Telomere length (TL) predicts the onset of cellular senescence in vitro but the diagnostic utility of TL measurement in clinical settings is not fully known. We tested the value of TL measurement by ...flow cytometry and FISH (flowFISH) in patients with mutations in telomerase and telomere maintenance genes. TL had a discrete and reproducible normal range with definable upper and lower boundaries. While TL above the 50th age-adjusted percentile had a 100% negative predictive value for clinically relevant mutations, the lower threshold in mutation carriers was age-dependent, and adult mutation carriers often overlapped with the lowest decile of controls. The extent of telomere shortening correlated with the age at diagnosis as well as the short telomere syndrome phenotype. Extremely short TL caused bone marrow failure and immunodeficiency in children and young adults, while milder defects manifested as pulmonary fibrosis-emphysema in adults. We prospectively examined whether TL altered treatment decisions for newly diagnosed idiopathic bone marrow failure patients and found abnormally short TL enriched for patients with mutations in some inherited bone marrow failure genes, such as RUNX1, in addition to telomerase and telomere maintenance genes. The result was actionable, altering the choice of treatment regimen and/or hematopoietic stem cell donor in one-fourth of the cases (9 of 38, 24%). We conclude that TL measurement by flowFISH, when used for targeted clinical indications and in limited settings, can influence treatment decisions in ways that improve outcome.
The multidisciplinary Epigenetics and Chromatin Clinic at Johns Hopkins provides comprehensive medical care for individuals with rare disorders that involve disrupted epigenetics. Initially centered ...on classical imprinting disorders, the focus shifted to the rapidly emerging group of genetic disorders resulting from pathogenic germline variants in epigenetic machinery genes. These are collectively called the Mendelian disorders of the epigenetic machinery (MDEMs), or more broadly, Chromatinopathies. In five years, 741 clinic visits have been completed for 432 individual patients, with 153 having confirmed epigenetic diagnoses. Of these, 115 individuals have one of 26 MDEMs with every single one exhibiting global developmental delay and/or intellectual disability. This supports prior observations that intellectual disability is the most common phenotypic feature of MDEMs. Additional common phenotypes in our clinic include growth abnormalities and neurodevelopmental issues, particularly hypotonia, attention-deficit/hyperactivity disorder (ADHD), and anxiety, with seizures and autism being less common. Overall, our patient population is representative of the broader group of MDEMs and includes mostly autosomal dominant disorders impacting writers more so than erasers, readers, and remodelers of chromatin marks. There is an increased representation of dual function components with a reader and an enzymatic domain. As expected, diagnoses were made mostly by sequencing but were aided in some cases by DNA methylation profiling. Our clinic has helped to facilitate the discovery of two new disorders, and our providers are actively developing and implementing novel therapeutic strategies for MDEMs. These data and our high follow-up rate of over 60% suggest that we are achieving our mission to diagnose, learn from, and provide optimal care for our patients with disrupted epigenetics.
Chronic obstructive pulmonary disease and pulmonary fibrosis have been hypothesized to represent premature aging phenotypes. At times, they cluster in families, but the genetic basis is not ...understood. We identified rare, frameshift mutations in the gene for nuclear assembly factor 1, NAF1, a box H/ACA RNA biogenesis factor, in pulmonary fibrosis-emphysema patients. The mutations segregated with short telomere length, low telomerase RNA levels, and extrapulmonary manifestations including myelodysplastic syndrome and liver disease. A truncated NAF1 was detected in cells derived from patients, and, in cells in which the frameshift mutation was introduced by genome editing, telomerase RNA levels were reduced. The mutant NAF1 lacked a conserved carboxyl-terminal motif, which we show is required for nuclear localization. To understand the disease mechanism, we used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) to generate Naf1(+/-) mice and found that they had half the levels of telomerase RNA. Other box H/ACA RNA levels were also decreased, but rRNA pseudouridylation, which is guided by snoRNAs, was intact. Moreover, first-generation Naf1(+/-) mice showed no evidence of ribosomal pathology. Our data indicate that disease in NAF1 mutation carriers is telomere-mediated; they show that NAF1 haploinsufficiency selectively disturbs telomere length homeostasis by decreasing the levels of telomerase RNA while sparing rRNA pseudouridylation.
The mechanisms that drive T cell aging are not understood. We report that children and adult telomerase mutation carriers with short telomere length (TL) develop a T cell immunodeficiency that can ...manifest in the absence of bone marrow failure and causes life-threatening opportunistic infections. Mutation carriers shared T cell-aging phenotypes seen in adults 5 decades older, including depleted naive T cells, increased apoptosis, and restricted T cell repertoire. T cell receptor excision circles (TRECs) were also undetectable or low, suggesting that newborn screening may identify individuals with germline telomere maintenance defects. Telomerase-null mice with short TL showed defects throughout T cell development, including increased apoptosis of stimulated thymocytes, their intrathymic precursors, in addition to depleted hematopoietic reserves. When we examined the transcriptional programs of T cells from telomerase mutation carriers, we found they diverged from older adults with normal TL. Short telomere T cells upregulated DNA damage and intrinsic apoptosis pathways, while older adult T cells upregulated extrinsic apoptosis pathways and programmed cell death 1 (PD-1) expression. T cells from mice with short TL also showed an active DNA-damage response, in contrast with old WT mice, despite their shared propensity to apoptosis. Our data suggest there are TL-dependent and TL-independent mechanisms that differentially contribute to distinct molecular programs of T cell apoptosis with aging.
