Heterozygous Variant Of Unknown Significance Research Articles

Congenital hallux valgus occurs in Fibrodysplasia Ossificans Progressiva and BMPR1B-associated dysplasia: an important distinction

BackgroundFibrodysplasia Ossificans Progressiva (FOP; OMIM #135100) is an ultrarare genetic disorder characterised by congenital bilateral hallux valgus (CBHV), intermittent soft tissue swellings and progressive heterotopic ossification. We report a three-month-old girl with great toe abnormalities similar to FOP, in whom comprehensive clinical workup and genetic investigations illustrates an alternative diagnosis.Case presentationA three-month-old girl presented with CBHV. The antenatal period was unremarkable, she was born by spontaneous vaginal delivery with an uneventful subsequent course, except for maternal concern of her bent toes which received reassurance from several health professionals. Her mother’s persisting concerns were explored via the internet and social media leading her to request referral to an expert bone centre for consideration of FOP. On examination, she was thriving, there was no dysmorphism, subcutaneous lumps, skeletal or extra-skeletal deformity except for shortened great toes with lateral deviation of the proximal and distal phalanges. FOP was a feasible diagnosis, for which CBHV is highlighted as an early sign. A cautionary potential diagnosis of FOP was counselled, including advice to defer intramuscular immunisations until genetic results available. Genetic investigation was undertaken through rapid whole genomic sequencing (WGS), with analysis of data from a skeletal dysplasia gene panel, which demonstrated no ACVR1variants. The only finding was a heterozygous variant of unknown significance in BMPR1B (c1460T>A, p.(Val487Asp)), which encodes a bone morphogenic receptor involved in brachydactyly syndromes A1, A2 and D and acromesomelic dysplasia 3 (only the latter being an autosomal recessive condition).ConclusionThis report highlights that CBHV serves as a vital diagnostic indicator of FOP and affected infants should be considered and investigated for FOP, including precautionary management whilst awaiting genetic studies. The second educational aspect is that CBHV may not represent a generalised skeletal disorder, or one much less significant than FOP. Receptor-ligand BMP and Activins mediated interactions are instrumental in the intricate embryology of the great toe. Recognition of non-FOP conditions caused by alterations in different genes are likely to increase with new genomic technology and large gene panels, enhancing understanding of bone signaling pathways.

Unusual Case of Young Age Onset Stroke Suspected to Be Associated with Pseudoxanthoma Elasticum Gene Mutation in ABCC6: A Case Report

Ischemic stroke in young adults can pose diagnostic challenges, particularly when the clinical features deviate from the common presentation. Here, we present the case of a 31-year-old female with transient left-sided hemiparesis. Brain magnetic resonance imaging and angiography revealed right frontoparietal lobe infarction and stenosis of the right internal carotid arteries. Genetic testing revealed a heterozygous variant of unknown significance in the ATP-binding cassette subfamily C member 6 gene NM_001171.6:c.3739C>A (p.Pro1247Thr) that is associated with pseudoxanthoma elasticum, which is a rare disorder characterized by abnormal mineralization of elastic tissues. Although variants of unknown significance can only be classified as causative conditions if all other possible causes are ruled out, this report suggests the importance of considering unusual genetic conditions in young patients with ischemic stroke. Further genetic research, functional studies, and genetic counseling are vital for a comprehensive understanding of these cases.

Two Hit Theory for the Pathogenesis of Type 3 Congenital Amegakaryocytic Thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is an autosomal recessive disorder characterized by thrombocytopenia with an absence of megakaryocytes. It can lead to aplastic anemia or myelodysplastic syndrome (MDS). CAMT is linked to a mutation in the c-MPL gene on chromosome 1, which encodes the thrombopoietin receptor. In 2005, CAMT was categorized into three types based on the severity of c-MPL gene loss. Type 1 is the most severe, leading to early-onset pancytopenia and bone marrow abnormalities. Type 2 shows a temporary increase in platelets during infancy, followed by bone marrow failure at 3-6 years due to partial gene function. Type 3 has normal c-MPL function but produces ineffective megakaryocytes. We report a case of a patient with CAMT-like symptoms, without a c-MPL gene mutation, suggesting the presence of an alternative genetic defect. Methods Multi-institutional collaboration and review of medical records in addition to PubMed search for thrombocytopenia, bleeding complications, macrocytosis, trisomy 6, GATA2 variant, inherited bone marrow failure. Case Description A 12-year-old male with severe thrombocytopenia initially observed at 2 years of age. Bleeding complications included scalp hematoma, excessive bleeding during adenoidectomy, widespread petechiae, purpura, epistaxis, and gum bleeding. During this time platelet count ranged between 30-80,000 x10 3/uL. At 10 years of age, bleeding worsened with platelet counts of 6-20,000 x10 3/uL. Mean platelet volume (MPV) remained within the normal range. Simultaneously there was a decline in hemoglobin and the emergence of macrocytosis. Mother had moderate thrombocytopenia during pregnancy, older sibling with similar thrombocytopenia but asymptomatic. Clinical presentation made immune thrombocytopenia, autoimmune disorders, and immune dysregulation-induced platelet destruction unlikely. Inherited thrombocytopenia panel testing for 42 germline variants was negative. Bone marrow biopsy showed 60% cellularity and was remarkable for a decrease in megakaryocytes. Cytogenetic analysis revealed trisomy 6 in 7/20 metaphases. Molecular hematopathology identified a GATA2 variant in 50% of the alleles. Inherited BMF panel revealed heterozygosity for multiple variants of unknown significance (VUS), including c.5096A>G (p.Asp1699Gly) in BRCA2 gene, c.3898A>G (p.Ile300Val) in LYST gene, 1408A>G (p.Thr470Ala) in PALB2 gene, and c.7240G>A (p.Val2414Met) in tVPS13B gene. Another BMF panel revealed two additional heterozygous VUS - NBEAL2 and ADAMTS13. Testing for Fanconi anemia and dyskeratosis congenita were negative. Whole exome sequencing revealed heterozygosity for a 1.57 Mb duplication on chromosome 3q29. Additional molecular pathology panel testing for 468 genes, germline genetic testing for lysosomal disorders and mucopolysaccharidoses, were also negative. Repeat bone marrow testing demonstrated a progressive decrease in cellularity to 5-30% with trilineage hypoplasia. With extensive yet inconclusive diagnostic workup, worsening clinical presentation, and progressive hypocellularity of the bone marrow, he underwent an ABO incompatible stem cell transplantation (9:10, A HR) from a mismatched unrelated donor. The conditioning regimen was myeloablative with busulfan, cyclophosphamide, anti-thymocyte globulin, and post-transplant cyclophosphamide. Currently, he is 150 days post-transplant with peripheral blood chimerism demonstrating 97.7% donor on day 98. Platelet counts have normalized, and he has not required a transfusion for nearly 100 days. Discussion This case report highlights the concept of a “double hit” hypothesis. While homozygous mutations of PALB2 and BRCA2 are individually associated with Fanconi anemia and BMF syndromes, no hematological pathology has been documented in their heterozygous forms, raising the question of whether the co-expression of these variants in a heterozygous state could result in a similar disease phenotype as their homozygous counterparts. This finding prompts further exploration into the potential impact of combined variants on disease manifestation.

