A novel IKZF1 variant in a family with autosomal dominant CVID: A case for expanding exon coverage in inborn errors of immunity.

Common variable immune deficiency (CVID) is a heterogenous group of disorders characterized by varying degrees of hypogammaglobulinemia, recurrent infections, and autoimmunity. Currently, pathogenic variants are identified in approximately 20-30% of CVID cases. Here we report a 3-generation family with autosomal dominant Common Variable Immunodeficiency (CVID) diagnosed in 9 affected individuals. Although primary immune deficiency panels and exome sequencing were non-diagnostic, whole genome sequencing revealed a novel, pathogenic c.499C > T: p.His167Tyr variant in IKZF1, a critical regulator of B cell development. Functional testing done through pericentromeric heterochromatin localization and light shift chemiluminescent electrophoretic mobility shift assay confirmed the variant's deleterious effect via a haploinsufficiency mechanism. Our findings expand the spectrum of known IKZF1 mutations and contribute to a more comprehensive understanding of CVID's genetic heterogeneity. Furthermore, this case underscores the importance of considering whole genome sequencing for comprehensive genetic diagnosis when concern for a monogenic inborn errors of immunity is high.

Clinical Practice Guidelines for the Immunological Management of Chromosome 22q11.2 Deletion Syndrome and Other Defects in Thymic Development.

Current practices vary widely regarding the immunological work-up and management of patients affected with defects in thymic development (DTD), which include chromosome 22q11.2 microdeletion syndrome (22q11.2del) and other causes of DiGeorge syndrome (DGS) and coloboma, heart defect, atresia choanae, retardation of growth and development, genital hypoplasia, ear anomalies/deafness (CHARGE) syndrome. Practice variations affect the initial and subsequent assessment of immune function, the terminology used to describe the condition and immune status, the accepted criteria for recommending live vaccines, and how often follow-up is needed based on the degree of immune compromise. The lack of consensus and widely varying practices highlight the need to establish updated immunological clinical practice guidelines. These guideline recommendations provide a comprehensive review for immunologists and other clinicians who manage immune aspects of this group of disorders.

Discovery of clinically relevant fusions in pediatric cancer.

BACKGROUND: Pediatric cancers typically have a distinct genomic landscape when compared to adult cancers and frequently carry somatic gene fusion events that alter gene expression and drive tumorigenesis. Sensitive and specific detection of gene fusions through the analysis of next-generation-based RNA sequencing (RNA-Seq) data is computationally challenging and may be confounded by low tumor cellularity or underlying genomic complexity. Furthermore, numerous computational tools are available to identify fusions from supporting RNA-Seq reads, yet each algorithm demonstrates unique variability in sensitivity and precision, and no clearly superior approach currently exists. To overcome these challenges, we have developed an ensemble fusion calling approach to increase the accuracy of identifying fusions. RESULTS: Our Ensemble Fusion (EnFusion) approach utilizes seven fusion calling algorithms: Arriba, CICERO, FusionMap, FusionCatcher, JAFFA, MapSplice, and STAR-Fusion, which are packaged as a fully automated pipeline using Docker and Amazon Web Services (AWS) serverless technology. This method uses paired end RNA-Seq sequence reads as input, and the output from each algorithm is examined to identify fusions detected by a consensus of at least three algorithms. These consensus fusion results are filtered by comparison to an internal database to remove likely artifactual fusions occurring at high frequencies in our internal cohort, while a "known fusion list" prevents failure to report known pathogenic events. We have employed the EnFusion pipeline on RNA-Seq data from 229 patients with pediatric cancer or blood disorders studied under an IRB-approved protocol. The samples consist of 138 central nervous system tumors, 73 solid tumors, and 18 hematologic malignancies or disorders. The combination of an ensemble fusion-calling pipeline and a knowledge-based filtering strategy identified 67 clinically relevant fusions among our cohort (diagnostic yield of 29.3%), including RBPMS-MET, BCAN-NTRK1, and TRIM22-BRAF fusions. Following clinical confirmation and reporting in the patient's medical record, both known and novel fusions provided medically meaningful information. CONCLUSIONS: The EnFusion pipeline offers a streamlined approach to discover fusions in cancer, at higher levels of sensitivity and accuracy than single algorithm methods. Furthermore, this method accurately identifies driver fusions in pediatric cancer, providing clinical impact by contributing evidence to diagnosis and, when appropriate, indicating targeted therapies.

