Resolution of SLC6A1 variable expressivity in a multi-generational family using deep clinical phenotyping and Drosophila models.

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.

Homozygous variants in WDR83OS lead to a neurodevelopmental disorder with hypercholanemia.

WD repeat domain 83 opposite strand (WDR83OS) encodes the 106-aa (amino acid) protein Asterix, which heterodimerizes with CCDC47 to form the PAT (protein associated with ER translocon) complex. This complex functions as a chaperone for large proteins containing transmembrane domains to ensure proper folding. Until recently, little was known about the role of WDR83OS or CCDC47 in human disease traits. However, biallelic variants in CCDC47 were identified in four unrelated families with trichohepatoneurodevelopmental syndrome, characterized by a neurodevelopmental disorder (NDD) with liver dysfunction. Three affected siblings in an additional family share a homozygous truncating WDR83OS variant and a phenotype of NDD, dysmorphic features, and liver dysfunction. Using family-based rare variant analyses of exome sequencing (ES) data and case matching through GeneMatcher, we describe the clinical phenotypes of 11 additional individuals in eight unrelated families (nine unrelated families, 14 individuals in total) with biallelic putative truncating variants in WDR83OS. Consistent clinical features include NDD (14/14), facial dysmorphism (13/14), intractable itching (9/14), and elevated bile acids (5/6). Whereas bile acids were significantly elevated in 5/6 of individuals tested, bilirubin was normal and liver enzymes were normal to mildly elevated in all 14 individuals. In three of six individuals for whom longitudinal data were available, we observed a progressive reduction in relative head circumference. A zebrafish model lacking Wdr83os function further supports its role in the nervous system, craniofacial development, and lipid absorption. Taken together, our data support a disease-gene association between biallelic loss-of-function of WDR83OS and a neurological disease trait with hypercholanemia.

Genomic Balancing Act: deciphering DNA rearrangements in the complex chromosomal aberration involving 5p15.2, 2q31.1, and 18q21.32.

Despite extensive research into the genetic underpinnings of neurodevelopmental disorders (NDD), many clinical cases remain unresolved. We studied a female proband with a NDD, mildly dysmorphic facial features, and brain stem hypoplasia on neuroimaging. Comprehensive genomic analyses revealed a terminal 5p loss and a terminal 18q gain in the proband while a diploid copy number for chromosomes 5 and 18 in both parents. Genomic investigations in the proband identified an unbalanced translocation t(5;18) with additional genetic material from chromosome 2 (2q31.3) inserted at the breakpoint, pointing to a complex chromosomal rearrangement (CCR) involving 5p15.2, 2q31.3, and 18q21.32. Breakpoint junction analyses enabled by long-read genome sequencing unveiled the presence of four distinct junctions in the father, who is a carrier of a balanced CCR. The proband inherited from the father both the abnormal chromosome 5 resulting in segmental aneusomies of chr5 (loss) and chr18 (gain) and a der(2) homologue. Evidences suggest a chromoplexy mechanism for this CCR derivation, involving double-strand breaks (DSBs) repaired by non-homologous end joining (NHEJ) or alternative end joining (alt-EJ). The complexity of the CCR and the segregation of homologues elucidate the genetic model for this family. This study demonstrates the importance of combining multiple genomic technologies to uncover genetic causes of complex neurodevelopmental syndromes and to better understand genetic disease mechanisms.

PSMD11 loss-of-function variants correlate with a neurobehavioral phenotype, obesity, and increased interferon response.

Primary proteasomopathies have recently emerged as a new class of rare early-onset neurodevelopmental disorders (NDDs) caused by pathogenic variants in the PSMB1, PSMC1, PSMC3, or PSMD12 proteasome genes. Proteasomes are large multi-subunit protein complexes that maintain cellular protein homeostasis by clearing ubiquitin-tagged damaged, misfolded, or unnecessary proteins. In this study, we have identified PSMD11 as an additional proteasome gene in which pathogenic variation is associated with an NDD-causing proteasomopathy. PSMD11 loss-of-function variants caused early-onset syndromic intellectual disability and neurodevelopmental delay with recurrent obesity in 10 unrelated children. Our findings demonstrate that the cognitive impairment observed in these individuals could be recapitulated in Drosophila melanogaster with depletion of the PMSD11 ortholog Rpn6, which compromised reversal learning. Our investigations in subject samples further revealed that PSMD11 loss of function resulted in impaired 26S proteasome assembly and the acquisition of a persistent type I interferon (IFN) gene signature, mediated by the integrated stress response (ISR) protein kinase R (PKR). In summary, these data identify PSMD11 as an additional member of the growing family of genes associated with neurodevelopmental proteasomopathies and provide insights into proteasomal biology in human health.

