Clinical and genetic characteristics and natural history of Finnish families with familial exudative vitreoretinopathy due to pathogenic FZD4 variants.

PURPOSE: To report clinical and genetic characteristics of familial exudative vitreoretinopathy (FEVR) in the Finnish population. METHODS: Detailed clinical and genetic data of 35 individuals with heterozygous pathogenic variants in FZD4 were gathered and analysed. RESULTS: Thirty-two individuals with FZD4 c.313A>G variant and three individuals with FZD4 c.40_49del were included in the study. The clinical phenotype was variable even among family members with the same FZD4 variant. Only 34% (N = 12/35) of variant-positive individuals had been clinically diagnosed with FEVR. The median age of the onset of symptoms was 2.3 years, ranging between 0 to 25 years. Median visual acuity was 0.1 logMAR (0.8 Snellen decimal), ranging between light perception and -0.1 logMAR (1.25 Snellen decimal). Most (N = 33/35, 94%) were classified as not visually impaired. Despite unilateral visual loss present in some, they did not meet the criteria of visual impairment according to the WHO classification. Two study patients (N = 2/35, 6%) had severe visual impairment. The most common FEVR stage in study patient's eyes (N = 28/70 eyes, 40%) was FEVR stage 1, that is, avascular periphery or abnormal vascularisation. Most of FZD4-variant-positive study patient's eyes (N = 31/50 eyes, 62%) were myopic. Two individuals presented with persistent hyperplastic primary vitreous expanding the phenotypic spectrum of FEVR. Shared haplotypes extending approximately 0.9 Mb around the recurrent FZD4 c.313A>G variant were identified. CONCLUSION: Most study patients were unaffected or had mild clinical manifestations by FEVR. Myopia seemed to be overly common in FZD4-variant-positive individuals.

Another Brick in the Wall of Tear Film Insights Added Through the Total Synthesis and Biophysical Profiling of anteiso-Branched Wax and Cholesteryl Esters.

The tear film lipid layer (TFLL) plays a vital part in maintenance of ocular health and represents a unique biological barrier comprising unusual and specialized lipid classes and species. The wax and cholesteryl esters (WEs and CEs) constitute roughly 80-90% of the TFLL. The majority of species in these lipid classes are branched and it is therefore surprising that the synthesis and properties of the second largest category of species, i.e., the anteiso-branched species, remain poorly characterized. In this study, we have developed a total synthesis route and completed a detailed NMR spectroscopic characterization of two common anteiso-branched species, namely: (22S)-22-methyltetracosanyl oleate and cholesteryl (22'S)-22'-methyltetracosanoate. In addition, we have studied their structural properties in the bulk state by wide-angle and small-angle X-ray scattering and their behavior at the aqueous interface using Langmuir monolayer techniques. A comparison to the properties displayed by iso-branched and straight-chain analogues indicate that branching patterns lead to distinct properties in the CE and WE lipid classes. Overall, this study complements the previous work in the field and adds another important brick in the tear film insights wall.

Evidence for the additivity of rare and common variant burden throughout the spectrum of intellectual disability.

Intellectual disability (ID) is a common disorder, yet there is a wide spectrum of impairment from mild to profoundly affected individuals. Mild ID is seen as the low extreme of the general distribution of intelligence, while severe ID is often seen as a monogenic disorder caused by rare, pathogenic, highly penetrant variants. To investigate the genetic factors influencing mild and severe ID, we evaluated rare and common variation in the Northern Finland Intellectual Disability cohort (n = 1096 ID patients), a cohort with a high percentage of mild ID (n = 550) and from a population bottleneck enriched in rare, damaging variation. Despite this enrichment, we found only a small percentage of ID was due to recessive Finnish-enriched variants (0.5%). A larger proportion was linked to dominant variation, with a significant burden of rare, damaging variation in both mild and severe ID. This rare variant burden was enriched in more severe ID (p = 2.4e-4), patients without a relative with ID (p = 4.76e-4), and in those with features associated with monogenic disorders. We also found a significant burden of common variants associated with decreased cognitive function, with no difference between mild and more severe ID. When we included common and rare variants in a joint model, the rare and common variants had additive effects in both mild and severe ID. A multimodel inference approach also found that common and rare variants together best explained ID status (ΔAIC = 16.8, ΔBIC = 10.2). Overall, we report evidence for the additivity of rare and common variant burden throughout the spectrum of intellectual disability.