Identification of 22 novel BTK gene variants in B cell deficiency with hypogammaglobulinemia.

X-linked agammaglobulinemia (XLA) is an inborn error of immunity caused by pathogenic variants in the BTK gene, resulting in impaired B cell differentiation and maturation. Over 900 variants have already been described in this gene, however, new pathogenic variants continue to be identified. In this report, we describe 22 novel variants in BTK, associated with B cell deficiency with hypo- or agammaglobulinemia in male patients or in asymptomatic female carriers. Genetic data was correlated with BTK protein expression by flow cytometry, and clinical and family history to obtain a comprehensive assessment of the clinico-pathologic significance of these new variants in the BTK gene. For one novel missense variant, p.Cys502Tyr, site-directed mutagenesis was performed to determine the impact of the sequence change on protein expression and stability. Genetic data should be correlated with protein and/or clinical and immunological data, whenever possible, to determine the clinical significance of the gene sequence alteration.

Fibrinogen Columbus II: A novel c.1075G>T mutation in the FGG gene causing hypodysfibrinogenemia and thrombosis in an adolescent male.

Hypodysfibrinogenemia, the least frequently reported congenital fibrinogen disorder is characterized by low circulating levels of a dysfunctional protein, and is associated with phenotypic features of both hypo- and dysfibrinogenemia. Herein, we report an adolescent male with unprovoked venous thromboembolism and hypodysfibrinogenemia. Patient had recurrent, progressive thrombosis despite therapeutic anticoagulation with both low molecular weight heparin and warfarin. He had clinical and radiological improvement after transition to a direct thrombin inhibitor. Sequencing of the FGG gene identified a novel heterozygous mutation, c.1075G>T. Structural visualization of the identified variant was pursued and suggested that the mutation likely destabilizes the Ca2+ -binding site of fibrinogen resulting in pathogenicity.

A novel SAMD9 variant identified in patient with MIRAGE syndrome: Further defining syndromic phenotype and review of previous cases.

We present here a case of MIRAGE syndrome due to novel variant (c.2318T>C) in the sterile α motif domain-containing protein 9 (SAMD9) gene. Previous reports have described the clinical phenotype, which includes myelodysplasia, recurrent infections, restriction of growth and development, adrenal insufficiency, genitourinary abnormalities, and enteropathies, often resulting in fatality within the first few years of life. This report illustrates the variability in phenotype by describing an 11-year-old male, diagnosed with MIRAGE at age 9 years when his novel variant was identified through whole exome sequencing. A brief review of previously published cases of MIRAGE syndrome and the genotypic and phenotypic spectrum are presented.

Defining the role of a genetic counselor within pediatric hematology and oncology comprehensive care teams: Perspectives of the provider team and patients.

As genetic testing is increasingly integrated into medical care, the genetic counselor (GC) has emerged as a key member of multidisciplinary (MD) teams. Prior research has demonstrated the importance of role clarification when subspecialties are introduced to these teams given the potential differences in team member expectations regarding the division of responsibilities in clinic. To assess perceptions of a GC's role in four pediatric hematology/oncology clinics, Accreditation Council for Genetic Counseling (ACGC) competencies were used to develop two separate surveys for providers and patients and their caregivers. Providers (n = 25) perceived roles related to genetic expertise and coordination of care to be primarily the role of a GC significantly more often than psychosocial roles (p < .0001). Several potential GC roles were perceived as a shared role or the role of another provider, such as eliciting psychosocial history. Patients/caregivers (n = 70) perceived genetic-centric roles as significantly more important than roles related to coordination of care (p = .03) and psychosocial skills (p < .0001). Our findings from a pediatric specialty department suggest that GCs may maximize their potential in MD clinics by functioning as genetic subject matter experts with care coordination responsibilities related to genetic testing. Further communication between team members may be indicated to clarify the division of responsibilities.

