Rationale: Mutations in telomere-related genes have been implicated as the most frequent monogenic cause of Familial Pulmonary Fibrosis (FPF) and are also identified in a subset of patients with sporadic cases of Idiopathic Pulmonary Fibrosis (IPF). The objective of this study was to determine how the clinical features and outcomes of FPF and IPF patients with telomerase mutations differed from those with other genetic risk mechanisms. Methods: DNA was extracted from whole blood collected from 1001 patients with familial IPF and 423 patients with sporadic IPF. They were evaluated for telomere restriction fragment length by Southern blot and for presence of protein altering rare variants (RVs) in telomerase genes by whole exome or whole genome sequencing. The age at diagnosis and longitudinal values of DLCO and FVC (in percent of predicted) were obtained from the medical record and were calculated as an average annualized change. Measurements and Main Results: Eighty-eight of 551 FPF families (16.0%) were found to carry likely/pathogenic RVs in TERT (n=84 individuals), RTEL1 (n=68 individuals), PARN (n=20 individuals) and TERC (n=3). 38% of subjects within the FPF cohort had a peripheral blood mononuclear cell (PBMC) telomere length <10th percentile. Compared to sporadic IPF patients without telomerase mutations (n=357), FPF patients with telomerase mutations were younger at diagnosis, with the average age of diagnosis for familial and sporadic cohorts being 61.5 and 65.9, respectively (P=0.00002). FPF patients were also more likely female and less likely to have used tobacco. Affected subjects in telomere mutation families had also shorter telomere length compared to subjects without telomere-related gene mutations. FPF subjects with likely/pathogenic RVs had accelerated FVC decline compared to subjects without telomere gene RVs (FPF+IPF (-7.7 and -2.4 percentage per year, respectively p=0.03)). There were no differences noted between change in DLCO% between patients with RVs vs non-RV variants when assessed as a group. However, nonsmoking individuals with likely/pathogenic telomere gene rare variants had a faster decline in DLCO when in compared to sporadic IPF patients without telomerase RVs with an average annualized change in DLCO% of –10.2 and –4.0, respectively, (p=0.03). Conclusion: FPF patients with telomerase mutations have a distinct clinical phenotype and molecular genotype, thus supporting the utility of telomere length measurements and genetic testing in patients with FPF.

Transcription factor TBX4 rare variants associate with pulmonary arterial hypertension (PAH), particularly in children, and are the second most common cause of heritable PAH. However, TBX4's down-stream targets and the molecular and cellular pathways these targets regulate remain largely unknown in PAH. We combined RNA-seq and ChIP-seq results to identify TBX4 direct targets in lung fibroblasts and pericytes, respectively. There were 555 genes with altered expression with TBX4 knockdown in both fibroblasts and pericytes by RNA-seq, and which also were found to be bound by TBX4 by ChIP-seq. Gene ontology analysis found that these were dominated by genes related to extracellular matrix, actin organization, and migration guidance, although there were also significant groups related to serine/threonine kinase signaling, GTPase mediated signaling, and glycoprotein metabolism. Migration and proliferation studies using TBX4 knockdown fibroblasts confirmed functional effects. These studies provide the first insights into how genes and pathways regulated by TBX4 are impacted and inform future studies about the key biological processes that lead to PAH in patients who carry pathologic TBX4 rare variants.