Does Corneal Topography Using 3-Dimensional Optical Coherence Tomography Suggest Different Subtypes of Terrien Marginal Degeneration?

PURPOSE: The aim of this study was to analyze corneal topography relative to astigmatism, higher order aberrations, and corneal curvatures in Terrien marginal degeneration using 3-dimensional anterior-segment optical coherence tomography. METHODS: Twenty-nine eyes of 15 Finnish patients from a tertiary referral center had topographic axial power maps classified into 4 patterns by visual grading: crab claw (CC), mixed (M), arcuate (A), and normal. Regular astigmatism, keratometry, higher order aberrations, maximal corneal thinning, apex thickness, and curvature changes relative to best fit sphere toward maximal peripheral thinning were compared. RESULTS: Four, 9, and 12 eyes were classified as CC, M, and A, respectively; 1 as normal with clinical disease; and 3 as normal with unilateral disease. Median follow-up was 2.3 (range, 0-7.2) years. Three eyes changed pattern. Patients with the CC pattern were the youngest when diagnosed, progressed more rapidly, exhibited cavities in superior quadrants with anterior bulging, and had greater higher order posterior aberrations. Patients with the M pattern were older, progressed slower, and showed superonasal asymmetric corneal steepening extending centrally, often with asymmetric bow tie. Patients with pattern A showed little progression and were the oldest when diagnosed, with maximal corneal thinning equally in all quadrants. According to the Wang classification, the median stage was 4, 2, and 2 in CC, M, and A patterns, respectively, whereas it was always 2 by the Süveges classification. CONCLUSIONS: Terrien marginal degeneration is characterized by distinct corneal topographic patterns that differ in tomographic features, suggesting existence of subtypes in addition to different stages of disease. Patients representing CC and M patterns might benefit from more frequent monitoring.

Early-Stage Development of an Anti-Evaporative Liposomal Formulation for the Potential Treatment of Dry Eyes.

Dry eye disease (DED), the most common ocular disorder, reduces the quality of life for hundreds of millions of people annually. In healthy eyes, the tear film lipid layer (TFLL) stabilizes the tear film and moderates the evaporation rate of tear fluid. In >80% of DED cases, these central features are compromised leading to tear film instability and excessive evaporation of tear fluid. Herein we assess the potential of liposomal formulations featuring phosphatidylcholines and tailored lipid species from the wax ester and O-acyl-ω-hydroxy fatty acid categories in targeting this defect. The developed lead formulation displays good evaporation-resistant properties and respreadability over compression-expansion cycles in our Langmuir model system and a promising safety and efficacy profile in vitro. Preclinical in vivo studies will in the future be required to further assess and validate the potential of this concept in the treatment of DED.

Exome sequencing identified rare recurrent copy number variants and hereditary breast cancer susceptibility.

Copy number variants (CNVs) are a major source of genetic variation and can disrupt genes or affect gene dosage. They are known to be causal or underlie predisposition to various diseases. However, the role of CNVs in inherited breast cancer susceptibility has not been thoroughly investigated. To address this, we performed whole-exome sequencing based analysis of rare CNVs in 98 high-risk Northern Finnish breast cancer cases. After filtering, selected candidate alleles were validated and characterized with a combination of orthogonal methods, including PCR-based approaches, optical genome mapping and long-read sequencing. This revealed three recurrent alterations: a 31 kb deletion co-occurring with a retrotransposon insertion (delins) in RAD52, a 13.4 kb deletion in HSD17B14 and a 64 kb partial duplication of RAD51C. Notably, all these genes encode proteins involved in pathways previously identified as essential for breast cancer development. Variants were genotyped in geographically matched cases and controls (altogether 278 hereditary and 1983 unselected breast cancer cases, and 1229 controls). The RAD52 delins and HSD17B14 deletion both showed significant enrichment among cases with indications of hereditary disease susceptibility. RAD52 delins was identified in 7/278 cases (2.5%, P = 0.034, OR = 2.86, 95% CI = 1.10-7.45) and HSD17B14 deletion in 8/278 cases (2.9%, P = 0.014, OR = 3.28, 95% CI = 1.31-8.23), the frequency of both variants in the controls being 11/1229 (0.9%). This suggests a role for RAD52 and HSD17B14 in hereditary breast cancer susceptibility. The RAD51C duplication was very rare, identified only in 2/278 of hereditary cases and 2/1229 controls (P = 0.157, OR = 4.45, 95% CI = 0.62-31.70). The identification of recurrent CNVs in these genes, and especially the relatively high frequency of RAD52 and HSD17B14 alterations in the Finnish population, highlights the importance of studying CNVs alongside single nucleotide variants when searching for genetic factors underlying hereditary disease predisposition.