Clinical Consequences of Rare Germline Variation in Genes Associated with Coagulation across 451,958 Individuals

Introduction Despite deep biological insights over the preceding decades, our ability to assess for inherited hypercoagulable states remains limited. In light of these challenges and the increasing relevance of precision medicine and next-generation sequencing (NGS) to clinical practice, the identification of rare germline variants in coagulation-focused NGS panels is increasingly becoming accepted practice. However, the discovery of variants of unknown significance (VUS) remains a common clinical conundrum in molecular testing, particularly because evidence for the thrombosis risk associated with many genes derives from small, uncontrolled studies, case reports, and pedigree analysis. Large-scale evaluation of the clinical consequences associated with loss of function in individual coagulation-related genes would contribute to the clinical assessment of patients presenting with heterozygous VUS in poorly characterized genes. Objectives Determine the risk of four thrombosis phenotypes associated with rare, germline loss-of-function variants in a major clinical coagulation-focused NGS panel. Methods The UK Biobank (UKBB) contains paired clinical and whole exome sequencing data for over 450,000 participants. We identified rare (minor allele frequency <0.1%) germline variants that were predicted in silico to alter protein activity (functional impact score ≥0.8) within the 54 evaluable genes of the Yale NGS coagulation panel. For each gene, parallel multivariable Firth's logistic regression models were generated to assess the association between rare qualifying variants and four thrombotic phenotypes as well as coagulopathic bleeding in a gene-level collapsing analysis (N=270 independent models). We adjusted for age, sex, ancestry, and sequencing batch. Phenotypes of interest included venous thromboembolism (VTE), myocardial infarction (MI), stroke (CVA), peripheral artery disease (PAD), and coagulopathic bleeding. The Bonferroni corrected p-value for each phenotype was 0.001. We validated functional impact score predictions in seven secreted proteins measured in a subset of 47,923 UKBB participants who underwent plasma proteomic assessment (Olink Explore 1536 panel). Results A total of 451,958 individuals in the UKBB with whole exome sequencing data were evaluated. Among the 54 genes in the Yale NGS coagulation panel, there were 18,975 qualifying variants (98.4% heterozygous). Function-altering variants in 4 of 54 genes were associated with a statistically significant increased risk of thrombosis after adjusting for multiple comparisons: PROC (OR = 5.7, 95% CI: 3.2 - 9.6, p = 1.6x10 -7), SERPINC1 (OR = 5.0, 95% CI: 2.7 - 8.7, p = 4.0x10 -6), PROS1 (OR = 3.2, 95% CI: 2.0 - 4.8, p = 6.0x10 -6), and STAB2 (OR = 1.4, 95% CI: 1.2 - 1.7, p = 5.3x10 -4) (Figure 1). Nominal significance was achieved for 7 genes in the VTE phenotype, 1 gene in CVA, 3 genes in MI, 5 genes in PAD, and 0 genes in coagulopathic bleeding. Variants in a total of 37 genes did not appear to be significantly associated with any of the evaluated phenotypes. To assess the reliability of variant functional impact scores derived in silico, plasma levels for 13 of the 53 coagulation genes were analyzed. Of these, 8 were identified as secreted proteins amenable to assessment by plasma levels, including ADAMTS13 ( ADAMTS13), factor IX ( F9), factor VII ( F7), tissue plasminogen activator ( PLAT), urokinase ( PLAU), protein C ( PROC), plasminogen activator inhibitor 1 ( SERPINE1), and von Willebrand factor ( VWF). For these proteins, average plasma levels consistently demonstrated a negative correlation with functional impact score (Pearson r = -0.98, P=0.0009), indicating that our in silico assessments in this context are broadly reliable (Figure 2). Conclusion Within the commonly encountered clinical context of heterozygous loss of function, most genes contained in coagulation-focused NGS panels likely have fairly weak effects on disease risk that are limited to venous thrombosis. However, our findings do not rule out the possibility of more profound effects from homozygous or compound heterozygous loss of function in these same genes. By defining the clinical consequence of heterozygous loss of function in specific coagulation-related genes across a large population, our findings may help inform the interpretation of molecular testing results in which patients are found to have a VUS.

Loss of Function MBD4 Mutation Impairs DNA Repair in Association with TET2 in Acute Myeloid Leukemia

