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Abstract Background The 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. Methods We 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. Results These 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. Conclusion The 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.

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.

Abstract Evidence from genetic disorders of CNP signalling suggests that plasma concentrations of CNP are subject to feedback regulation. In subjects with Achondroplasia (Ach), CNP intracellular activity is suppressed and plasma concentrations are raised but the therapeutic impact of exogenous CNP agonists on endogenous CNP is unknown. In this exploratory dose finding and extension study of 28 Ach children receiving Vosoritide over a 5 year period of treatment, endogenous CNP production was assessed using measurements of plasma aminoterminal proCNP (NTproCNP) adjusted for age and sex and normalised as standard deviation score (SDS), and then related to skeletal growth. Before treatment NTproCNP SDS was raised. Within the first 3 months of accelerating growth, levels were significantly reduced. Across the 5 years of sustained growth, levels varied widely and were markedly increased in some subjects during adolescence. Plasma NTproCNP was suppressed at 4 h post-injection in proportion to the prevailing level of hormone resistance as reflected by SDS before injection. We conclude CNP remains subject to regulation during growth promoting doses of Vosoritide. Fall in CNP during accelerating growth is consistent with an indirect feedback whereas the fall at 4 h is likely to be a direct effect from removal of intra cellular CNP resistance.

Summary: Whole-exome sequencing (WES) in the clinic has identified several rare monogenic developmental and epileptic encephalopathies (DEE) caused by ion channel variants. However, WES often fails to provide actionable insight for rare diseases, such as DEEs, due to the challenges of interpreting variants of unknown significance (VUS). Here, we describe a “personalized structural biology” (PSB) approach that leverages recent innovations in the analysis of protein 3D structures to address this challenge. We illustrate this approach in an Undiagnosed Diseases Network (UDN) individual with DEE symptoms and a de novo VUS in KCNC2 (p.V469L), the Kv3.2 voltage-gated potassium channel. A nearby KCNC2 variant (p.V471L) was recently suggested to cause DEE-like phenotypes. Computational structural modeling suggests that both affect protein function. However, despite their proximity, the p.V469L variant is likely to sterically block the channel pore, while the p.V471L variant is likely to stabilize the open state. Biochemical and electrophysiological analyses demonstrate heterogeneous loss-of-function and gain-of-function effects, as well as differential response to 4-aminopyridine treatment. Molecular dynamics simulations illustrate that the pore of the p.V469L variant is more constricted, increasing the energetic barrier for K+ permeation, whereas the p.V471L variant stabilizes the open conformation. Our results implicate variants in KCNC2 as causative for DEE and guide the interpretation of a UDN individual. They further delineate the molecular basis for the heterogeneous clinical phenotypes resulting from two proximal pathogenic variants. This demonstrates how the PSB approach can provide an analytical framework for individualized hypothesis-driven interpretation of protein-coding VUS.

Rare genetic disorders, when considered together, are relatively common. Despite advancements in genetics and genomics technologies as well as increased understanding of genomic function and dysfunction, many genetic diseases continue to be difficult to diagnose. The goal of this Review is to increase the familiarity of genetic testing strategies for non-genetics providers. As genetic testing is increasingly used in primary care, many subspecialty clinics, and various inpatient settings, it is important that non-genetics providers have a fundamental understanding of the strengths and weaknesses of various genetic testing strategies as well as develop an ability to interpret genetic testing results. We provide background on commonly used genetic testing approaches, give examples of phenotypes in which the various genetic testing approaches are used, describe types of genetic and genomic variations, cover challenges in variant identification, provide examples in which next-generation sequencing (NGS) failed to uncover the variant responsible for a disease, and discuss opportunities for continued improvement in the application of NGS clinically. As genetic testing becomes increasingly a part of all areas of medicine, familiarity with genetic testing approaches and result interpretation is vital to decrease the burden of undiagnosed disease.