Nizon-Isidor syndrome is a rare disorder caused by heterozygous variants in MED12L, with only eight documented cases in the literature. Here, we present three additional cases of this syndrome. Proband 1 was a 7-year-old female who presented with developmental delay, right-leg hemihypertrophy, laryngeal cleft, esotropia, abnormal skin pigmentation, sectoral iris hypopigmentation, dysphagia, periventricular nodular heterotopia, seizures, morbid obesity, and a pelvic kidney. Genome sequencing (GS) revealed a MED12L variant, NM_053002.5:c.3559+2T>G. Both computational models and transcriptomic analysis confirmed that this variant induced splice loss of MED12L exon 25. Probands 2 and 3 presented with overlapping phenotypes of developmental delay; sequencing confirmed c.3441_3444dup; p.(G1149Nfs*13) and seq[GRCh37] del(3)(q25.1q25.1) chr3:g.?_151075120 variants affecting MED12L. Further investigation found diploid-triploid mosaicism in Proband 1, supporting the hypothesis that loss of MED12L function may increase risk for other cytogenetic abnormalities. Probands 2 and 3 did not harbor evidence of additional cytogenetic aberrations. In Proband 1, caloric restriction and semaglutide-pramlintide combination therapy were started at age eight and were effective in weight reduction. Overall, this report expands the phenotypic spectrum of Nizon-Isidor syndrome, highlights a potential link between MED12L and cytogenetic abnormalities, and demonstrates a case of weight loss through GLP-1 therapy in a child with a genetic obesity syndrome.

Rationale: Approximately 1 in 5 cases of idiopathic pulmonary fibrosis (IPF) are familial, and families with highly penetrant PF have increased likelihood of a monogenic disease. We recently described a large multiplex family with >8 affected members who shared a rare variant in the gene encoding for the orphan G-protein coupled receptor GPR87. In these studies, we sought to investigate the mechanism through which GPR87 contributes to the pathogenesis of PF. Methods: Variant allele status was determined by whole-exome and/or Sanger sequencing. Human single-cell RNA-sequencing data from GSE227136 were reanalyzed. GPR87 localization in human tissue-was performed using RNA-ISH (RNAScope). Gpr87 mutant mice were generated using CRISPR-Cas9 and crossed with Scgb1a1-CreERTtDtomato or Krt5-CreERTtDtomato reporter mice for single-dose or repetitive IT bleomycin studies. RNA-ISH and immunofluorescence data were quantified by automated image analysis (HALO, Indica Labs). GPR87 null HBEC3KT cells were generated using CRISPR-Cas9 and WT/mutant rescue studies performed by lentiviral transduction. Results: GPR87 expressed was increased in basal and KRT5-/KRT17+ aberrant basaloid cells in IPF lungs compared to controls and localized to remodeled airways in areas of active collagen production. Gpr87 mutant mice had increased lung fibrosis following single-dose IT bleomycin. Lineage-tracing studies revealed Gpr87+ cells arise from Scgb1a1-labeled airway progenitors (not Krt5+ airway basal cells or AT2 cells) following repetitive bleomycin, with mutant Gpr87 promoting an Krt5+ basal-like fate and restraining alveolar differentiation of distal-airway progenitors. HBEC3-KT cells demonstrated GPR87-dependent adoption of “aberrant basaloid” features upon LPA stimulation. Conclusion: This FPF-associated GPR87 mutation promotes lung fibrosis through regulation of distal airway progenitor cell fate at least in part via acting as an epithelial LPA receptor. GPR87 represents a novel potential epithelial antifibrotic target for IPF.