Evaluating the role of CHEK2 p.(Asp438Tyr) allele in inherited breast cancer predisposition.

CHEK2 is a well-established breast cancer susceptibility gene. The most frequent pathogenic CHEK2 variant is 1100delC, a loss-of-function mutation conferring 2-fold risk for breast cancer. This gene also harbors other rare variants encountered in the clinical gene panels for hereditary cancer. One of these is CHEK2 c.1312 G > T, p.(Asp438Tyr) in the kinase domain of the protein, but due to its rarity its clinical significance for breast cancer predisposition has remained unclear. Here, we tested the prevalence of CHEK2 p.(Asp438Tyr) allele showing enrichment in the Northern Finnish population, in a total of 2284 breast cancer patients from this geographical region. Genotyping was performed for DNA samples extracted from peripheral blood using high-resolution melt analysis. Fourteen CHEK2 p.(Asp438Tyr) carriers were identified (14/2284, 0.6%, P = 0.67): two in the cohort of breast cancer cases with the indication of inherited disease susceptibility (2/281, 0.7%, P = 1.00) and twelve in the breast cancer cohort unselected for the family history of disease and age at disease onset (12/2003, 0.6%, P = 0.66). This frequency did not differ from the frequency in the general population (10/1299, 0.8%). No CHEK2 p.(Asp438Tyr) homozygotes were identified. Our results indicate that CHEK2 p.(Asp438Tyr) carriers do not have an increased risk for breast cancer and the classification of the CHEK2 p.(Asp438Tyr) variant can be changed from the variant of uncertain significance (VUS) to likely benign for breast cancer.

Biophysical profiling of synthetic ultra-long tear film lipids.

The tear film lipid layer (TFLL) is a unique biological membrane of importance to the maintenance of ocular surface health. The underlying factors at play, e.g. the ability to retard evaporation and offer protection from the environment, are all closely connected to the properties of individual lipid components and their interplay. The TFLL contains unique ultra-long polar lipid species such as O-acyl-ω-hydroxy fatty acids, type I-St diesters and type II diesters, which are considered important for its proper function. Herein, we have synthesized model compounds from these categories and studied their biophysical and surface rheological properties at the aqueous interface. Altogether, we provide insights on the distinct biophysical profiles of these lipid classes and discuss how their interplay may affect the structure and function of the TFLL.

Psychiatric symptoms in Salla disease.

Salla disease (SD) is a rare lysosomal storage disorder characterised by intellectual disability ataxia, athetosis, nystagmus, and central nervous system demyelination. Although the neurological spectrum of SD's clinical phenotype is well defined, psychotic symptoms in SD remain unreported. We reviewed the presence of psychiatric symptoms in patients diagnosed with SD. Medical records of all SD patients at Oulu University Hospital during the years 1982-2015 were systematically reviewed to evaluate the presence of psychiatric symptoms. Psychiatric symptoms were frequently associated with SD (10/24, 42%), and two patients were described as developing psychosis as adolescents. We reported their clinical characteristics in detail and assessed the prevalence of psychiatric symptoms in a cohort of 24 patients. Other psychiatric factors associated with SD were sleeping disorders (8/24, 32%), aggressive behaviour disorders or restlessness (6/24, 25%), and off-label antipsychotic medication (4/24, 17%). This report expands the knowledge of the phenotypic spectrum of SD and demonstrates the importance of recognising the possibility of psychiatric symptoms, including psychosis, in persons with SD.

Truncating TINF2 p.Tyr312Ter variant and inherited breast cancer susceptibility.

