Low-frequency somatic copy number alterations in normal human lymphocytes revealed by large-scale single-cell whole-genome profiling

Genomic-scale somatic copy number alterations in healthy humans are difficult to investigate because of low occurrence rates and the structural variations’ stochastic natures. Using a Tn5-transposase-assisted single-cell whole-genome sequencing method, we sequenced over 20,000 single lymphocytes from 16 individuals. Then, with the scale increased to a few thousand single cells per individual, we found that about 7.5% of the cells had large-size copy number alterations. Trisomy 21 was the most prevalent aneuploid event among all autosomal copy number alterations, whereas monosomy X occurred most frequently in over-30-yr-old females. In the monosomy X single cells from individuals with phased genomes and identified X-inactivation ratios in bulk, the inactive X Chromosomes were lost more often than the active ones.

Monosomy X in isogenic human iPSC-derived trophoblast model impacts expression modules preserved in human placenta

Mammalian sex chromosomes encode homologous X/Y gene pairs that were retained on the male Y and escape X chromosome inactivation (XCI) in females. Inferred to reflect X/Y-pair dosage sensitivity, monosomy X is a leading cause of miscarriage in humans with near full penetrance. This phenotype is shared with many other mammals but not the mouse, which offers sophisticated genetic tools to generate sex chromosomal aneuploidy but also tolerates its developmental impact. To address this critical gap, we generated X-monosomic human induced pluripotent stem cells (hiPSCs) alongside otherwise isogenic euploid controls from male and female mosaic samples. Phased genomic variants of these hiPSC panels enable systematic investigation of X/Y dosage-sensitive features using in vitro models of human development. Here, we demonstrate the utility of these validated hiPSC lines to test how X/Y-linked gene dosage impacts a widely-used model for the human syncytiotrophoblast. While these isogenic panels trigger a GATA2/3 and TFAP2A/C -driven trophoblast gene circuit irrespective of karyotype, differential expression implicates monosomy X in altered levels of placental genes, and in secretion of placental growth factor (PlGF) and human chorionic gonadotropin (hCG). Remarkably, weighted gene co-expression network modules that significantly reflect these changes are also preserved in first-trimester chorionic villi and term placenta. Our results suggest monosomy X may skew trophoblast cell type composition, and that the pseudoautosomal region likely plays a key role in these changes, which may facilitate prioritization of haploinsufficient drivers of 45,X extra-embryonic phenotypes.

A high-throughput amplicon screen for somatic UBA1 variants in Cytopenic and Giant Cell Arteritis cohorts

Somatic mutations in the gene encoding the major E1 ubiquitin ligase, UBA1, were recently identified as a cause of VEXAS, a late-onset acquired auto-inflammatory syndrome. Differential diagnoses for patients subsequently found to have VEXAS include relapsing polychondritis, Sweet’s syndrome, myelodysplastic syndrome (MDS), giant cell arteritis (GCA) and undifferentiated systemic autoinflammatory disease (uSAID). We therefore sought to screen DNA from individuals with a non-diagnostic cytopenia or GCA, for known VEXAS-associated mutations. To this end, we developed a multiplexed UBA1 amplicon sequencing assay, allowing quick screening of large cohorts while also providing sufficient sequencing depth to identify somatic mutations to an allele frequency <1%. Using this assay, we screened genomic DNA from 612 males diagnosed with GCA, and bone marrow DNA from 1,055 cases with an undiagnosed cytopenia. No GCA cases were found to have UBA1 mutations, however 4 different mutations in the cytopenic cohort were identified in 7 individuals. Furthermore, we describe a female case identified in the screen with a UBA1 mutation and all VEXAS-associated phenotypes, but without Monosomy X. Our study suggests that, despite the overlap in clinical features, VEXAS is rarely misdiagnosed as GCA, but identified in 1.0% of males with an undiagnosed cytopenia. The identification of a UBA1 variant in a female case adds further evidence that VEXAS should not be ruled out as a differential diagnosis in females with VEXAS-like symptoms.Key points-Mutations in UBA1 exon 3 have been associated with VEXAS syndrome-UBA1 exon 3 was screened in 1650 patients with cytopenia or GCA by amplicon sequencing.-6 males were identified from the non-diagnostic cytopenia cohort (1.0%) with UBA1 mutations.-A female with a somatic UBA1 mutation was identified without Monosomy X

Low frequency somatic copy number alterations in normal human lymphocytes revealed by large scale single-cell whole genome profiling

Genomic-scale somatic copy number alterations in healthy humans are difficult to investigate because of low occurrence rates and the structural variations' stochastic natures. Using a Tn5-transposase assisted single-cell whole genome sequencing method, we sequenced over 20,000 single lymphocytes from 16 individuals. Then, with the scale increased to a few thousand single cells per individual, we found that about 7.5% of the cells had large-size copy number alterations. Trisomy 21 was the most prevalent aneuploid event among all autosomal copy number alterations, while monosomy X occurred most frequently in over-30-year-old females. In the monosomy X single cells from individuals with phased genomes and identified X- inactivation ratios in bulk, the inactive X Chromosomes were lost more often than were the active ones.

Longitudinal Health Care for Girls and Women With Turner Syndrome

Background Turner Syndrome (TS) is the most common chromosomal abnormality seen in females and is often diagnosed in childhood. The transition of patients with TS from pediatric to adult providers is characterized by inconsistency. As such, limited data are available to assess long term health outcomes of women with TS. Objectives: 1) To evaluate the long term health outcomes in women with TS followed at a single academic institution and 2) to establish clinical recommendations for evaluation, counseling, and planned transition of this population. Methods: A retrospective pilot study was performed by examining medical records of women with TS who presented with short stature or delayed puberty and were diagnosed with TS on the basis of cytogenetic analysis. Patients with monosomy X, mosaic monosomy X, or X chromosome structural abnormalities consistent with TS or mosaic TS diagnosis were included. Medical records from an academic children’s hospital and an affiliated women’s hospital were reviewed. Results: To date, 15 females aged 26-32 years were identified. Electronic records were available for 12/15 with age at diagnosis ranging from in utero to 15 years of age. Those with pediatric information available had documentation addressing use of growth hormone and pubertal hormone replacement therapies. Of the 12 patients, 11 had cardiac imaging performed; only one had an MRI. Consistent planned transition to adult health care providers was not evident. Of the 12 patients, 3 had consultation with a Reproductive Endocrinology and Infertility specialist; 3 had a diagnosis of anxiety or depression; 3 had a DEXA scan done (1 had osteopenia); 5 had dyslipidemia; and 2 developed insulin resistance. Conclusions: This pilot study confirms heterogeneity in practice patterns and variable interactions of women with TS with the healthcare system, especially as patients enter adulthood. Although some women were referred to subspecialists, our initial data uncover patient uncertainty about healthcare and transition recommendations. Our preliminary data indicate the need for early patient education in a collaborative, multi-disciplinary fashion. We plan to validate and extend our initial findings by reviewing additional medical records. Ultimately, we plan for expanded education, consistent surveillance recommendations, and planned transition of patients with TS from pediatrics to adult caregivers.