Background: Methyl-CpG binding domain 4 (MBD4) is a known DNA glycosylase involved in critical steps of base excision repair (BER) and DNA mismatch repair (MMR) and is essential for reducing genotoxic stress in normal cells. MBD4 binds to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) - oxidation products of TET2 dioxygenases - and may initiate a multi-step BER/MMR around mCpG/hmCpG DNA. Loss of function (LOF) mutations in MBD4 (MBD4 MT) result in compromised BER/MMR, leading to genomic instability. Certain germline MBD4 variants have been reported to be predisposing factors for early onset AML, among other cancers. 1,2 In cases screened at our institution, biallelic MBD4 p.R431* & p.L563* tracked with AML in brother and sister. 3 This inspired us to hypothesize that partial or complete loss of MBD4 may result in reduced BER/MMR function and ultimately in genomic instability and faster accumulation of somatic lesions favoring malignant evolution. Methods: We performed NGS and epigenetic studies coupled with detailed biochemical analyses in CRISPR engineered cell lines and primary AML cells and in vivo model systems to dissect the mechanism of clonal evolution of MBD4 MT hematopoietic stem and progenitor cells (HSPCs). Results: We analyzed WES data from 1,692 patients with myeloid neoplasms (MN, 732 MDS; 960 AML). After adequate filtering based on frequency in healthy population (odds ratio of >1.5), 16 variants in MBD4 were identified in 24 MN cases (MDS=9, AML=15). Among them, 4 heterozygous variants of unknown significance of suspected germline nature (p.R425Q, p.N461S, p.R486K, p.D504H) mapped in the glycosylase domain. Comparing MBD4 MT vs. wild-type ( MBD4 WT) cases, MBD4 MT were found in younger patients (median 55 vs. 71 yrs; p = 0.012) with TET2 MT co-occurring at a lower frequency (8 vs. 24%). Since MBD4 is known to preferentially bind with 5hmCpG DNA, a stable epigenetic mark induced by TET2, we investigated the biochemical basis of MBD4 dysfunction and its connection to LOF TET2 MT. We show that MBD4 hydrolase activity levels affect the genomic accumulation of 5hmC in a TET2-dependent manner. MBD4 KO led to more than 2-fold increased 5hmC in TET2WT cells but not in TET2KO cells, suggesting a functional connection between MBD4 and TET2 in BER/MMR. To test the function and effects of the 4 variants (p.R425Q, p.N461S, p.R486K, p.D504H) identified in our cohort on the activity and function of MBD4, we generated these mutations in a pcDNA plasmid using site directed mutagenesis. The mutants were ectopically expressed in isogenic CRISPR-engineered MBD4 KO HEK293 cells and its effects on 5hmC accumulation were tested. The impact on the functional outcomes were compared to WT controls as described earlier. 4 We found that these 4 variants lead to MBD4 protein instability, and thereby lower MBD4 DNA glycosylase activity resulting in increased (1.6 to 3.3-fold) genomic accumulation of TET2-dependent oxidation products. To establish the effect of MBD4 MT in primary cells, we generated CRISPR-engineered human CD34 + HSPCs and assessed their clonal evolution via colony forming assay. Loss of MBD4 led to clonal advantage reflected in persistent colonies in MBD4KO CD34 + HSPCs that demonstrated myeloid differentiation bias similar to what was observed in patients, suggesting that MBD4 MT may favor clonal evolution to fully blown MN. Finally, transcriptomics analysis of MBD4KO in K562 cells in the presence and absence of functional TET2 demonstrated loss of BER genes compared to WT. The co-immunoprecipitation of MBD4 followed by western blot analysis identified XRCC1, a key component of the BER complex, as a part of MBD4 interactome. Comprehensive characterization of protein complexes with LCMS/MS in K562 cells suggests that MBD4 is involved in maintaining genomic integrity. Consistent with our hypothesis, MBD4 KO in K562 and THP1 cells resulted in a 2- and 3-fold increase in spontaneous mutations respectively, as observed via HPRT assay. Conclusions: Genomic instability due to MMR/BER dysfunction in MBD4 MT cells may result in a higher rate of clonal progression. Interestingly, MBD4 deficiency may provide opportunity for therapeutic targeting of DNA repair pathways in a subset of MBD4 MT-associated MN. Indeed, our findings indicate the possibility of leveraging MBD4 as an actionable target to regulate DNA methylation in association with TET2 during leukemogenesis.

Clinical Findings and Genetic Analysis of Nine Mexican Families with Bartter Syndrome

Bartter's syndrome (BS) is a group of salt-wasting tubulopathies characterized by hypokalemia, metabolic alkalosis, hypercalciuria, secondary hyperaldosteronism, and low or normal blood pressure. Loss-of-function variants in genes encoding for five proteins expressed in the thick ascending limb of Henle in the nephron, produced different genetic types of BS. Clinical and genetic analysis of families with Antenatal Bartter syndrome (ABS) and with Classic Bartter syndrome (CBS). Nine patients from unrelated non-consanguineous Mexican families were studied. Massive parallel sequencing of a gene panel or whole-exome sequencing was used to identify the causative gene. Proband 1 was homozygous for the pathogenic variant p.Arg302Gln in the SLC12A1 gene encoding for the sodium-potassium-chloride NKCC2 cotransporter. Proband 3 was homozygous for the nonsense variant p.Cys308* in the KCNJ1 gene encoding for the ROMK potassium channel. Probands 7, 8, and 9 showed variants in the CLCKNB gene encoding the chloride channel ClC-Kb: proband 7 was compound heterozygous for the deletion of the entire gene and the missense change p.Arg438Cys; proband 8 presented a homozygous deletion of the whole gene and proband 9 was homozygous for the nonsense mutation p.Arg595*. A heterozygous variant of unknown significance was detected in the SLC12A1 gene in proband 2, and no variants were found in SLC12A1, KCNJ1, BSND, CLCNKA, CLCNKB, and MAGED2 genes in probands 4, 5, and 6. Genetic analysis identified loss-of-function variants in the SLC12A1, KCNJ1, and CLCNKB genes in four patients with ABS and in the CLCNKB gene in two patients with CBS.

High-throughput microfluidic blood testing to phenotype genetically linked platelet disorders: an aid to diagnosis

AbstractLinking the genetic background of patients with bleeding diathesis and altered platelet function remains challenging. We aimed to assess how a multiparameter microspot-based measurement of thrombus formation under flow can help identify patients with a platelet bleeding disorder. For this purpose, we studied 16 patients presenting with bleeding and/or albinism and suspected platelet dysfunction and 15 relatives. Genotyping of patients revealed a novel biallelic pathogenic variant in RASGRP2 (splice site c.240-1G>A), abrogating CalDAG-GEFI expression, compound heterozygosity (c.537del, c.571A>T) in P2RY12, affecting P2Y12 signaling, and heterozygous variants of unknown significance in the P2RY12 and HPS3 genes. Other patients were confirmed to have Hermansky-Pudlak syndrome type 1 or 3. In 5 patients, no genetic variant was found. Platelet functions were assessed via routine laboratory measurements. Blood samples from all subjects and day controls were screened for blood cell counts and microfluidic outcomes on 6 surfaces (48 parameters) in comparison with those of a reference cohort of healthy subjects. Differential analysis of the microfluidic data showed that the key parameters of thrombus formation were compromised in the 16 index patients. Principal component analysis revealed separate clusters of patients vs heterozygous family members and control subjects. Clusters were further segregated based on inclusion of hematologic values and laboratory measurements. Subject ranking indicated an overall impairment in thrombus formation in patients carrying a (likely) pathogenic variant of the genes but not in asymptomatic relatives. Taken together, our results indicate the advantages of testing for multiparametric thrombus formation in this patient population.