TINF2 is a critical subunit of the shelterin complex, which protects and maintains the length of telomeres. Pathogenic missense and truncating TINF2 mutations are causative for dyskeratosis congenita (DC), a rare, dominantly inherited bone marrow failure syndrome characterized by mucocutaneous abnormalities and cancer predisposition. Recent reports indicate that specific TINF2 truncating mutations act as high penetrance cancer predisposition alleles outside DC context, including breast cancer in their tumor spectrum. Here, we have evaluated the role of germline mutations in TINF2 and other shelterin genes in inherited breast cancer susceptibility using exome sequencing data from 98 Northern Finnish breast cancer cases with indication of inherited disease predisposition as a discovery cohort. A single protein truncating variant, TINF2 p.Tyr312Ter, was identified in one of the cases (1/98), and four more carriers were observed in the subsequently genotyped unselected breast cancer cohort (4/1904). None of the carriers were reported to have DC. TINF2 p.Tyr312Ter resulted in stable short form of mRNA transcript, and normal telomere length has been indicated by a recent report. Although recurrent in cases (total of 5/2095), TINF2 p.Tyr312Ter is also present in Finnish population controls (8/12,517), and the observed 4-fold higher frequency in cases falls at most into the range of moderate breast cancer risk alleles (OR 3.74, 95% CI 1.22-11.45, p = 0.029). Current results indicate that not all TINF2 truncating variants are high cancer risk alleles and add further evidence that different TINF2 mutations can have very diverse effects on the disease phenotype.

Sex Steroid Hormone Analysis in Human Tear Fluid Using a Liquid Chromatography-Mass Spectrometry Method.

The marked sexual dimorphism prevalent in inflammatory/autoimmune diseases is mostly due to sex hormone actions. One common eye disease that disproportionately affects women is dry eye. Thus, our aim was to optimise our highly sensitive liquid chromatography-tandem mass spectrometry method for steroid hormone quantification in tear fluid (TF). We used tears and matched serum samples from 10 heathy individuals. Estrone, estradiol testosterone, progesterone, androstenedione, and dehydroepiandrosterone, were quantified with an HPLC coupled with a Triple Quad 5500 MS. Estrone was measured in 80% of female and 20% of male TF samples (mean ± SD, 68.9 ± 62.2 pmol/L), whereas estradiol was undetectable in tears. Progesterone was identified in half of the female tear samples (2.91 ± 3.47 nmol/L) but in none of the male samples, whereas testosterone was quantifiable only in male tears (0.24 ± 0.1 nmol/L). TF hormone levels were, on average, from 1.4% to 55% of systemic values. Estrone, progesterone, and testosterone levels in tears correlated with the matching serum samples (r = 0.82, 0.79, and 0.85, respectively), but androstenedione and dehydroepiandrosterone showed no correlations. Our LC-MS/MS method could detect five out of the six steroid hormones studied in individual human TF samples and could therefore be used to analyse the role of sex steroids in eye diseases.

Clinical and Genetic Characteristics of Finnish Patients with Autosomal Recessive and Dominant Non-Syndromic Hearing Loss Due to Pathogenic TMC1 Variants.

Sensorineural hearing loss (SNHL) is one of the most common sensory deficits worldwide, and genetic factors contribute to at least 50−60% of the congenital hearing loss cases. The transmembrane channel-like protein 1 (TMC1) gene has been linked to autosomal recessive (DFNB7/11) and autosomal dominant (DFNA36) non-syndromic hearing loss, and it is a relatively common genetic cause of SNHL. Here, we report eight Finnish families with 11 affected family members with either recessively inherited homozygous or compound heterozygous TMC1 variants associated with congenital moderate-to-profound hearing loss, or a dominantly inherited heterozygous TMC1 variant associated with postlingual progressive hearing loss. We show that the TMC1 c.1534C>T, p.(Arg512*) variant is likely a founder variant that is enriched in the Finnish population. We describe a novel recessive disease-causing TMC1 c.968A>G, p.(Tyr323Cys) variant. We also show that individuals in this cohort who were diagnosed early and received timely hearing rehabilitation with hearing aids and cochlear implants (CI) have reached good speech perception in noise. Comparison of the genetic data with the outcome of CI rehabilitation increases our understanding of the extent to which underlying pathogenic gene variants explain the differences in CI rehabilitation outcomes.

On the importance of chain branching in tear film lipid layer wax and cholesteryl esters.

