, which is known to associated with TSC. The established hiPSC lines will enable proper in vitro disease modelling of TSC.

Abstract #1377932: An Inherited Metabolic Disorder That Masquerades as Osteoporosis

Hypophosphatasia is a rare genetic disorder caused by loss-of-function mutations in the ALPL gene, which encodes tissue non-specific alkaline phosphatase. The enzyme defect results in the accumulation of substrates like inorganic pyrophosphate, thereby inhibiting mineralization of bones and teeth. Clinical features include premature loss of deciduous teeth, bowed legs, lower extremity stress fractures, bone pain and impaired mobility. Serum alkaline phosphatase (ALP) is a marker of bone turnover. Low ALP, high serum vitamin B6 and high urine phosphoethanolamine are diagnostic hallmarks of hypophosphatasia. A 54-year-old lady was referred to the endocrinology clinic for severe osteoporosis, characterized by an olecranon fracture and low bone mineral density (T score -3.0 at the left femoral neck). Bone disease risk factors included premature menopause at age 38 years and maternal history of hip fracture. There was no personal or family history of recurrent fractures, impaired mobility, or unexplained tooth loss. Despite treatment with ibandronate, zoledronic acid and teriparatide, her bone mineral density declined over the course of a decade. Physical examination revealed low BMI (18.07 kg/m2), normal gait and intact muscle strength. Lab testing disclosed low serum alkaline phosphatase (ALP) 18 units/L [reference 45 to 117 units/L], high serum vitamin B6 131.8 nmol/L [reference 20.0 to 125.0 nmol/L] and high urine phosphoethanolamine 104 nmol/mg Cr [reference < 48]. ALP results prior to bisphosphonate exposure were unavailable. Vitamin D status, thyroid function, parathyroid hormone, corrected serum calcium, ceruloplasmin and zinc levels were normal. Exome sequencing identified a pathogenic heterozygous nonsense ALPL gene mutation, c.1282C >T (p.Arg428X). She was counselled to avoid bisphosphonate therapy indefinitely. ALP measurement was advised for her blood relatives since hypophosphatasia is an autosomal recessive or dominant disorder. With asfotase alfa, the only FDA-approved therapy for juvenile-onset hypophosphatasia, there have been no further fractures to date, and her mobility remains unimpaired. Hypophosphatasia can be mistaken for osteoporosis since both conditions present with low bone mineral density on DEXA scan. Furthermore, bisphosphonate therapy and hypophosphatasia are mutually exclusive entities that can present with low ALP. Thus, bisphosphonate exposure may pose a diagnostic quandary when distinguishing between osteoporosis and hypophosphatasia. This case highlights the importance of assessing ALP when evaluating osteoporosis with atypical features such as age of onset under fifty years, remarkably low bone mineral density, fracture(s), and inadequate response to anti-osteoporosis agents. Moreover, if ALP is low, some index of suspicion for hypophosphatasia ought to be maintained, even if dental or lower extremity bone symptoms are absent. Genetic testing is confirmatory. Bisphosphonate therapy and parathyroid hormone analogs are not established treatment modalities for hypophosphatasia. Instead, enzyme replacement with asfotase alfa is recommended.

386 A novel truncating variant of EBF2 disrupts human adipocyte differentiation in lipodystrophy syndromes: an example of a discovery from a clinical translational pipeline

OBJECTIVES/GOALS: Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy (PL), we initiated whole-exome sequencing for our patients with PL syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel truncating EBF2 variant. METHODS/STUDY POPULATION: Using 3T3-L1 and human primary subcutaneous preadipocytes, we performed loss-of-function and gene rescue experiments. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen) at 5% CO2. After lentivirus transfection, cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone (0.25µM), insulin (1µg/ml) and isobutyl methylxanthine (0.5 mM). Total RNA was extracted using RNeasy Mini Kit (Qiagen) and cDNA was synthesized using IScript (Bio-Rad). Real-time qPCR was performed using TaqMan probes for Pparg and Fabp4, two key adipogenesis markers. RESULTS/ANTICIPATED RESULTS: Patient was found to carry a heterozygous nonsense mutation in exon 6 of EBF2, causing the premature termination of the protein at amino acid position 165. Adipogenesis was significantly suppressed in 3T3L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed marked reduction of intracellular lipid content and Pparg and Fabp4 expression (&gt;80% reduction). With lentiviral gene transfer, EBF2 fully rescued adipogenic potential, whereas the truncated variant EBF2 did not. Of note, 3T3-L1 cells transfected with the EBF2 variant displayed impaired adipogenesis, suggesting a dominant-negative effect of the EBF2 variant on adipogenesis. We confirmed the dominant effect of the EBF2 variant in human adipocyte differentiation. DISCUSSION/SIGNIFICANCE: Our data suggest that EBF2 is indispensable for adipogenesis. The loss of function and dominant-negative effect of the truncating variant of EBF2 likely plays a pathogenic role in PL. Whole exome sequencing of PL patients and ex-vivo functional analysis help identify novel gene variants and better understand the molecular pathogenesis of PL.