Genome Sequencing for Diagnosing Rare Diseases.

BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).

Clinical and genetic delineation of autosomal recessive and dominant ACTL6B-related developmental brain disorders.

PURPOSE: This study aims to comprehensively delineate the phenotypic spectrum of ACTL6B-related disorders, previously associated with both autosomal recessive and autosomal dominant neurodevelopmental disorders. Molecularly, the role of the nucleolar protein ACTL6B in contributing to the disease has remained unclear. METHODS: We identified 105 affected individuals, including 39 previously reported cases, and systematically analysed detailed clinical and genetic data for all individuals. Additionally, we conducted knockdown experiments in neuronal cells to investigate the role of ACTL6B in ribosome biogenesis. RESULTS: Biallelic variants in ACTL6B are associated with severe-to-profound global developmental delay/intellectual disability (GDD/ID), infantile intractable seizures, absent speech, autistic features, dystonia, and increased lethality. De novo monoallelic variants result in moderate-to-severe GDD/ID, absent speech, and autistic features, while seizures and dystonia were less frequently observed. Dysmorphic facial features and brain abnormalities, including hypoplastic corpus callosum, parenchymal volume loss/atrophy, are common findings in both groups. We reveal that in the nucleolus, ACTL6B plays a crucial role in ribosome biogenesis, in particular in pre-rRNA processing. CONCLUSION: This study provides a comprehensive characterization of the clinical spectrum of both autosomal recessive and dominant forms of ACTL6B-associated disorders. It offers a comparative analysis of their respective phenotypes provides a plausible molecular explanation and suggests their inclusion within the expanding category of 'ribosomopathies'.

KCNT2-Related Disorders: Phenotypes, Functional, and Pharmacological Properties.

OBJECTIVE: Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. METHODS: Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. RESULTS: The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. INTERPRETATION: We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype-phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023;94:332-349.

The constitutional gain-of-function variant p.Glu1099Lys in NSD2 is associated with a novel syndrome.

NSD2 dimethylates histone H3 at lysine 36 (H3K36me2) and is located in the Wolf-Hirschhorn syndrome (WHS) critical region. Recent descriptions have delineated loss-of-function (LoF) variants in NSD2 with a distinct disorder. The oncogenic missense variant p.Glu1099Lys occurs somatically in leukemia and has a gain-of-function (GoF) effect. We describe two individuals carrying p.Glu1099Lys as heterozygous de novo germline variant identified by exome sequencing (ES) of blood DNA and subsequently confirmed in two ectodermal tissues. Clinically, these individuals are characterized by intellectual disability, coarse/ square facial gestalt, abnormalities of the hands, and organomegaly. Public cell lines with NSD2 GoF variants had increased K36me2, DNA promoter methylation, and dysregulated RNA expression. NSD2 GoF caused by p.Glu1099Lys is associated with a novel phenotype different from WHS and Rauch-Steindl syndrome (RAUST).

Genetic and phenotypic spectrum in the NONO-associated syndromic disorder.

The non-POU domain-containing octamer-binding (NONO) protein is involved in multiple steps of gene regulation such as RNA metabolism and DNA repair. Hemizygous pathogenic variants in the NONO gene were confirmed to cause a rare X-linked syndromic disorder. Through our in-house diagnostics and subsequent matchmaking, we identified six unrelated male individuals with pathogenic or likely pathogenic NONO variants. For a detailed comparison, we reviewed all published characterizations of the NONO-associated disorder. The combined cohort consists of 16 live-born males showing developmental delay, corpus callosum anomalies, non-compaction cardiomyopathy and relative macrocephaly as leading symptoms. Seven prenatal literature cases were characterized by cardiac malformations. In this study, we extend the phenotypic spectrum through two more cases with epilepsy as well as two more cases with hematologic anomalies. By RNA expression analysis and structural modeling of a new in-frame splice deletion, we reinforce loss-of-function as the pathomechanism for the NONO-associated syndromic disorder.

Compound-heterozygous GRIN2A null variants associated with severe developmental and epileptic encephalopathy.