The tear film lipid layer (TFLL) is important to the maintenance of ocular surface health. Surprisingly, information on the individual roles of the myriad of unique lipids found therein is limited. The most abundant lipid species are the wax esters (WE) and cholesteryl esters (CE), and, especially their branched analogs. The isolation of these lipid species from the TFLL has proved to be tedious, and as a result, insights on their biophysical profiles and role in the TFLL is currently lacking. Herein, we circumvent these issues by a total synthesis of the most abundant iso-methyl branched WEs and CEs found in the TFLL. Through a detailed characterization of the biophysical properties, by the use of Langmuir monolayer and wide-angle X-ray scattering techniques, we demonstrate that chain branching alters the behavior of these lipid species on multiple levels. Taken together, our results fill an important knowledge gap concerning the structure and function of the TFLL on the whole.

A novel variant in SMG9 causes intellectual disability, confirming a role for nonsense-mediated decay components in neurocognitive development.

Biallelic loss-of-function variants in the SMG9 gene, encoding a regulatory subunit of the mRNA nonsense-mediated decay (NMD) machinery, are reported to cause heart and brain malformation syndrome. Here we report five patients from three unrelated families with intellectual disability (ID) and a novel pathogenic SMG9 c.551 T > C p.(Val184Ala) homozygous missense variant, identified using exome sequencing. Sanger sequencing confirmed recessive segregation in each family. SMG9 c.551T > C p.(Val184Ala) is most likely an autozygous variant identical by descent. Characteristic clinical findings in patients were mild to moderate ID, intention tremor, pyramidal signs, dyspraxia, and ocular manifestations. We used RNA sequencing of patients and age- and sex-matched healthy controls to assess the effect of the variant. RNA sequencing revealed that the SMG9 c.551T > C variant did not affect the splicing or expression level of SMG9 gene products, and allele-specific expression analysis did not provide evidence that the nonsense mRNA-induced NMD was affected. Differential gene expression analysis identified prevalent upregulation of genes in patients, including the genes SMOX, OSBP2, GPX3, and ZNF155. These findings suggest that normal SMG9 function may be involved in transcriptional regulation without affecting nonsense mRNA-induced NMD. In conclusion, we demonstrate that the SMG9 c.551T > C missense variant causes a neurodevelopmental disorder and impacts gene expression. NMD components have roles beyond aberrant mRNA degradation that are crucial for neurocognitive development.

A comprehensive model for measuring real-life cost-effectiveness in eyecare: automation in care and evaluation of system (aces-rwm™).

This paper describes a holistic, yet simple and comprehensible, ecosystem model to deal with multiple and complex challenges in eyecare. It aims at producing the best possible wellbeing and eyesight with the available resources. When targeting to improve the real-world cost-effectiveness, what gets done in everyday practice needs be measured routinely, efficiently and unselectively. Collection of all real-world data of all patients will enable evaluation and comparison of eyecare systems and departments between themselves nationally and internationally. The concept advocates a strategy to optimize real-life effectiveness, sustainability and outcomes of the service delivery in ophthalmology. The model consists of three components: (1) resource-governing principles (i.e., to deal with increasing demand and limited resources), (2) real-world monitoring (i.e., to collect structured real-world data utilizing automation and visualization of clinical parameters, health-related quality of life and costs), and (3) digital innovation strategy (i.e., to evaluate and benchmark real-world outcomes and cost-effectiveness). The core value and strength of the model lies in the consensus and collaboration of all Finnish university eye clinics to collect and evaluate the uniformly structured real-world outcomes data. In addition to ophthalmology, the approach is adaptable to any medical discipline to efficiently generate real-world insights and resilience in health systems.

Tear Film Lipid Layer Structure: Self-Assembly of O-Acyl-ω-hydroxy Fatty Acids and Wax Esters into Evaporation-Resistant Monolayers.

In healthy eyes, the tear film lipid layer (TFLL) is considered to act as an evaporation resistant barrier, which prevents eyes from drying. Seeking to understand the mechanisms behind the evaporation resistance of the TFLL, we studied mixtures of lipid layer wax esters and O-acyl-ω-hydroxy fatty acids. Analyzing their self-assembly and biophysical properties led to new discoveries concerning the structure and function of the TFLL. We discovered how these lipids self-assemble at the air-water interface and form an efficient antievaporative barrier, demonstrating for the first time how the interaction of different tear film lipid species can improve the evaporation resistance compared with individual lipid classes on their own. These results provide a potential mechanism for the evaporation resistance of the lipid layer. In addition, the results serve as a base for the future development of improved dry eye treatments and other applications where the evaporation of water represents a significant challenge.