Method-dependent Discrepancies in Fetal Hemoglobin Quantification in Patients With Hemoglobin S.

Hemoglobin F (HbF) concentration is used in the diagnosis of certain hemoglobinopathies and accurate quantification is central to treatment of patients with sickle cell disease. The 2 most commonly used methods to quantify HbF are high performance liquid chromatography and capillary zone electrophoresis. This study reports discrepancies in HbF quantification between these methods when hemoglobin S is present in the sample. Clinicians and investigators should be mindful of the method used for HbF quantification when evaluating and treating patients who produce hemoglobin S.

SHH desmoplastic/nodular medulloblastoma and Gorlin syndrome in the setting of Down syndrome: case report, molecular profiling, and review of the literature.

INTRODUCTION: Individuals with Down syndrome (DS) have an increased risk of acute leukemia compared to a markedly decreased incidence of solid tumors. Medulloblastoma, the most common malignant brain tumor of childhood, is particularly rare in the DS population, with only one published case. As demonstrated in a mouse model, DS is associated with cerebellar hypoplasia and a decreased number of cerebellar granule neuron progenitor cells (CGNPs) in the external granule cell layer (EGL). Treatment of these mice with sonic hedgehog signaling pathway (Shh) agonists promote normalization of CGNPs and improved cognitive functioning. CASE REPORT: We describe a 21-month-old male with DS and concurrent desmoplastic/nodular medulloblastoma (DNMB)-a tumor derived from Shh dysregulation and over-activation of CGNPs. Molecular profiling further classified the tumor into the new consensus SHH molecular subgroup. Additional testing revealed a de novo heterozygous germ line mutation in the PTCH1 gene encoding a tumor suppressor protein in the Shh pathway. DISCUSSION: The developmental failure of CGNPs in DS patients offers a plausible explanation for the rarity of medulloblastoma in this population. Conversely, patients with PTCH1 germline mutations experience Shh overstimulation resulting in Gorlin (Nevoid Basal Cell Carcinoma) syndrome and an increased incidence of malignant transformation of CGNPs leading to medulloblastoma formation. This represents the first documented report of an individual with DS simultaneously carrying PTCH1 germline mutation. CONCLUSION: We have observed a highly unusual circumstance in which the PTCH1 mutation appears to "trump" the effects of DS in causation of Shh-activated medulloblastoma.

MTHFR: Addressing Genetic Counseling Dilemmas Using Evidence-Based Literature.

The 5, 10 methylenetetrahydrofolate reductase (MTHFR) enzyme is a catalyst in the folate metabolism pathway, the byproducts of which are involved in the remethylation of homocysteine to methionine. Methionine is a precursor for a major DNA methyl donor and is important for DNA methylation and gene regulation. Rare mutations in the MTHFR gene have been associated with autosomal recessive MTHFR deficiency leading to homocystinuria. In addition, two polymorphic variants in this gene (C677T and A1298C) have been implicated in a mild form of MTHFR deficiency associated with hyperhomocysteinemia. Mild to moderate hyperhomocysteinemia has been previously implicated as a risk factor for cardiovascular disease. Further, the presence of these variants, with and without mildly elevated levels of homocysteine, has been studied in relation to several multifactorial disorders including recurrent pregnancy loss, neural tube defects and congenital anomalies, cancer, and neurodevelopmental disorders. Given this wide spectrum of purported clinical implications and the prevalence of these polymorphisms, genetic counselors may encounter questions regarding the significance of MTHFR polymorphisms in a variety of settings. Here we present a brief background of the MTHFR polymorphisms, review of the literature regarding clinical considerations, and discussion of relevant genetic counseling aspects through case vignettes. Educational resources for patients and providers are also included.