We present a case study of a 20-year-old male with an unknown neurodegenerative disease who was referred to the Undiagnosed Diseases Network Vanderbilt Medical Center site. A previous metabolic panel showed that the patient had a critical deficiency in nicotinamide intermediates that are generated during the biosynthesis of NAD(H). We followed up on these findings by evaluating the patient’s ability to metabolize nicotinamide. We performed a global metabolic profiling analysis of plasma samples that were collected: (1) under normal fed conditions (baseline), (2) after the patient had fasted, and (3) after he was challenged with a 500 mg nasogastric tube bolus of nicotinamide following the fast. Our findings showed that the patient’s nicotinamide N-methyltransferase (NNMT), a key enzyme in NAD(H) biosynthesis and methionine metabolism, was not functional under normal fed or fasting conditions but was restored in response to the nicotinamide challenge. Altered levels of metabolites situated downstream of NNMT and in neighboring biochemical pathways provided further evidence of a baseline defect in NNMT activity. To date, this is the only report of a critical defect in NNMT activity manifesting in adulthood and leading to neurodegenerative disease. Altogether, this study serves as an important reference in the rare disease literature and also demonstrates the utility of metabolomics as a diagnostic tool for uncharacterized metabolic diseases.

Abstract Background Resources within the Undiagnosed Diseases Network (UDN), such as genome sequencing (GS) and model organisms aid in diagnosis and identification of new disease genes, but are currently difficult to access by clinical providers. While these resources do contribute to diagnoses in many cases, they are not always necessary to reach diagnostic resolution. The UDN experience has been that participants can also receive diagnoses through the thoughtful and customized application of approaches and resources that are readily available in clinical settings. Methods The UDN Genetic Counseling and Testing Working Group collected case vignettes that illustrated how clinically available methods resulted in diagnoses. The case vignettes were classified into three themes; phenotypic considerations, selection of genetic testing, and evaluating exome/GS variants and data. Results We present 12 participants that illustrate how clinical practices such as phenotype‐driven genomic investigations, consideration of variable expressivity, selecting the relevant tissue of interest for testing, utilizing updated testing platforms, and recognition of alternate transcript nomenclature resulted in diagnoses. Conclusion These examples demonstrate that when a diagnosis is elusive, an iterative patient‐specific approach utilizing assessment options available to clinical providers may solve a portion of cases. However, this does require increased provider time commitment, a particular challenge in the current practice of genomics.

Abstract Background Next-generation sequencing of individuals with genetic diseases often detects candidate rare variants in numerous genes, but determining which are causal remains challenging. We hypothesized that the spatial distribution of missense variants in protein structures contains information about function and pathogenicity that can help prioritize variants of unknown significance (VUS) and elucidate the structural mechanisms leading to disease. Results To illustrate this approach in a clinical application, we analyzed 13 candidate missense variants in regulator of telomere elongation helicase 1 (RTEL1) identified in patients with Familial Interstitial Pneumonia (FIP). We curated pathogenic and neutral RTEL1 variants from the literature and public databases. We then used homology modeling to construct a 3D structural model of RTEL1 and mapped known variants into this structure. We next developed a pathogenicity prediction algorithm based on proximity to known disease causing and neutral variants and evaluated its performance with leave-one-out cross-validation. We further validated our predictions with segregation analyses, telomere lengths, and mutagenesis data from the homologous XPD protein. Our algorithm for classifying RTEL1 VUS based on spatial proximity to pathogenic and neutral variation accurately distinguished 7 known pathogenic from 29 neutral variants (ROC AUC = 0.85) in the N-terminal domains of RTEL1. Pathogenic proximity scores were also significantly correlated with effects on ATPase activity (Pearson r = −0.65, p = 0.0004) in XPD, a related helicase. Applying the algorithm to 13 VUS identified from sequencing of RTEL1 from patients predicted five out of six disease-segregating VUS to be pathogenic. We provide structural hypotheses regarding how these mutations may disrupt RTEL1 ATPase and helicase function. Conclusions Spatial analysis of missense variation accurately classified candidate VUS in RTEL1 and suggests how such variants cause disease. Incorporating spatial proximity analyses into other pathogenicity prediction tools may improve accuracy for other genes and genetic diseases.