#5158 AUTOSOMAL DOMINANT TUBULAR KIDNEY DISEASE: FROM PHENOTYPE TO GENOTYPE

Abstract Background and Aims Autosomal dominant tubulointerstitial kidney disease (ADTKD) represent a recently described group of kidney diseases, characterized by chronic interstitial nephritis (CIN), in most cases hereditary, with an autosomal dominant inheritance pattern, subclassified according to its genetic cause, when identified. Mutations in UMOD, MUC1, REN and HNF1B genes have been implicated in their pathogenesis. Recent molecular diagnostic technologies, such as Next-Generation Sequencing (NGS), and particularly the use of restriction and Snapshot method for the detection of a cytosine insertion on the coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1, have allowed the nephrologists to diagnose renal diseases of previously unknown cause, with a real prevalence probably underestimated. We present the cohort of patients identified with ADTKD in the Nephrogenetics’ clinic from the Nephrology department of our hospital from 2015 to 2022. Method Patients with chronic kidney disease (CKD) of unknown etiology and renal phenotype suggesting CIN (bland urine analysis, hyperuricemia and/or anemia unrelated to the degree of CKD, hypo/hypercalcemia, acidemia, urinary concentration defects, absent hypertension, renal cysts), especially with a family history of similar CKD, were referred for the Nephrogenetics’ clinic and ADTKD was investigated. In these cases, we performed a genetic study of ADTKD by the following steps: 1) UMOD, REN, HNF1B mutations studied through NGS; 2) if negative, search for the insertion of a single cytosine of the repeat unit comprising the extremely long (∼1.5-5 kb), GC-rich (>80%) coding variable-number tandem repeat (VNTR) sequence in the MUC1 gene encoding mucin 1 (see Kirby et al Nat Genet. 2013). It is used a restriction and Snapshot method. 3) if negative, search for HNF1B Large deletion, detected by MLPA (kit P241-D2, MRC-Holland). Pre-genetic counselling was performed by the nephrologist, and post-genetic counselling was performed by the nephrologist and geneticist, namely to proceed with family screening of potentially affected family members when a pathogenic mutation was identified or when segregation studies were necessary. Results 31 families were studied and a confirmed genetic diagnosis was obtained in 11 (35%), allowing the identification of 30 patients with ADTKD; the mutations identified were in MUC1 in 4 families (13 patients), UMOD in 4 families (11 patients), HNF1B (2 families, 5 patients) and REN: 1 patient with a phenotype compatible with ADTKD but with a genotype compatible with renal tubular dysgenesis (two mutations in REN gene from each of the parents). One patient with HNF1B deletion also presented with a heterozygous variant of unknown significance in PKD1 gene, and a pathogenic variant of SLC12A3, heterozygous. Three families are still under study: 2 families are being studied with segregation studies to evaluate the pathogenicity of mutations in UMOD (1 family) and HNF1B (1 family), and 1 family with a phenotype of ADTKD (3 young patients with positive family history) with negative results for mutations in ADTKD genes is in study, with a negative result in WES. The study will be pursued in a specialized laboratory to better study MUC1, but at this moment we classify this family as ADTKD-NOS. If positive results are obtained for these 3 families, the percentage of families identified using this protocol will increase to 43,3% of the families studied through this model. Conclusion In our cohort, a genetic diagnosis was established in 35% of the families studied. MUC1 and UMOD are the most prevalent genes implicated in ADTKD at our center, similarly to what has been reported in other cohorts of ADTKD. Given the rarity of these diseases and the fact that they require specific molecular techniques for accurate diagnosis, we believe that Nephrogenetics’ clinics with dedicated nephrologists and geneticists in close cooperation are critical for an accurate diagnosis.

Abnormal kinetics of contraction caused by an α-actinin 2 missense variant in the EF-3-4 hand domain in human myocardium.

ACTN2 gene encodes α-actinin 2 protein, which binds actin. In cardiac muscle, α-actinin 2 is located in the Z-disc of the sarcomere, where it anchors myofibrillar actin filaments. A 48-years old female presented with out-of-hospital ventricular fibrillation arrest. Echocardiogram showed stage II diastolic dysfunction, elevated RVSP, and abnormal global longitudinal strain consistent with early LV systolic dysfunction. Cardiopulmonary testing indicated AHA functional class IV HF. Genetic testing identified a missense heterozygous variant of unknown significance in ACTN2 (gene previously associated with HCM and DCM), and SCNB2 (β-2 subunit of type II voltage-gated sodium channel). Histopathology demonstrated mild interstitial fibrosis of the LV free wall. We investigated the effects of the heterozygous A868T variant in α-actinin 2 on cardiac muscle mechanics. LV free wall samples were obtained from the patient's explanted heart at the time of cardiac transplantation. Permeabilized cardiac muscle preparations (CMPs) containing A868T and WT proteins displayed increased myofilament Ca2+-sensitivity of isometric force and lower maximal isometric specific force when compared to human donor samples. Sinusoidal stiffness was decreased at all levels of Ca2+-activation in A8686T CMPs. The rate of tension redevelopment (kTR) was faster at all levels of Ca2+-activation in A8686T CMPs. Computational modeling suggests that faster kTR in A868T CMPs is mainly due to faster cross-bridge detachment (g). The kinetic changes are consistent with decreased force generation by A868T CMPs being due to abnormalities in the cross-bridge interaction. Molecular dynamics simulation did not find significant differences in the interaction of A868T variant with titin and α-actinin. This project establishes the importance of α-actinin 2 variant in the Z-disc mechanosensitive interaction of thin- and thick-filament distal from its structural role in the sarcomere.