We report on an 8-year-old girl with severe developmental and epileptic encephalopathy due to the compound heterozygous null variants p.(Gln661*) and p.(Leu830Profs*2) in GRIN2A resulting in a knockout of the human GluN2A subunit of the N-methyl-D-aspartate receptor. Both parents had less severe GRIN2A-related phenotypes and were heterozygous carriers of the respective null variant. Functional investigations of both variants suggested a loss-of-function effect. This is the first description of an autosomal recessive, biallelic type of GRIN2A-related disorder. Nonetheless, there are marked parallels to two previously published families with severe epileptic encephalopathy due to homozygous null variants in GRIN1 as well as various knockout animal models. Compared to heterozygous null variants, biallelic knockout of either GluN1 or GluN2A is associated with markedly more severe phenotypes in both humans and mice. Furthermore, recent findings enable a potential precision medicine approach targeting GRIN-related disorders due to null variants.

Heterozygous loss-of-function variants significantly expand the phenotypes associated with loss of GDF11.

PURPOSE: Growth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants. METHODS: We present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality. RESULTS: Patients with variants in GDF11 presented with craniofacial (5/6), vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6), and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients' variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) allele whereas the missense variants in our cohort are partial LOF variants. CONCLUSION: GDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues.

GRIN2A-related disorders: genotype and functional consequence predict phenotype.

Alterations of the N-methyl-d-aspartate receptor (NMDAR) subunit GluN2A, encoded by GRIN2A, have been associated with a spectrum of neurodevelopmental disorders with prominent speech-related features, and epilepsy. We performed a comprehensive assessment of phenotypes with a standardized questionnaire in 92 previously unreported individuals with GRIN2A-related disorders. Applying the criteria of the American College of Medical Genetics and Genomics to all published variants yielded 156 additional cases with pathogenic or likely pathogenic variants in GRIN2A, resulting in a total of 248 individuals. The phenotypic spectrum ranged from normal or near-normal development with mild epilepsy and speech delay/apraxia to severe developmental and epileptic encephalopathy, often within the epilepsy-aphasia spectrum. We found that pathogenic missense variants in transmembrane and linker domains (misTMD+Linker) were associated with severe developmental phenotypes, whereas missense variants within amino terminal or ligand-binding domains (misATD+LBD) and null variants led to less severe developmental phenotypes, which we confirmed in a discovery (P = 10-6) as well as validation cohort (P = 0.0003). Other phenotypes such as MRI abnormalities and epilepsy types were also significantly different between the two groups. Notably, this was paralleled by electrophysiology data, where misTMD+Linker predominantly led to NMDAR gain-of-function, while misATD+LBD exclusively caused NMDAR loss-of-function. With respect to null variants, we show that Grin2a+/- cortical rat neurons also had reduced NMDAR function and there was no evidence of previously postulated compensatory overexpression of GluN2B. We demonstrate that null variants and misATD+LBD of GRIN2A do not only share the same clinical spectrum (i.e. milder phenotypes), but also result in similar electrophysiological consequences (loss-of-function) opposing those of misTMD+Linker (severe phenotypes; predominantly gain-of-function). This new pathomechanistic model may ultimately help in predicting phenotype severity as well as eligibility for potential precision medicine approaches in GRIN2A-related disorders.

Full ablative versus fractional ablative laser therapy for Dowling-Degos disease.

Dowling-Degos disease (DDD) is a rare autosomal-dominant genodermatosis with limited treatment possibilities. Although the efficacy of ablative laser therapy has been reported, we sought to examine the efficacy of fractional versus full ablative laser therapy in a female patient with DDD in a split-side report. We treated the lesions on the right side of the patient's upper abdomen with an ablative fractional CO2 laser and the lesions on the left side of the upper abdomen with a full ablative Er:YAG laser (erbium-doped yttrium aluminium garnet laser) three times at monthly intervals. After three laser sessions, the lesions treated with the Er:YAG laser showed a complete response, whereas the fractional CO2 laser treatment was less effective. After the three treatments were performed, the right side of the patient's upper abdomen and portions of her lower abdomen and chest were also treated with the Er:YAG laser in full ablation mode with the same settings. After 1 year of follow up, there was no recurrence observed. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

GestaltMatcher Database - A global reference for the facial phenotypic variability of rare human diseases.