Deep vein thrombosis (DVT) and pulmonary embolism (PE): awareness and prophylaxis practices reported by patients with cancer.

Patients with cancer are at increased risk for venous thromboembolism (VTE). An online survey to measure PE/DVT terminology awareness and understanding of VTE risks revealed 24% and 15% of the 500 cancer patients surveyed had heard of term DVT/PE; 19% and 17% could name signs/ symptoms of DVT/PE; 3% recognized cancer treatments as risk factors for DVT/PE. Only 25% of the patients received prevention education from providers; <50% received VTE prophylaxis. Cancer patient awareness of VTE terminology and cancer and/or its treatment as risk for VTE is low. More effective patient/physician dialogue about VTE risk and thromboprophylaxis is needed.

Novel inherited CDX2 variant segregating in a family with diverse congenital malformations of the genitourinary system.

Anorectal malformations (ARMs) constitute a group of congenital defects of the gastrointestinal and urogenital systems. They affect males and females, with an estimated worldwide prevalence of 1 in 5000 live births. These malformations are clinically heterogeneous and can be part of a syndromic presentation (syndromic ARM) or as a nonsyndromic entity (nonsyndromic ARM). Despite the well-recognized heritability of nonsyndromic ARM, the genetic etiology in most patients is unknown. In this study, we describe three siblings with diverse congenital anomalies of the genitourinary system, anemia, delayed milestones, and skeletal anomalies. Genome sequencing identified a novel, paternally inherited heterozygous Caudal type Homeobox 2 (CDX2) variant (c.722A > G (p.Glu241Gly)), that was present in all three affected siblings. The variant identified in this family is absent from population databases and predicted to be damaging by most in silico pathogenicity tools. So far, only two other reports implicate variants in CDX2 with ARMs. Remarkably, the individuals described in these studies had similar clinical phenotypes and genetic alterations in CDX2 CDX2 encodes a transcription factor and is considered the master regulator of gastrointestinal development. This variant maps to the homeobox domain of the encoded protein, which is critical for interaction with DNA targets. Our finding provides a potential molecular diagnosis for this family's condition and supports the role of CDX2 in anorectal anomalies. It also highlights the clinical heterogeneity and variable penetrance of ARM predisposition variants, another well-documented phenomenon. Finally, it underscores the diagnostic utility of genomic profiling of ARMs to identify the genetic etiology of these defects.

Can Factor V Leiden and prothrombin G20210A testing in women with recurrent pregnancy loss result in improved pregnancy outcomes?: Results from a targeted evidence-based review.

Women with recurrent pregnancy loss are offered Factor V Leiden (F5) and/or prothrombin G20210A (F2) testing to identify candidates for anticoagulation to improve outcomes. A systematic literature review was performed to estimate test performance, effect sizes, and treatment effectiveness. Electronic searches were performed through April 2011, with review of references from included articles. English-language studies addressed analytic validity, clinical validity, and/or clinical utility and satisfied predefined inclusion criteria. Adequate evidence showed high analytic sensitivity and specificity for F5 and F2 testing. Evidence for clinical validity was adequate. The summary odds ratio for association of recurrent pregnancy loss with F5 in case-controlled studies was 2.02 (95% confidence interval, 1.60-2.55), with moderate heterogeneity and suggestion of publication bias. Longitudinal studies in women with recurrent pregnancy loss or unselected cohorts showed F5 carriers were more likely to have a subsequent loss than noncarriers (odds ratios: 1.93 and 2.03, respectively). Results for F2 testing were similar. For clinical utility, evidence was adequate that anticoagulation treatments were ineffective (except in antiphospholipid antibody syndrome) and had treatment-associated harms. The certainty of evidence is moderate (high, moderate, and low) that anticoagulation of women with recurrent pregnancy loss and F5/F2 variants would currently lead to net harms.

You want to do what? My mother's choice to have direct-to-consumer genetic testing.