The BMPR2 gene encodes the BMPR-II (bone morphogenetic protein receptor type-II) and is a known regulator of endothelial proliferation, apoptosis, and translational stress responses. While these effects are generally attributed to the actions of BMPR-II protein, we used circular RNA profiling to identify circ3218 and circ5078 as new BMPR2-derived functional RNAs. Circular RNAs were profiled by ultradeep RNA sequencing of human pulmonary artery endothelial cells. Novel BMPR2-derived circular RNAs were assessed for their effects on endothelial proliferation, apoptosis, and translational stress responses in human pulmonary artery endothelial cells and endothelial cells from patients with pulmonary arterial hypertension with heterozygous loss-of-function BMPR2 mutations. circ5078 to linear BMPR2 mRNA ratios were quantified in cultured lymphocytes from patients with pulmonary arterial hypertension with BMPR2 mutations versus unaffected mutation carriers. Depletion of circ3218 enhanced human pulmonary artery endothelial cell apoptosis, whereas circ5078 silencing increased proliferation. Enhanced proliferation with circ5078 depletion was eliminated by cosilencing of circ5078 with either linear BMPR2 mRNA or the stress granule protein, Caprin-1, indicating a potential interdependence of BMPR2 transcripts in the regulation of endothelial function. Patients with pulmonary arterial hypertension with BMPR2 mutations exhibited increased circ5078 to linear BMPR2 mRNA ratios, alongside impaired stress responses in patient endothelial cells that were deficient in linear BMPR2 transcripts but not circ5078. circ5078 depletion enhanced stress responses in human pulmonary artery endothelial cells and rescued stress granule formation in patient endothelial cells, independent of BMPR-II protein levels. Assessment of translational regulation by polysome profiling did not identify any impact of linear or circular BMPR2 transcript loss on global protein synthesis or stress-induced eIF2α (eukaryotic initiation factor 2α) phosphorylation but did identify the enhanced translational efficiency of select nuclear-encoded mitochondrial ribosome proteins with circ5078 depletion, offering a link between mitochondrial function and the circ5078-deficient endothelial phenotype. The identification of circ3218 and circ5078 as novel BMPR2-derived gene products reveals interdependent roles for coding and noncoding BMPR2 transcripts as regulators of endothelial function.

Familial Adult Myoclonic Epilepsy type 3 (FAME3) is a rare autosomal dominant disorder characterized by cortical tremor and epilepsy, caused by a noncoding pentanucleotide repeat expansion (TTTTA/TTTCA)n in the MARCHF6 gene. Conventional genetic testing often fails to detect this expansion due to its repetitive structure and intronic location. We evaluated a 61-year-old woman with refractory myoclonic and generalized tonic-clonic seizures, whose prior genetic testing-including exome and genome sequencing-was non-diagnostic. Using PacBio HiFi long-read whole-genome sequencing and the tandem repeat genotyping tool TRGT, we identified a pathogenic MARCHF6 intronic expansion. The proband harbored one allele with 15 TTTTA repeats and a second allele with a compound expansion of 661 TTTTA and 12 TTTCA repeats. Three affected relatives shared similarly expanded alleles, but with increasing repeat size in the latter generations. Importantly, analysis using TRGT-instability revealed repeat mosaicism in all affected individuals, reflected by variability in motif counts across individual sequencing reads. This somatic heterogeneity may contribute to the phenotypic penetrance, variable expressivity and pleiotropism seen in FAME3 disease expression. To our knowledge, this is the first clinical diagnosis of FAME3 using a commercially available long-read sequencing platform, underscoring its diagnostic utility in resolving complex repeat expansion disorders and uncovering biologically relevant mosaicism.

PURPOSE:The multicenter NIH-funded Undiagnosed Diseases Network (UDN) exists to diagnose puzzling and newly discovered conditions. We report the UDN's assistance in diagnosing perplexing ocular disorders along with 6 case illustrations. DESIGN:Retrospective Interventional Case Series. SUBJECTS:Participants with ocular phenotypes who had applied and were accepted into the UDN with detailed supporting letters written by ophthalmologists or other clinicians when clinically indicated genetic and laboratory testing results were not diagnostic. METHODS:Human Phenotype Ontology Codes were used to identify and categorize subjects with ocular phenotypes. Advanced genomic technologies (exome, genome, mitochondrial and RNA sequencing, X-inactivation analysis, immunoblot analysis) were available for diagnoses. MAIN OUTCOME MEASURE:Proportion of cases solved by the UDN in subjects manifesting an ophthalmic component of their undiagnosed disorder. RESULTS:The national UDN diagnostic rate for subjects with an eye phenotype was 40.2% (452 of 1123); the diagnostic rate for the other subjects (without an eye) phenotype was 27.8% (276 of 992). In univariate analysis, having an eye phenotype was significantly associated with receiving a diagnosis (odds ratio [OR] = 1.75; CI = 1.45-2.10; P = 2.28e-09). Of 58 eye diagnosed cases/104 total diagnosed cases at the Vanderbilt UDN site, 6 will be discussed more fully. Vanderbilt UDN cases include an autosomal dominant glaucoma with a variant in TEK/TIE2; a de novo heterozygous variant in PRPS1 causing microcornea and glaucoma with skewed X-inactivation affecting a female; a homozygous variant in NADK2 causing optic nerve atrophy; autosomal recessive variants in EPG5 resulting in optic nerve atrophy and cone-rod dystrophy; and a rare de novo variant in COG4 causing a cataract/ retinitis pigmentosa/ nystagmus phenotype. EPG5 and COG4 are not present on inherited retinal disease panels. CONCLUSIONS:The UDN is a national resource available to increase solving undiagnosed diseases including those with ocular phenotypes, facilitate research on undiagnosed diseases, and create a collaboration to improve care options for patients with undiagnosed diseases. Clinicians including ophthalmologists can collaborate with the UDN to solve challenging ocular mysteries using genomic technologies.