The Role of Heterozygous Variants in SAMD9/9L and Recessive Bone Marrow Failure-Related Genes in Adults with Severe Aplastic Anemia

Background Genetic reports of patients with severe AA (SAA) often identify heterozygous variants of unknown significance in recessive genes associated with inherited bone marrow failure syndromes (IBMFS), such as Fanconi anemia (FA) and telomere biology diseases (TBD). More recently, heterozygous SAMD9 and SAMD9L (SAMD9/9L) variants have also been reported in children and young adults with BMF, but whether all variants are gain-of-function (GoF) and pathogenic is unknown. In a large cohort of SAA patients, we investigated the prevalence of heterozygous variants in SAMD9/9L and FA/TBD genes and correlated them to disease characteristics and clinical outcomes. Methods SAA patients (n=198; median age [range]: 32 [3-82]) enrolled on a phase II trial for treatment with horse anti-thymocyte globulin, cyclosporine, and eltrombopag (NCT01623167) at the National Institutes of Health from 2012 - 2022 were included (Table 1). All patients were screened for germline mutations in IBMFS related genes. SAMD9/9L variants were segregated by rarity according to frequency in the gnomAD database as common (<0.1% in overall population, including variants with maximum population frequency [MaxPopFreq] >1%), rare (MaxPopFreq <0.1%), very rare (MaxPopFreq <0.01%), ultra-rare (MaxPopFreq <0.005%), and novel (absent in gnomAD). Results We first investigated whether patients had an undiagnosed SAMD9/9L disorder. Although 40/198 patients (20%) had a heterozygous SAMD9/9L variant, including 8 (20%) with 2 variants, these were mostly classed as common (73%; n=35), followed by rare, very rare, ultra-rare and novel variants seen in 17% (n=8), 4% (n=2), 2% (n=1), and 4% (n=2), respectively. Most variants were missense (n=39; 81%), followed by nonsense (n=6; 13%) and frameshift mutations (n=3; 6%). Except for ultra-rare and novel, SAMD9/9L often co-occurred with typical features of immune AA (6p-LOH, PNH clones, HLA and PIGA mutations). None of the 40 patients with germline SAMD9/9L variants had primary somatic genetic reversions (SGR) such as somatic loss of function (LoF) mutations (VAF>0.5%) in SAMD9/9L or UPD7q. Since GoF SAMD9/9L variants are located on chr7 and they associate with development of monosomy 7, we investigated whether SAMD9/9L variants in our cohort were associated with clonal evolution after treatment. However, only 2/40 patients (5%) with chr7 abnormalities had these variants, both common. Patients with SAMD9/9L variants, both common and rare, were significantly younger (35 years vs. 24 years, p=0.026) and more frequently had VSAA (defined as absolute neutrophil count <0.2 K/uL) than those without these variants (62% vs. 41%, p=0.02); they had lower median neutrophil counts (p=0.038), reticulocyte counts (p=0.024), and platelet counts (p=0.047). The presence of rarer (MaxPopFreq<0.1%; n=11) or common SAMD9/9L variants (n=29) did not impact disease severity, or correlate with molecular characteristics of immune AA, response to immunosuppressive therapy, and overall survival (Figure 1). In addition to SAMD9/9L variants, 82 patients (41%) had 1 or more heterozygous variants in FA/TBD, but these were not associated with disease severity or with clinical outcomes, even when co-occurring with SAMD9/9L variants. Twenty five patients (13%) had co-occurring heterozygous mutations in both SAMD9/9L and FA/TBD genes, but these were not correlated with disease severity or response to therapy when compared to patients without co-occurring mutations. Conclusions As opposed to classical SAMD9/9L disorders in pediatric cohorts, SAMD9/9L variants found in our primarily adult patients with SAA are not disease-causing germline GoF mutations, even the rarest variants. Most patients with variants responded to immunosuppressive therapy, and the less rare variants were associated with molecular features of immune AA but not SGR, both findings suggestive of immune pathophysiology. Interpretation of genetic reports, including SAMD9/9L, in adults appears to be different from mostly pediatric cohorts with SAMD9/9L disorders. Heterozygous SAMD9/9L variants do affect AA severity but are not predictive of response to therapy or prognosis. FA/TBD heterozygous variants did not impact disease characteristics or outcomes. We did not observe a higher rate of clonal evolution in patients with SAMD9/9L after EPAG treatment compared to patients carrying FA/TBD variants and others without variants. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

RARE MUTATION IN AIRE GENE IDENTIFIED IN PATIENT WITH VERY EARLY ONSET IBD

<h3>Introduction</h3> The etiology of very early-onset inflammatory bowel disease (VEO IBD) is thought to have a greater likelihood of being due to a monogenic inborn error of immunity than later-onset IBD. <h3>Case Description</h3> We present the case of a 6-year-old female with VEO IBD who was found to have a variant of unknown significance (VUS) in the AIRE gene. She was diagnosed with VEO IBD at 2 years old after she was found to have failure to thrive, diarrhea, and blood in stool. She was initially treated with mesalamine, azathioprine, and steroids, but never went into remission. She does not have history of recurrent infections or other symptoms of autoimmune disease. There is no significant known family history of autoimmune disease. Labs were notable for normal antibody responses to Hep A, Hep B, and VZV vaccines, but low S. pneumoniae titers despite vaccination with Prevnar. She had normal T and B-cell counts, including T-cell subsets, and a normal oxidative burst test. Invitae PID panel was notable for a rare heterozygous VUS in AIRE gene. Given mutations in AIRE gene are associated with the autoimmune disease APECED, plan was made to test family for VUS in AIRE and evaluate for immunologic defects seen in AIRE, such as low Tregs and IL-17, as well as elevated ANA and autoantibodies to GAD65 and thyroid. <h3>Discussion</h3> While various organs are affected in APECED, the intestinal tract is not usually affected. Nonetheless, given early onset of IBD, strong suspicion remains for monogenic disorder, either in AIRE or other genes.

Adult-Onset Alexander Disease: New Causal Sequence Variant in the GFAP Gene.