Epidemiological, clinical, and genetic characteristics of paediatric genetic white matter disorders in Northern Finland.

AIM: To examine the epidemiological, clinical, and genetic characteristics of paediatric patients with genetic white matter disorders (GWMDs) in Northern Finland. METHOD: A longitudinal population-based cohort study was conducted in the tertiary catchment area of Oulu University Hospital from 1990 to 2019. Patients were identified retrospectively by International Statistical Classification of Diseases and Related Health Problems codes in hospital records and prospectively by attending physicians. Inclusion criteria were children younger than 18 years with defined GWMDs or genetic disorders associated with white matter abnormalities (WMAs) on brain magnetic resonance imaging. RESULTS: Eighty patients (mean age [SD] at the end of the study 11y [8y 6mo], range 0-35y; 45 males, 35 females) were diagnosed with a defined GWMD. The cumulative childhood incidence was 30 per 100 000 live births. Regarding those patients with 49 distinct GWMDs, 20% had classic leukodystrophies and 80% had genetic leukoencephalopathies. The most common leukodystrophies were cerebral adrenoleukodystrophy, Krabbe disease, and Salla disease. Additionally, 29 patients (36%) had genetic aetiologies not previously associated with brain WMAs or they had recently characterised GWMDs, including SAMD9L- and NHLRC2-related neurological disorders. Aetiology was mitochondrial in 21% of patients. The most common clinical findings were motor developmental delay, intellectual disability, hypotonia, and spasticity. INTERPRETATION: The cumulative childhood incidence of childhood-onset GWMDs was higher than previously described. Comprehensive epidemiological and natural history data are needed before future clinical trials are undertaken. What this paper adds Forty-nine distinct genetic white matter disorders (GWMDs) were identified, with 20% of cases being classic leukodystrophies. The cumulative childhood incidence of GWMDs was higher than described previously. A considerable proportion (36%) of GWMDs were previously undefined or recently characterised GWMDs. Mitochondrial aetiology was more common (21%) than previously reported.

The Properties and Role of O-Acyl-ω-hydroxy Fatty Acids and Type I-St and Type II Diesters in the Tear Film Lipid Layer Revealed by a Combined Chemistry and Biophysics Approach.

The tear film lipid layer (TFLL) that covers the ocular surface contains several unique lipid classes, including O-acyl-ω-hydroxy fatty acids, type I-St diesters, and type II diesters. While the TFLL represents a unique biological barrier that plays a central role in stabilizing the entire tear film, little is known about the properties and roles of individual lipid species. This is because their isolation from tear samples in sufficient quantities is a tedious task. To provide access to these species in their pure form, and to shed light on their properties, we here report a general strategy for the synthesis and structural characterization of these lipid classes. In addition, we study the organization and behavior of the lipids at the air-tear interface. Through these studies, new insights on the relationship between structural features, such as number of double bonds and the chain length, and film properties, such as spreading and evaporation resistance, were uncovered.

Alternative genomic diagnoses for individuals with a clinical diagnosis of Dubowitz syndrome.

Dubowitz syndrome (DubS) is considered a recognizable syndrome characterized by a distinctive facial appearance and deficits in growth and development. There have been over 200 individuals reported with Dubowitz or a "Dubowitz-like" condition, although no single gene has been implicated as responsible for its cause. We have performed exome (ES) or genome sequencing (GS) for 31 individuals clinically diagnosed with DubS. After genome-wide sequencing, rare variant filtering and computational and Mendelian genomic analyses, a presumptive molecular diagnosis was made in 13/27 (48%) families. The molecular diagnoses included biallelic variants in SKIV2L, SLC35C1, BRCA1, NSUN2; de novo variants in ARID1B, ARID1A, CREBBP, POGZ, TAF1, HDAC8, and copy-number variation at1p36.11(ARID1A), 8q22.2(VPS13B), Xp22, and Xq13(HDAC8). Variants of unknown significance in known disease genes, and also in genes of uncertain significance, were observed in 7/27 (26%) additional families. Only one gene, HDAC8, could explain the phenotype in more than one family (N = 2). All but two of the genomic diagnoses were for genes discovered, or for conditions recognized, since the introduction of next-generation sequencing. Overall, the DubS-like clinical phenotype is associated with extensive locus heterogeneity and the molecular diagnoses made are for emerging clinical conditions sharing characteristic features that overlap the DubS phenotype.