Autosomal dominant congenital disorder of glycosylation (CDG) type Iw (OMIM# 619714) is caused by a heterozygous mutation in the STT3A gene. Most CDGs have an autosomal recessive (AR) mode of inheritance, but several cases with an autosomal dominant (AD) form of an AR CDG have been recently identified. This report describes a 17-year-old male who was referred to the Undiagnosed Diseases Network (UDN) with a history of macrocephaly, failure to thrive, short stature, epilepsy, autism, attention-deficit/hyperactivity disorder, mild developmental delay, intermittent hypotonia, dysmorphic features, and mildly enlarged aortic root. Trio exome sequencing was negative. His biochemical workup included normal plasma amino acids, ammonia, acylcarnitine profile and urine organic and amino acids. His UDN genome sequencing (GS) identified a previously unreported de novo STT3A variant (c.1631A > G: p.Asn544Ser). This variant removes a glycosylation site and was predicted to be destabilizing by structural biology modeling. The patient was formally diagnosed by the UDN Metabolomics Core as having an abnormal transferrin profile indicative of CDG type Iw through metabolomic profiling. We report here an affected male with phenotypic, molecular, and metabolic findings consistent with CDG type Iw due to a heterozygous STT3A variant. This case highlights the importance of further testing of individuals with the phenotypic and metabolic findings of an AR disorder who are heterozygous for a single disease-causing allele and can be shown to have a new AD form of the disorder that represents clinical heterogeneity.

BackgroundThe number of known inherited metabolic diseases (IMDs) has been expanding, and the rate of diagnosis is improving with the development of innovative approaches including next generation sequencing (NGS). However, a substantial proportion of IMDs remain undetected by traditional diagnostic approaches. We aim to highlight the spectrum of IMDs diagnosed by the Undiagnosed Diseases Network (UDN) and to learn from the UDN diagnostic processes that were able to detect IMDs.MethodsWe conducted a retrospective analysis of 757 UDN participants diagnosed from 2015 until 2023 using the cohort database, which were divided into a cohort with IMDs (n = 194; 27%) and a cohort whose phenotypes were not explained by an IMD (n = 563; 73%), based on the International Classification of Inherited Metabolic Disorders (ICIMD). Then, we divided the causes of the metabolic 194 diagnoses into seven groups that included all the ICIMD categories. We inspected which clinical and laboratory approaches contributed to a final UDN diagnosis. We also present a UDN case example from each group to highlight the diagnostic yields that resulted from combining newer diagnostic approaches in the UDN and illustrate potential pitfalls of current NGS methods.ResultsThese 194 cases of IMDs included examples from 21/25 (84%) of the ICIMD categories. Of the UDN subjects 164/194 (85%) were diagnosed with IMDs through NGS.ConclusionThe spectrum of IMDs detected in the UDN cohort is large and growing and appropriate use of newer multiple diagnostic approaches should further increase diagnosis of IMDs that are presently missed by the traditional laboratory screening methods.