ObjectivesAlexander disease (AD) is a rare disorder of the CNS. Diagnosis is based on clinical symptoms, typical MRI findings, and mutations in the glial fibrillary acid protein (GFAP) gene. In this case study, we describe a new mutation (p.L58P) in GFAP that caused a phenotype of adult-onset AD (AOAD).MethodsIn our outpatient clinic, a patient presented with cerebellar and bulbar symptoms after brain concussion. We used MRI and performed next-generation exome sequencing (NGS) to find mutations in GFAP to diagnose AD. The mutation was then transfected into HeLa cell lines to prove its pathogenicity.ResultsThe brain MRI finding showed typical AD alterations. The NGS found a heterozygous variant of unknown significance in GFAP (c.173T>C; p.L58P). After transfecting HeLa cell lines with this mutation, we showed that GFAP-L58P formed pathogenic clusters of cytoplasmic aggregates.DiscussionWe have found a new mutation that causes AOAD. We recommend that AOAD is included in the diagnostic workup in adult patients with gait ataxia and cerebellar and bulbar symptoms in association with a traumatic head injury.

Targeted Exome Sequencing of Genes Involved in Rare CNVs in Early-Onset Severe Obesity.

Context: Rare copy number variants (CNVs) have been associated with the development of severe obesity. However, the potential disease-causing contribution of individual genes within the region of CNVs is often not known. Objective: Screening of rare variants in genes involved in CNVs in Finnish patients with severe early-onset obesity to find candidate genes linked to severe obesity. Methods: Custom-made targeted exome sequencing panel to search for rare (minor allele frequency <0.1%) variants in genes affected by previously identified CNVs in 92 subjects (median age 14 years) with early-onset severe obesity (median body mass index (BMI) Z-score + 4.0). Results: We identified thirteen rare heterozygous variants of unknown significance in eleven subjects in twelve of the CNV genes. Two rare missense variants (p.Pro405Arg and p.Tyr232Cys) were found in SORCS1, a gene highly expressed in the brain and previously linked to diabetes risk. Four rare variants were in genes in the proximal 16p11.2 region (a frameshift variant in TAOK2 and missense variants in SEZ6L2, ALDOA and KIF22) and three rare missense variants were in genes in the 22q11.21 region (AIFM3, ARVCF and KLHL22). Conclusion: We report several rare variants in CNV genes in subjects with childhood obesity. However, the role of the individual genes in the previously identified rare CNVs to development of obesity remains uncertain. More studies are needed to understand the potential role of the specific genes within obesity associated CNVs.

Case Series: Bone Marrow Failure in Teen Siblings with Unique RPS19 Variant

Aplastic anemia occurs when there is a lack of hematopoiesis in the bone marrow leading to peripheral pancytopenia. It is a rare entity and is often idiopathic. However, upon presentation, inherited bone marrow failure syndromes and acquired etiologies must be considered. Investigating causality is particularly important when multiple family members are affected, especially in a short amount of time. It is also essential to identify novel causative genetic variants of bone marrow failure in order to direct treatment in these patients.In our case series, we describe two siblings who presented two weeks apart with severe pancytopenia. The first patient is a 13 year-old non-binary female (prefers pronouns they/them/theirs) who presented from clinic after getting routine labs with white blood cell (WBC) 1.83 x10(3)/mcL, absolute neutrophil count (ANC) 0.16 x10(3)/mcL, hemoglobin (Hgb) 4.8 gm/dL, platelet 13 x10(3)/mcL. Bone marrow biopsy revealed marked hypocellularity (0-10%) with hypoplasia. The second patient is their 16 year-old brother who presented two weeks later with new-onset petechial rash and was found to have WBC 4 x10(3)/mcL, ANC 2.19 x10(3)/mcL, Hgb 6.3 gm/dL, platelet 16 x10(3)/mcL. His bone marrow biopsy demonstrated variable cellularity (10-70%), but after months of transfusion-dependence he met criteria for severe aplastic anemia. Laboratory evaluation for acquired etiologies such as infection was negative. Both patients had a shared medical history of depression briefly treated with fluoxetine, but otherwise no potential medical triggers or environmental exposures were identified. Telomere length analysis was normal and chromosomal breakage analysis was negative. Upon genetic evaluation, both patients were found to have a heterozygous variant of unknown significance of RPS19 (c.-163>T), which substitutes a moderately conserved nucleotide in the noncoding exon 1 in 5‘ untranslated region of RPS19. Although this variant has not been classified as pathogenic, three other variants in the 5‘ untranslated region of RPS19 have been reported in patients with Diamond-Blackfan Anemia (DBA). In contrast to classic DBA, these patients did not present in infancy or early childhood. Likewise, they lacked congenital anomalies or other classic phenotypic characteristics of disease. Lab studies showed normal erythrocyte adenosine deaminase levels, though it should be noted that these levels could not be obtained prior to transfusion in both patients. Given lack of other identified etiologies, however, an inherited bone marrow failure syndrome, possibly due to this variant, was presumed. With suspected but potentially unidentifiable genetic predisposition, matched sibling donor transplant was deferred and both patients underwent a matched unrelated donor bone marrow transplant with reduced-intensity conditioning.With these cases, we aim to share a unique presentation of aplastic anemia that reveals a potentially novel pathogenic variant as well as to provide our approach to medical management in pediatric aplastic anemia in the setting of uncertainty. Identification of other patients with bone marrow failure and this genetic variant will be important to determine its pathogenicity. DisclosuresNo relevant conflicts of interest to declare.