Dysmorphologists sometimes encounter challenges in recognizing disorders due to phenotypic variability influenced by factors such as age and ethnicity. Moreover, the performance of Next Generation Phenotyping Tools such as GestaltMatcher is dependent on the diversity of the training set. Therefore, we developed GestaltMatcher Database (GMDB) - a global reference for the phenotypic variability of rare diseases that complies with the FAIR-principles. We curated dysmorphic patient images and metadata from 2,224 publications, transforming GMDB into an online dynamic case report journal. To encourage clinicians worldwide to contribute, each case can receive a Digital Object Identifier (DOI), making it a citable micro-publication. This resulted in a collection of 2,312 unpublished images, partly with longitudinal data. We have compiled a collection of 10,189 frontal images from 7,695 patients representing 683 disorders. The web interface enables gene- and phenotype-centered queries for registered users (https://db.gestaltmatcher.org/). Despite the predominant European ancestry of most patients (59%), our global collaborations have facilitated the inclusion of data from frequently underrepresented ethnicities, with 17% Asian, 4% African, and 6% with other ethnic backgrounds. The analysis has revealed a significant enhancement in GestaltMatcher performance across all ethnic groups, incorporating non-European ethnicities, showcasing a remarkable increase in Top-1-Accuracy by 31.56% and Top-5-Accuracy by 12.64%. Importantly, this improvement was achieved without altering the performance metrics for European patients. GMDB addresses dysmorphology challenges by representing phenotypic variability and including underrepresented groups, enhancing global diagnostic rates and serving as a vital clinician reference database.

GestaltMatcher Database - A global reference for facial phenotypic variability in rare human diseases.

The most important factor that complicates the work of dysmorphologists is the significant phenotypic variability of the human face. Next-Generation Phenotyping (NGP) tools that assist clinicians with recognizing characteristic syndromic patterns are particularly challenged when confronted with patients from populations different from their training data. To that end, we systematically analyzed the impact of genetic ancestry on facial dysmorphism. For that purpose, we established the GestaltMatcher Database (GMDB) as a reference dataset for medical images of patients with rare genetic disorders from around the world. We collected 10,980 frontal facial images - more than a quarter previously unpublished - from 8,346 patients, representing 581 rare disorders. Although the predominant ancestry is still European (67%), data from underrepresented populations have been increased considerably via global collaborations (19% Asian and 7% African). This includes previously unpublished reports for more than 40% of the African patients. The NGP analysis on this diverse dataset revealed characteristic performance differences depending on the composition of training and test sets corresponding to genetic relatedness. For clinical use of NGP, incorporating non-European patients resulted in a profound enhancement of GestaltMatcher performance. The top-5 accuracy rate increased by +11.29%. Importantly, this improvement in delineating the correct disorder from a facial portrait was achieved without decreasing the performance on European patients. By design, GMDB complies with the FAIR principles by rendering the curated medical data findable, accessible, interoperable, and reusable. This means GMDB can also serve as data for training and benchmarking. In summary, our study on facial dysmorphism on a global sample revealed a considerable cross ancestral phenotypic variability confounding NGP that should be counteracted by international efforts for increasing data diversity. GMDB will serve as a vital reference database for clinicians and a transparent training set for advancing NGP technology.

Clinical Impact of Polygenic Risk Score for Breast Cancer Risk Prediction in 382 Individuals with Hereditary Breast and Ovarian Cancer Syndrome.

Single nucleotide polymorphisms are currently not considered in breast cancer (BC) risk predictions used in daily practice of genetic counselling and clinical management of familial BC in Germany. This study aimed to assess the clinical value of incorporating a 313-variant-based polygenic risk score (PRS) into BC risk calculations in a cohort of German women with suspected hereditary breast and ovarian cancer syndrome (HBOC). Data from 382 individuals seeking counselling for HBOC were analysed. Risk calculations were performed using the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm with and without the inclusion of the PRS. Changes in risk predictions and their impact on clinical management were evaluated. The PRS led to changes in risk stratification based on 10-year risk calculations in 13.6% of individuals. Furthermore, the inclusion of the PRS in BC risk predictions resulted in clinically significant changes in 12.0% of cases, impacting the prevention recommendations established by the German Consortium for Hereditary Breast and Ovarian Cancer. These findings support the implementation of the PRS in genetic counselling for personalized BC risk assessment.

CERT1 mutations perturb human development by disrupting sphingolipid homeostasis.

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Generalized Epilepsy and Myoclonic Seizures in 22q11.2 Deletion Syndrome.