The Undiagnosed Disease Network (UDN) is comprised of clinical and research experts collaborating to diagnose rare disease. The UDN is funded by the National Institutes of Health and includes 12 different clinical sites (About Us, 2022). Here we highlight the success of collaborative efforts within the UDN Clinical Site at Vanderbilt University Medical Center (VUMC) in utilizing a cohort of experts in bioinformatics, structural biology, and genetics specialists in diagnosing rare disease. Our UDN team identified a de novo mosaic CACNA1D variant c.2299T>C in a 5-year-old female with a history of global developmental delay, dystonia, dyskinesis, and seizures. Using a collaborative multidisciplinary approach, our VUMC UDN team diagnosed the participant with Primary Aldosteronism, Seizures, and Neurologic abnormalities (PASNA) OMIM: 615474 due to a rare mosaic CACNA1D variant (O'Neill, 2013). Interestingly, this patient was mosaic, a phenotypic trait previously unreported in PASNA cases. This report highlights the importance of a multidisciplinary approach in diagnosing rare disease.

Purpose:Variants in SLC6A1 result in a rare neurodevelopmental disorder characterized by a variable clinical presentation of symptoms including developmental delay, epilepsy, motor dysfunction, and autism spectrum disorder. SLC6A1 haploinsufficiency has been confirmed as the predominant pathway of SLC6A1-related neurodevelopmental disorders (NDDs), however, the molecular mechanism underlying the variable clinical presentation remains unclear. Methods:Here, through work of the Undiagnosed Diseases Network, we identify an undiagnosed individual with an inherited p.(A334S) variant of uncertain significance. To resolve this case and better understand the variable expressivity with SLC6A1, we assess the phenotypes of the proband with a cohort of cases diagnosed with SLC6A1-related NDDs. We then create an allelic series in the Drosophila melanogaster to functionally characterize case variants. Results:We identify significant clinical overlap between the unsolved case and confirmed cases of SLC6A1-related NDDs and find a mild to severe clinical presentation associated with missense variants. We confirm phenotypes in flies expressing SLC6A1 variants consistent with a partial loss-of-function mechanism. Conclusion:We conclude that the p.(A334S) variant is a hypomorphic allele and begin to elucidate the underlying variability in SLC6A1-related NDDs. These insights will inform clinical diagnosis, prognosis, treatment and inform therapeutic design for those living with SLC6A1-related NDDs.

"Peripheral Blood Telomere Attrition in Persons at Risk for Familial Pulmonary Fibrosis." American Journal of Respiratory and Critical Care Medicine, 0(ja), pp. –

The contribution of mosaicism to diagnosed genetic disease and presumed de novo variants (DNV) is under investigated. We determined the contribution of mosaic genetic disease (MGD) and diagnosed parental mosaicism (PM) in parents of offspring with reported DNV (in the same variant) in the (1) Undiagnosed Diseases Network (UDN) (N = 1946) and (2) in 12,472 individuals electronic health records (EHR) who underwent genetic testing at an academic medical center. In the UDN, we found 4.51% of diagnosed probands had MGD, and 2.86% of parents of those with DNV exhibited PM. In the EHR, we found 6.03% and 2.99% and (of diagnosed probands) had MGD detected on chromosomal microarray and exome/genome sequencing, respectively. We found 2.34% (of those with a presumed pathogenic DNV) had a parent with PM for the variant. We detected mosaicism (regardless of pathogenicity) in 4.49% of genetic tests performed. We found a broad phenotypic spectrum of MGD with previously unknown phenotypic phenomena. MGD is highly heterogeneous and provides a significant contribution to genetic diseases. Further work is required to improve the diagnosis of MGD and investigate how PM contributes to DNV risk.