A Novel Quali-Quantitative Defect of VWF

BackgroundVon Willebrand Factor (VWF) is a multimeric protein largely involved in both primary and secondary hemostasis. The diagnosis and classification of von Willebrand Disease (VWD) patients can be challenging. In this poster we explore the genetic defects and their structural consequences in a VWD patient with a disproportionately high bleeding phenotype for her VWD severity. A 31-year old female, initially diagnosed with VWD type 1, presented herself with a bleeding tendency (ISTH-BAT 13) disproportionate to the severity of her VWD with antigen levels of 36%. Additional analysis showed decreased FVIII-binding at 28%. The combination of both quantitative and functional defects of VWF was an indication for further genetic analysis in order to better define the subtype of VWD.AimsGenetic and structural analysis of VWF in a patient with a disproportionally high bleeding phenotype with regard to a mild decrease in VWF antigen.MethodsRoutine laboratory analysis for VWD was performed. Genetic screening was performed by exome sequencing of hemostasis related genes. VWF mRNA analysis was carried out by RT-PCR and Sanger sequencing. The X-ray structure of furin in complex with a peptide-based inhibitor (PDB ID: 6YD7) was used as a template to construct furin-VWF (759HR(R760S)SKRS764) complex. The derived structures (furin in complex with WT/R760S-VWF) were subjected to molecular dynamics (MD) simulations (200ns) and binding free energy (BFE) calculations by using standard parameters and protocols implemented in AMBER20 program.ResultsRoutine analysis showed PFA-ADP and PFA EPI >300 seconds, VWF:ACT of 37% with a VWF:AG of 36%. Collagen binding and FVIII-binding were 46% and 28% respectively. Genetic analysis of the VWF gene disclosed 2 heterozygous variants of unknown significance (VUS): c.2771 G>A (exon 21, p.Arg924Gln) has a 1-2.5% population prevalence and has been previously described in type 1 and 2N VWD. The other VUS (c.2278 C>A; exon 17) is a novel mutation predicting a major amino acid substitution (p.Arg760Ser) in the D2-domain of VWF. Sequencing of exons 17 and 21 in the patient's VWF mRNA revealed homozygosity for the mutated allele at both mutation sites, indicating that the two variants are in cis and that the ‘normal’ allele is not expressed at mRNA level. Molecular dynamics simulations of the novel c.2278 C>A mutation (Arg760Ser) predicts a markedly decreased binding of furin to its VWF binding site, possibly decreasing or preventing VWF pro-peptide cleavage. This in turn has been shown to lead to reduced FVIII-binding of VWF.ConclusionGenetic analysis shows one polymorphism (c.2771 G>A) and one variation of unknown significance (c.2278 C>A) in the patient's VWF-gene. The polymorphism is known to be of low pathogenicity. The c. 2278 C>A mutation was not known in any of the mutation databases and is a novel VWF mutation. Both mutations were shownto be present on the same allele. As the wild-type allele was not expressed on mRNA level, all of the patient's VWF protein includes both amino acid substitutions. Modeling and molecular dynamics simulations show a markedly decreased affinity of furin to its cleavage site on the VWF protein due to the Arg760Ser substitution, likely resulting in a persistent pro-peptide binding to the mature VWF protein. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Arrhythmias caused by mutations in SCN5A in children

Abstract Pathogenic genetic variants of the sodium channel alpha-subunit gene (SCN5A) is the leading cause of familial bradyarrhythmias. These diseases often manifest with syncope, have family segregation, and needs antiarrhythmic devices implantation, even in children. However, phenotypically these mutations can manifest as sick sinus syndrome (SSS), atrial fibrillation (AF), Brugada syndrome, and long QT syndrome. There are no studies on the prevalence of SCN5A mutations in children with arrhythmias. The aim is to study phenotypic and electrocardiographic manifestations of SCN5A-mutations in children with bradyarrhythmias. Methods We analyzed the data of 26 patients with bradyarrhythmias, who underwent genetic testing, from the database of the Russian Pediatric Arrhythmia Center. Seventeen were the probands, and nine - family members. The average age was 15.4±4.5 (from 2 to 17). Whole-exome sequencing was performed for 18 patients, cardiovascular panels for others. Results Variants of the sodium channel gene were detected in 3 family cases of arrhythmias and one sporadic case. In total, variants in SCN5A gene were found in 9 patients out of 26 (35%). In the first family, two sisters aged 6 and 17 have two biallelic variants of unknown significance in SCN5A gene. Both girls periodically have a Brugada marker on their ECG. The sisters both had syncope before the implantation of defibrillators. The girls' parents and older sister are clinically healthy, but the father previously had syncope. In the second family, a boy with a binodal disease, syncope, and an implanted pacemaker has two biallelic likely pathogenic variants in SCN5A gene. The boy's sister inherited only the mother's of SCN5A variant, showed a normal ECG in the first year of life, but then developed bradycardia within one year, and then joined the AVB of the 1st degree. Both parents had no clinical manifestations and no complaints. The third family included a mother and daughter with SSS and the heterozygous missense variant of unknown significance in SCN5A gene. Both of them do not need pacemaker implantation. The 12-year-old girl with two heterozygous variants of unknown significance in SCN5A gene had SSS, AF and underwent pacemaker implantation. Conclusion Genetic variants of SCN5A were detected in 35% of patients with bradyarrhythmias. Electrocardiographic manifestations of arrhythmias in our patients with SCN5A variants are diverse – SSS, Brugada syndrome, atrial fibrillation. Phenotypic variability in the manifestation of the SCN5A mutation requires a more thorough study of bradyarrhythmias' genetic causes in children. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Clinical Research Institute of Pediatrics named after acad. Y.E.Veltishev at the Pirogov Russian National Research Medical University, Moscow, Russia

Collagen remodelling and plasma ascorbic acid levels in patients suspected of inherited bleeding disorders harbouring germline variants in collagen-related genes.

IntroductionVariants in collagen‐related genes COL1A1, COL3A1, COL5A1 and COL5A2 are associated with Ehlers‐Danlos syndrome (EDS), a heterogeneous group of connective tissue disorders strongly associated with increased bleeding. Of patients with incompletely explained bleeding diathesis, a relatively high proportion were shown to harbour at least one heterozygous variant of unknown significance (VUS) in one of these genes, the vast majority without meeting the clinical criteria for EDS.AimTo investigate the functional consequences of the identified variants by assessing the formation and degradation of types I, III and V collagen, in addition to plasma levels of ascorbic acid (AA).MethodsA total of 31 patients harbouring at least one heterozygous VUS in COL1A1, COL3A1, COL5A1 or COL5A2 and 20 healthy controls were assessed using monoclonal antibodies targeting neo‐epitopes specific for collagen formation and degradation. Plasma AA levels were measured in patients using high‐performance liquid chromatography.ResultsSerum levels of C5 M (degradation of type V collagen) were decreased in patients compared with healthy controls (p = .033). No significant differences were found in biomarkers for remodelling of types I and III collagen. A significant negative correlation between bleeding (ISTH‐BAT score) and plasma AA levels was shown (r = −.42; r2 = .17; p = .020). Suboptimal or marginally deficient AA status was found in 8/31 patients (26%).ConclusionFunctional investigations of collagen remodelling were not able to identify any clear associations between the identified variants and increased bleeding. The negative correlation between plasma AA levels and ISTH‐BAT score motivates further investigations.