Abstract Background Family screening of a 48‐year‐old male with recently diagnosed IgG4‐related disease (IgG4‐RD) revealed unanticipated elevations in plasma IgG4 in his two healthy teenaged sons. Methods We performed gene sequencing, immune cell studies, HLA typing, and analyses of circulating cytotoxic CD4+ T lymphocytes and plasmablasts to seek clues to pathogenesis. DNA from a separate cohort of 99 patients with known IgG4‐RD was also sequenced for the presence of genetic variants in a specific gene, FGFBP2. Results The three share a previously unreported heterozygous single base deletion in fibroblast growth factor binding protein type 2 (FGFBP2), which causes a frameshift in the coding sequence. The FGFBP2 protein is secreted by cytotoxic T‐lymphocytes and binds fibroblast growth factor. The variant sequence in the FGFBP2 protein is predicted to form a disordered random coil rather than a helical‐turn‐helix structure, unable to adopt a stable conformation. The proband and the two sons had 5–10‐fold higher numbers of circulating cytotoxic CD4 + T cells and plasmablasts compared to matched controls. The three members also share a homozygous missense common variant in FGFBP2 found in heterozygous form in ~40% of the population. This common variant was found in 73% of an independent, well characterized IgG4‐RD cohort, showing enrichment in idiopathic IgG4‐RD. Conclusions The presence of a shared deleterious variant and homozygous common variant in FGFBP2 in the proband and sons strongly implicates this cytotoxic T cell product in the pathophysiology of IgG4‐RD. The high prevalence of a common FGFBP2 variant in sporadic IgG4‐RD supports the likelihood of participation in disease.

Abstract Background Rare variants (RV) in immunoglobulin mu‐binding protein 2 (IGHMBP2) [OMIM 600502] can cause an autosomal recessive type of Charcot‐Marie‐Tooth (CMT) disease [OMIM 616155], an inherited peripheral neuropathy. Over 40 different genes are associated with CMT, with different possible inheritance patterns. Methods and Results An 11‐year‐old female with motor delays was found to have distal atrophy, weakness, and areflexia without bulbar or sensory findings. Her clinical evaluation was unrevealing. Whole exome sequencing (WES) revealed a maternally inherited IGHMBP2 RV (c.1730T>C) predicted to be pathogenic, but no variant on the other allele was identified. Deletion and duplication analysis was negative. She was referred to the Undiagnosed Disease Network (UDN) for further evaluation. Whole genome sequencing (WGS) confirmed the previously identified IGHMBP2 RV and identified a paternally inherited non‐coding IGHMBP2 RV. This was predicted to activate a cryptic splice site perturbing IGHMBP2 splicing. Reverse transcriptase polymerase chain reaction (RT‐PCR) analysis was consistent with activation of the cryptic splice site. The abnormal transcript was shown to undergo nonsense‐mediated decay (NMD), resulting in halpoinsufficiency. Conclusion This case demonstrates the deficiencies of WES and traditional molecular analyses and highlights the advantages of utilization of WGS and functional studies.

Thermoregulatory processes were compared among three-size groups of free-ranging Komodo dragons (Varanus komodoensis) comprising small (5–20 kg), medium (20–40 gm) and large (40–70 kg) lizards. While all size groups maintained a similar preferred body temperature of ≈35∘C, they achieved this end point differently. Small dragons appeared to engage in sun shuttling behavior more vigorously than large dragons as represented by their greater frequency of daily ambient temperature and light intensity changes as well as a greater activity and overall exposure to the sun. Large dragons were more sedentary and sun shuttled less. Further, they appear to rely to a greater extent on microhabitat selection and employed mouth gaping evaporative cooling to maintain their preferred operational temperature and prevent overheating. A potential ecological consequence of size-specific thermoregulatory habits for dragons is separation of foraging areas. In part, differences in thermoregulation could contribute to inducing shifts in predatory strategies from active foraging in small dragons to more sedentary sit-and-wait ambush predators in adults.