As a genetic counselor, I had mixed opinions when my mother told me of her intent to undergo genomewide, SNP-based direct-to-consumer (DTC) genetic testing. I cautioned her that results could be misleading, could increase anxiety and were often of limited clinical validity or utility. I warned of the possibility of learning unintended health information and expressed concerns about how the information might be used by a private company. I told her about the variability in results among companies. Yet, she persisted in her desire, reminding me that she was an informed consumer. After reviewing her goals and understanding of the information she might receive, she elected to proceed. Despite my insistence that I would not be her personal genetic counselor, when the results came back, I found myself immersed in her genetic data. In this manuscript, I will examine how this personal experience challenged my perceptions of DTC testing.

Direct-to-consumer genetic testing for factor V Leiden and prothrombin 20210G>A: the consumer experience.

BACKGROUND: Clinical genetic testing for inherited predisposition to venous thromboembolism (VTE) is common among patients and their families. However, there is incomplete consensus about which individuals should receive testing, and the relative risks and benefits. METHODS: We assessed outcomes of receiving direct-to-consumer (DTC) results for the two most common genetic risk factors for VTE, factor V Leiden in the F5 gene (FVL) and prothrombin 20210G>A in the F2 gene (PT). Two thousand three hundred fifty-four customers (1244 variant-positive and 1110 variant-negative individuals) of the personal genetics company 23andMe, Inc., who had received results online for F5 and F2 variants, participated in an online survey-based study. Participants responded to questions about perception of VTE risk, discussion of results with healthcare providers (HCPs) and recommendations received, actions taken to control risk, emotional responses to receiving risk results, and perceived value of the information. RESULTS: Most participants (90% of variant-positive individuals, 99% of variant-negative individuals) had not previously been tested for F5 and/or F2 variants. The majority of variant-positive individuals correctly perceived that they were at higher than average risk for developing VTE. These individuals reported moderate rates of discussing results with HCPs (41%); receiving prevention advice from HCPs (31%), and making behavioral changes to control risk (e.g., exercising more, 30%). A minority (36%) of variant-positive individuals worried more after receiving VTE results. Nevertheless, most participants reported that knowing their risk had been an advantage (78% variant-positive and 58% variant-negative) and were satisfied knowing their genetic probability for VTE (81% variant-positive and 67% variant-negative). CONCLUSION: Consumers reported moderate rates of behavioral change and perceived personal benefit from receiving DTC genetic results for VTE risk.

A Novel Pathogenic Variant in CARMIL2 (RLTPR) Causing CARMIL2 Deficiency and EBV-Associated Smooth Muscle Tumors.

CARMIL2 deficiency is a rare combined immunodeficiency (CID) characterized by defective CD28-mediated T cell co-stimulation, altered cytoskeletal dynamics, and susceptibility to Epstein Barr Virus smooth muscle tumors (EBV-SMTs). Case reports associated with EBV-SMTs are limited. We describe herein a novel homozygous CARMIL2 variant (c.1364_1393del) in two Saudi Arabian male siblings born to consanguineous parents who developed EBV-SMTs. CARMIL2 protein expression was significantly reduced in CD4+ T cells and CD8+ T cells. T cell proliferation on stimulation with soluble (s) anti-CD3 or (s) anti-CD3 plus anti-CD28 antibodies was close to absent in the proband, confirming altered CD28-mediated co-signaling. CD28 expression was substantially reduced in the proband's T cells, and was diminished to a lesser degree in the T cells of the younger sibling, who has a milder clinical phenotype. Defects in both T and B cell compartments were observed, including absent central memory CD8+ T cells, and decreased frequencies of total and class-switched memory B cells. FOXP3+ regulatory T cells (Treg) were also quantitatively decreased, and furthermore CD25 expression within the Treg subset was substantially reduced. These data confirm the pathogenicity of this novel loss-of-function (LOF) variant in CARMIL2 and expand the genotypic and phenotypic spectrum of CIDs associated with EBV-SMTs.