Genetic predisposition to neural crest-derived tumors: revisiting the role of KIF1B.

ObjectiveWe previously described a family in which predisposition to pheochromocytoma (PCC) segregates with a germline heterozygous KIF1B nucleotide variant (c.4442G>A, p.Ser1481Asn) in three generations. During the clinical follow-up, one proband’s brother, negative for the KIF1B nucleotide variant, developed a bilateral PCC at 31 years. This prompted us to reconsider the genetic analysis.Design and methodsGermline DNA was analyzed by next-generation sequencing (NGS) using a multi-gene panel plus MLPA or by whole exome sequencing (WES). Tumor-derived DNA was analyzed by SnapShot, Sanger sequencing or NGS to identify loss-of-heterozygosity (LOH) or additional somatic mutations.ResultsA germline heterozygous variant of unknown significance in MAX (c.145T>C, p.Ser49Pro) was identified in the proband’s brother. Loss of the wild-type MAX allele occurred in his PCCs thus demonstrating that this variant was responsible for the bilateral PCC in this patient. The proband and her affected grandfather also carried the MAX variant but no second hit could be found at the somatic level. No other pathogenic mutations were detected in 36 genes predisposing to familial PCC/PGL or familial cancers by WES of the proband germline. Germline variants detected in other genes, TFAP2E and TMEM214, may contribute to the multiple tumors of the proband.ConclusionIn this family, the heritability of PCC is linked to the MAX germline variant and not to the KIF1B germline variant which, however, may have contributed to the occurrence of neuroblastoma (NB) in the proband.

SAT-057 A Novel IGSF1 Variant in a Boy with Central Hypothyroidism and Epiphyseal Dysplasia

Background: IGSF1 deficiency syndrome, also known as X-linked central hypothyroidism and testicular enlargement (CHTE) syndrome, is caused by mutations in the immunoglobulin superfamily, member 1 gene. Recently recognized as the most common genetic cause of isolated central hypothyroidism (CH), its cardinal features include CH and adult macroorchidism. We describe a boy with CH and epiphyseal dysplasia found to have a novel IGSF1 variant.Clinical case: A male with attention deficit hyperactivity disorder (ADHD), oppositional defiant disorder, obsessive compulsive disorder and obesity was evaluated at age 9y for high total and LDL cholesterol and low T4 with normal TSH. He had short stature for family, high BMI, high sitting height ratio, and short arm span. Laboratory investigation revealed persistently mildly high total and LDL cholesterol, low T4, normal cortisol response to low-dose Cortrosyn stimulation, and normal IGF-1 and IGF-BP3. Bone age at 9y1m chronological age was 8y0m. Skeletal survey showed abnormal epiphyses of the femoral heads, knees, and humeral capitella suggesting primary epiphyseal dysplasia; he was referred to genetics. Growth improved after starting levothyroxine for CH. Multiple epiphyseal dysplasia gene panel (with reflex to clinical exome) did not find any variants known to be pathogenic for his condition. He was found to be a carrier of autosomal recessive Bartter syndrome. Heterozygous variants of unknown significance were found in RMRP, FGFR1, CDT1 and APOB. Whole exome sequencing showed hemizygosity for the p.Q743X (c.2227C>T) variant in IGSF1.Discussion: The p.Q743X IGSF1 variant has not been reported in the literature and is not present in population databases. It is predicted to cause loss of normal protein function and is considered pathogenic. CH is found in all males with IGSF1 deficiency syndrome. Macroorchidism, another defining feature, develops in adulthood; age of testicular growth onset is normal, but pubertal testosterone rise is delayed. Our patient remains prepubertal at age 11y. Other features sometimes present include hypoprolactinemia (which was found in this child) and transient partial GHD (not seen in this case). Overweight and the metabolic syndrome are common; this child’s cholesterol abnormalities may be due to weight and/or APOB variant found on genetic testing. ADHD has been seen in some patients with this syndrome; this child also has extensive psychiatric/behavioral problems, which have not been described. The skeletal findings in this case have not been previously noted in IGSF1 deficiency syndrome; whether this is a rare feature of IGSF1 deficiency syndrome or a separate entity is unclear. This case adds to the growing list of disease-causing variants in IGSF1. Endocrinologists should consider IGSF1 deficiency syndrome when diagnosing isolated CH in boys, as the characteristic macroorchidism is not present in childhood.

Genome and RNA sequencing in patients with methylmalonic aciduria of unknown cause

PurposeOur laboratory has classified patients with methylmalonic aciduria using somatic cell studies for over four decades. We have accumulated 127 fibroblast lines from patients with persistent elevated methylmalonic acid (MMA) levels in which no genetic cause could be identified. Cultured fibroblasts from 26 of these patients had low [14C]propionate incorporation into macromolecules, possibly reflecting decreased methylmalonyl-CoA mutase function. MethodsGenome sequencing (GS), copy-number variation (CNV) analysis, and RNA sequencing were performed on genomic DNA and complementary DNA (cDNA) from these 26 patients. ResultsNo patient had two pathogenic variants in any gene associated with cobalamin metabolism. Nine patients had heterozygous variants of unknown significance previously identified by a next-generation sequencing (NGS) panel targeting cobalamin metabolic genes. Three patients had pathogenic changes in genes not associated with cobalamin metabolism (PCCA, EPCAM, and a 17q12 duplication) that explain parts of their phenotypes other than elevated MMA. ConclusionGenome and RNA sequencing did not detect any additional putative causal genetic defects in known cobalamin genes following somatic cell studies and the use of a targeted NGS panel. They did detect pathogenic variants in other genes in three patients that explained some aspects of their clinical presentation.