We report biallelic missense and frameshift pathogenic variants in the gene encoding human nucleoporin NUP214 causing acute febrile encephalopathy. Clinical symptoms include neurodevelopmental ...regression, seizures, myoclonic jerks, progressive microcephaly, and cerebellar atrophy. NUP214 and NUP88 protein levels were reduced in primary skin fibroblasts derived from affected individuals, while the total number and density of nuclear pore complexes remained normal. Nuclear transport assays exhibited defects in the classical protein import and mRNA export pathways in affected cells. Direct surface imaging of fibroblast nuclei by scanning electron microscopy revealed a large increase in the presence of central particles (known as “plugs”) in the nuclear pore channels of affected cells. This observation suggests that large transport cargoes may be delayed in passage through the nuclear pore channel, affecting its selective barrier function. Exposure of fibroblasts from affected individuals to heat shock resulted in a marked delay in their stress response, followed by a surge in apoptotic cell death. This suggests a mechanistic link between decreased cell survival in cell culture and severe fever-induced brain damage in affected individuals. Our study provides evidence by direct imaging at the single nuclear pore level of functional changes linked to a human disease.
Chromosomal microarray has been widely adopted as the first-tier clinical test for individuals with multiple congenital anomalies, developmental delay, intellectual disability, and autism spectrum ...disorders. Although chromosomal microarray has been extensively shown to provide a higher diagnostic yield than conventional cytogenetic methods, some health insurers refuse to provide coverage for this test, claiming that it is experimental and does not affect patients' clinical management.
We retrospectively reviewed the electronic medical records of all patients who had abnormal chromosomal microarray findings reported by our laboratory over a 3-year period and quantified the management recommendations made in response to these results.
Abnormal chromosomal microarray findings were reported for 12.7% of patients (227/1,780). For patients with clinical follow-up notes available, these results had management implications for 54.5% of patients in the entire abnormal cohort (102/187) and for 42.1% of patients referred for isolated neurodevelopmental disorders (16/38). Recommendations included pharmacological treatment, cancer-related screening or exclusion of screening, contraindications, and referrals for further evaluation.
These results empirically demonstrate the clinical utility of chromosomal microarray by providing evidence that management was directly affected for the majority of patients in our cohort with abnormal chromosomal microarray findings.
BackgroundThe list of Mendelian disorders of the epigenetic machinery has expanded rapidly during the last 5 years. A few missense variants in the chromatin remodeler CHD1 have been found in several ...large-scale sequencing efforts focused on uncovering the genetic aetiology of autism.ObjectivesTo explore whether variants in CHD1 are associated with a human phenotype.MethodsWe used GeneMatcher to identify other physicians caring for patients with variants in CHD1. We also explored the epigenetic consequences of one of these variants in cultured fibroblasts.ResultsHere we describe six CHD1 heterozygous missense variants in a cohort of patients with autism, speech apraxia, developmental delay and facial dysmorphic features. Importantly, three of these variants occurred de novo. We also report on a subject with a de novo deletion covering a large fraction of the CHD1 gene without any obvious neurological phenotype. Finally, we demonstrate increased levels of the closed chromatin modification H3K27me3 in fibroblasts from a subject carrying a de novo variant in CHD1.ConclusionsOur results suggest that variants in CHD1 can lead to diverse phenotypic outcomes; however, the neurodevelopmental phenotype appears to be limited to patients with missense variants, which is compatible with a dominant negative mechanism of disease.
To investigate the utility of whole-exome sequencing (WES) to define a molecular diagnosis for patients clinically diagnosed with congenital anomalies of kidney and urinary tract (CAKUT).
WES was ...performed in 62 families with CAKUT. WES data were analyzed for single-nucleotide variants (SNVs) in 35 known CAKUT genes, putatively deleterious sequence changes in new candidate genes, and potentially disease-associated copy-number variants (CNVs).
In approximately 5% of families, pathogenic SNVs were identified in PAX2, HNF1B, and EYA1. Observed phenotypes in these families expand the current understanding about the role of these genes in CAKUT. Four pathogenic CNVs were also identified using two CNV detection tools. In addition, we found one deleterious de novo SNV in FOXP1 among the 62 families with CAKUT. The clinical database of the Baylor Miraca Genetics laboratory was queried and seven additional unrelated individuals with novel de novo SNVs in FOXP1 were identified. Six of these eight individuals with FOXP1 SNVs have syndromic urinary tract defects, implicating this gene in urinary tract development.
We conclude that WES can be used to identify molecular etiology (SNVs, CNVs) in a subset of individuals with CAKUT. WES can also help identify novel CAKUT genes.
Genet Med19 4, 412–420.
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