Prompted by the observations of juvenile myoclonic epilepsy (JME) in 22q11.2 deletion syndrome (22q11DS) and recurrent copy number variants in genetic generalized epilepsy (GGE), we searched for further evidence supporting a possible correlation of 22q11DS with GGE and with myoclonic seizures. Through routine diagnostics, we identified 3 novel individuals with the seemingly uncommon combination of 22q11DS and JME. We subsequently screened the literature for reports focussing on the epilepsy phenotype in 22q11DS. We additionally screened a database of 173 22q11DS patients and identified a fourth individual with JME as well as 2 additional cases with GGE. We describe 6 novel and 22 published cases with co-occurrence of 22q11DS and GGE. In many patients, GGE was associated with myoclonic seizures allowing for a diagnosis of JME in at least 6 individuals. Seventeen of the 173 22q11DS cases (10%) had a diagnosis of either focal or generalized epilepsy. In these cases, focal epilepsy could often be attributed to syndrome-associated hypocalcaemia, cerebral bleeds, or structural brain anomalies. However, the cause of GGE remained unclear. In this study, we describe and review 28 individuals with 22q11DS and GGE (especially JME), showing that both disorders frequently co-occur. Compared to the reported prevalence of 15-21%, in our case series only 10% of 22q11DS individuals were found to have epilepsy, often GGE. Since 22q11.2 does not contain convincing GGE candidate genes, we discuss the possibility of an aetiological correlation through a possibly disturbed interaction with the GABAB receptor.

Development of LbL biopolymer capsules as a delivery system for the multilayer-assembled anti-inflammatory substance α1-antitrypsin.

The treatment of chronic inflammation requires new concepts since recent approaches are mostly accompanied by massive side effects. Layer-by-layer (LbL) microcapsules, functionalized with anti-inflammatory substances such as α1-antitrypsin (AT), may avoid major side- and off-target effects thanks to their local application and the sustained delivery of defined amounts of the active agents into polymorphonuclear leukocytes (PMNs). However, LbL microcapsule application in inflamed tissues requires specific design and preparation. High biocompatibility has to be guaranteed by using biopolymers and ensuring the complete dissolution of the particle core. Moreover, off-target effects - such as macrophage-mediated pro-inflammatory signaling - have to be avoided. In our approach, biopolymer-coated CaCO3 particles were used and the core dissolution process was optimized to obtain highly biocompatible, non-aggregated, long-time stable and clearly calcium-free capsules. A fast verification tool was applied to monitor the remaining Ca2+ content. The incubation with macrophages shows reduced pro-inflammatory signaling compared to microparticles. Regarding their performance as a drug delivery system, AT-functionalized capsules showed a high inhibiting capacity towards neutrophil elastase, a major degradative enzyme in chronic inflammation. Consequently, the optimized design and preparation methods described in this study provide the basis for the development of medically applicable LbL carrier systems for active agents in the treatment of inflammatory processes.

Inhibition of human neutrophil elastase by α1-antitrypsin functionalized colloidal microcarriers.

Layer-by-layer (LbL)-coated microcarriers offer a good opportunity as transport systems for active agents into specific cells and tissues. The assembling of oppositely charged polyelectrolytes enables a modular construction of the carriers and therefore an optimized integration and application of drug molecules. Here, we report the multilayer incorporation and transport of α(1)-antitrypsin (AT) by colloidal microcarriers. AT is an anti-inflammatory agent and shows inhibitory effects toward its pro-inflammatory antagonist, human neutrophil elastase (HNE). The highly proteolytic enzyme HNE is released by polymorphonuclear leukocytes (PMNs) during inflammatory processes and can cause host tissue destruction and pain. The high potential of this study is based on a simultaneous intra- and extracellular application of AT-functionalized LbL carriers. Carrier application in PMNs results in significant HNE inhibition within 21 h. Microcarriers phagocytosed by PMNs were time dependently decomposed inside phagolysosomes, which enables the step-by-step release of AT. Here, AT inactivates HNE before being released, which avoids a further HNE concentration increase in the extracellular space and, subsequently, reduces the risk of further tissue destruction. Additionally, AT surface-functionalized microcarriers allow the inhibition of already released HNE in the extracellular space. Finally, this study demonstrates the successful application of LbL carriers for a concurrent extra- and intracellular HNE inhibition aiming the rebalancing of protease and antiprotease concentrations and the subsequent termination of chronic inflammations.