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PIK3CA -related overgrowth spectrum (PROS) disorders are caused by somatic mosaic variants that result in constitutive activation of the phosphatidylinositol-3-kinase/AKT/mTOR pathway. Promising responses to molecularly targeted therapy have been reported, although identification of an appropriate agent can be hampered by the mosaic nature and corresponding low variant allele frequency of the causal variant. Moreover, our understanding of the molecular consequences of these variants—for example how they affect gene expression profiles—remains limited. Here we describe in vitro expansion of a human capillary malformation followed by molecular characterization using exome sequencing, single cell gene expression, and targeted long-read single cell RNA-sequencing in a patient with clinical features consistent with Megalencephaly-Capillary Malformation Syndrome (MCAP, a PROS condition). These approaches identified a targetable PIK3CA variant with expression restricted to PAX3+ fibroblast and undifferentiated keratinocyte populations. This study highlights the innovative combination of next-generation single cell sequencing methods to better understand unique transcriptomic profiles and cell types associated with MCAP, revealing molecular intricacies of this genetic syndrome.

Background Liquid biopsy assays using cerebrospinal fluid (CSF) can revolutionize care for children with central nervous system (CNS) tumors by enabling precise monitoring of therapeutic responses and detecting recurrence or measurable residual disease (MRD). These assays can detect cell-free, circulating tumor DNA (ctDNA) via somatic alterations, though accurately measuring low-abundance ctDNA in CSF is challenging. Methods Our research focused on the optimization of next-generation sequencing library preparation from cell-free DNA (cfDNA), evaluating four commercial kits to address the low nucleic acid yield in CSF-derived cfDNA. The selected kit minimized false positives and detected somatic variants at 5% variant allele frequency using 0.1 ng input of synthetic cfDNA, suitable for low-volume CSF samples. Results We then applied our optimized workflow to six children with CNS tumors using a personalized hybrid-capture sequencing strategy (“MRD4U”), in which individualized panels were designed based on each patient’s tumor sequencing. Using MRD4U, we identified ctDNA in two samples, even though neither patient had radiographic or clinical evidence of disease at the time of liquid biopsy. Notably, one ctDNA-positive patient developed radiographic recurrence four months later, demonstrating the assay’s potential to detect molecular relapse ahead of conventional clinical measures. Conclusions These findings demonstrate applicability of our personalized MRD4U assay in early detection of disease recurrence. Unlike non-targeted or tumor-agnostic CSF liquid biopsy approaches, MRD4U leverages patient-specific genomic information to enable sensitive, tumor-informed monitoring that can be deployed across a wide range of pediatric CNS tumors. Our approach is broadly applicable to any tumor type with existing genomic data, enabling ctDNA detection across diverse diagnoses. Ultimately, this strategy may inform clinical decision-making and enable earlier therapeutic intervention.

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 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 and expand the genotypic and phenotypic spectrum of CIDs associated with EBV-SMTs.

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. We assessed the consumer experience receiving direct‐to‐consumer (DTC) genetic risk for venous thromboembolism (VTE). 2354 customers (1244 variant‐positive and 1110 variant‐negative individuals) of the personal genetics company 23andMe, Inc., who had received results online for risk variants in clotting factor genes F2 and F5, participated in an online survey‐based study. Participants responded to questions about perception of VTE risk, discussion of results with healthcare providers and recommendations received, actions taken to control risk, emotional responses to receiving risk results, and perceived value of the information.

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.

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.

Next-generation sequencing (NGS) technology is increasingly utilized to identify therapeutic targets for patients with malignancy. This technology also has the capability to reveal the presence of constitutional genetic alterations, which may have significant implications for patients and their family members. Here we present the case of a 23 year old Caucasian patient with recurrent undifferentiated sarcoma who had NGS-based tumor analysis using an assay which simultaneously analyzed the entire coding sequence of 236 cancer-related genes (3769 exons) plus 47 introns from 19 genes often rearranged or altered in cancer. Pathogenic alterations were reported in tumor as the predicted protein alterations, BRCA2 “R645fs*15″ and MLH1 “E694*”. Because constitutional BRCA2 and MLH1 gene mutations are associated with Hereditary Breast Ovarian Cancer Syndrome (HBOCS) and Lynch syndrome respectively, sequence analysis of DNA isolated from peripheral blood was performed. The presence of the alterations, BRCA2 c.1929delG and MLH1 c.2080G>T, corresponding to the previously reported predicted protein alterations, were confirmed by Sanger sequencing in the constitutional DNA. An additional DNA finding was reported in this analysis, MLH1 c.2081A>C at the neighboring nucleotide. Further evaluation of the family revealed that all alterations were paternally inherited and the two MLH1 substitutions were in cis , more appropriately referred to as MLH1 c.2080_2081delGAinsTC, which is classified as a variant of uncertain significance. This case illustrates important considerations related to appropriate interpretation of NGS tumor results and follow-up of patients with potentially deleterious constitutional alterations.

Genetic testing for inherited thrombophilia, including mutation analysis for factor V Leiden and prothrombin G20210A, is commonly performed. Yet, tests for inherited thrombophilia are frequently ordered inappropriately, and without proper counseling about the risks, benefits and limitations of testing. Genetic counselors are uniquely trained to help people understand and adapt to medical, psychological and familial implications of genetic contributions to disease. In the context of thrombophilia, genetic counselors may serve as a resource to other clinicians to: (a) identify individuals and families at increased risk for inherited thrombophilia, (b) offer and explain testing to patients and families, as appropriate, (c) facilitate patient-focused decision-making and informed consent prior to testing, (d) interpret test results, (e) explain inheritance patterns and discuss implications of thrombophilia for family members and (f) provide education and support resources. This article will provide insight into the training and roles of genetic counselors, review indications for thrombophilia testing, and highlight specific issues related to genetic testing, including genetic discrimination concerns.

Traditionally, genetics is defined as the study of single-gene and chromosomal disorders, inheritance patterns and variation in organisms. Recent developments in molecular genetics have increased our understanding of how genetic traits contribute to common disease. As a result, it is anticipated that genetic technologies will increasingly be integrated into medical practice. Therefore, it is essential that all medical practitioners have an understanding of basic genetic concepts and terminology. The goal of this manuscript is to provide an overview of genetic and genomic terminology in order that health care practitioners may gain a better appreciation of how these concepts may be applied to medical practice. Factor V Leiden, a genetic alteration that results in a hypercoagulable state, is utilized to explore relevant genetic concepts.