Nuclear receptor subfamily 2 group F member 2 (NR2F2 or COUP-TF2) encodes a transcription factor which is expressed at high levels during mammalian development. Rare heterozygous Mendelian variants in NR2F2 were initially identified in individuals with congenital heart disease (CHD), then subsequently in cohorts of congenital diaphragmatic hernia (CDH) and 46,XX ovotesticular disorders/differences of sexual development (DSD); however, the phenotypic spectrum associated with pathogenic variants in NR2F2 remains poorly characterized. Currently, less than 40 individuals with heterozygous pathogenic variants in NR2F2 have been reported. Here, we review the clinical and molecular details of 17 previously unreported individuals with rare heterozygous NR2F2 variants, the majority of which were de novo. Clinical features were variable, including intrauterine growth restriction (IUGR), CHD, CDH, genital anomalies, DSD, developmental delays, hypotonia, feeding difficulties, failure to thrive, congenital and acquired microcephaly, dysmorphic facial features, renal failure, hearing loss, strabismus, asplenia, and vascular malformations, thus expanding the phenotypic spectrum associated with NR2F2 variants. The variants seen were predicted loss of function, including a nonsense variant inherited from a mildly affected mosaic mother, missense and a large deletion including the NR2F2 gene. Our study presents evidence for rare, heterozygous NR2F2 variants causing a highly variable syndrome of congenital anomalies, commonly associated with heart defects, developmental delays/intellectual disability, dysmorphic features, feeding difficulties, hypotonia, and genital anomalies. Based on the new and previous cases, we provide clinical recommendations for evaluating individuals diagnosed with an NR2F2-associated disorder.

CADM3 has been recently reported causing a rare axonal Charcot-Marie-Tooth disease in three independent Caucasian families carrying a recurrent change. We describe the first alternative causative mutation in CADM3 in a family from black African and also observed de novo in a patient of Caucasian ancestry. The disease inheritance was consistent with autosomal dominant and sporadic patterns, respectively. Eight patients and their relatives were enroled from both families. The mean age at diagnosis was 33.9 years, and walking difficulty was commonly the first symptom. Neurological examination showed distal muscle weakness and atrophy, sensory loss and foot and hand deformities. A high clinical variability was noted, but as seen in CADM3-associated neuropathy, symptoms were more pronounced in the arms in some patients. Nerve conduction studies showed no response in most of the examined nerves, and an axonal type of neuropathy, where recorded. Whole exome sequencing revealed a novel missense variant (c.1102G>T; Gly368Cys) in CADM3, segregating with the disease. Functional analyses showed a significant decrease in CADM3-Gly368Cys protein levels in the membrane and major structural changes in its predicted secondary structure. Therefore, we extend the genotype spectrum of CADM3, underlining the need for genetic studies in underrepresented populations like in Africa.

Rationale and Objectives: Up to 20% of idiopathic interstitial lung disease is familial, referred to as familial pulmonary fibrosis (FPF). An integrated analysis of FPF genetic risk was performed by comprehensively evaluating for genetic rare variants (RVs) in a large cohort of FPF kindreds. Methods: Whole-exome sequencing and/or candidate gene sequencing from affected individuals in 569 FPF kindreds was performed, followed by cosegregation analysis in large kindreds, gene burden analysis, gene-based risk scoring, cell-type enrichment analysis, and coexpression network construction. Measurements and Main Results: It was found that 14.9-23.4% of genetic risk in kindreds could be explained by RVs in genes previously linked to FPF, predominantly telomererelated genes. New candidate genes were identified in a small number of families-including SYDE1, SERPINB8, GPR87, and NETO1-and tools were developed for evaluation and prioritization of RV-containing genes across kindreds. Several pathways were enriched for RV-containing genes in FPF, including focal adhesion and mitochondrial complex I assembly. By combining single-cell transcriptomics with prioritized candidate genes, expression of RV-containing genes was discovered to be enriched in smooth muscle cells, type II alveolar epithelial cells, and endothelial cells. Conclusions: In the most comprehensive FPF genetic study to date, the prevalence of RVs in known FPF-related genes was defined, and new candidate genes and pathways relevant to FPF were identified. However, new RV-containing genes shared across multiple kindreds were not identified, thereby suggesting that heterogeneous genetic variants involving a variety of genes and pathways mediate genetic risk in most FPF kindreds.