Variants of Unknown Significance in Maturity-Onset Diabetes of the Young: High Rate of Conundrum Resolution via Variants of Unknown Significance Reanalysis.

INTRODUCTION: In the era of next-generation sequencing, clinicians frequently encounter variants of unknown significance (VUS) in genetic testing. VUS may be reclassified over time as genetic knowledge grows. We know little about how best to approach VUS in the maturity-onset diabetes of the young (MODY). Therefore, our study aimed to determine the utility of reanalysis of previous VUS results in genetic confirmation of MODY. METHODS: A single-center retrospective chart review identified 85 subjects with a MODY clinical diagnosis. We reanalyzed genetic testing in 10 subjects with 14 unique VUS on MODY genes that was performed >3 years before the study. Demographic, clinical, and biochemical data was collected for those individuals. RESULTS: After reanalysis, 43% (6/14) of the gene variants were reclassified to a different category: 7% (1/14) were "likely pathogenic" and 36% (5/14) were "benign" or "likely benign." The reclassified pathogenic variant was in HNF1A and all reclassified benign variants were in HNF1A, HNF1B and PDX1. The median time between MODY testing and reclassification was 8 years (range: 4-10 years). CONCLUSION: In sum, iterative reanalyzing the genetic data from VUS found during MODY testing may provide high-yield diagnostic information. Further studies are warranted to identify the optimal time and frequency for such analyses.

Exome sequencing implicates ancestry-related Mendelian variation at SYNE1 in childhood-onset essential hypertension.

Childhood-onset essential hypertension (COEH) is an uncommon form of hypertension that manifests in childhood or adolescence and, in the United States, disproportionately affects children of African ancestry. The etiology of COEH is unknown, but its childhood onset, low prevalence, high heritability, and skewed ancestral demography suggest the potential to identify rare genetic variation segregating in a Mendelian manner among affected individuals and thereby implicate genes important to disease pathogenesis. However, no COEH genes have been reported to date. Here, we identify recessive segregation of rare and putatively damaging missense variation in the spectrin domain of spectrin repeat containing nuclear envelope protein 1 (SYNE1), a cardiovascular candidate gene, in 3 of 16 families with early-onset COEH without an antecedent family history. By leveraging exome sequence data from an additional 48 COEH families, 1,700 in-house trios, and publicly available data sets, we demonstrate that compound heterozygous SYNE1 variation in these COEH individuals occurred more often than expected by chance and that this class of biallelic rare variation was significantly enriched among individuals of African genetic ancestry. Using in vitro shRNA knockdown of SYNE1, we show that reduced SYNE1 expression resulted in a substantial decrease in the elasticity of smooth muscle vascular cells that could be rescued by pharmacological inhibition of the downstream RhoA/Rho-associated protein kinase pathway. These results provide insights into the molecular genetics and underlying pathophysiology of COEH and suggest a role for precision therapeutics in the future.

Implementing evidence-based assertions of clinical actionability in the context of secondary findings: Updates from the ClinGen Actionability Working Group.

PURPOSE: The ClinGen Actionability Working Group (AWG) developed an evidence-based framework to generate actionability reports and scores of gene-condition pairs in the context of secondary findings from genome sequencing. Here we describe the expansion of the framework to include actionability assertions. METHODS: Initial development of the actionability rubric was based on previously scored adult gene-condition pairs and individual expert evaluation. Rubric refinement was iterative and based on evaluation, feedback, and discussion. The final rubric was pragmatically evaluated via integration into actionability assessments for 27 gene-condition pairs. RESULTS: The resulting rubric has a 4-point scale (limited, moderate, strong, and definitive) and uses the highest-scoring outcome-intervention pair of each gene-condition pair to generate a preliminary assertion. During AWG discussions, predefined criteria and factors guide discussion to produce a consensus assertion for a gene-condition pair, which may differ from the preliminary assertion. The AWG has retrospectively generated assertions for all previously scored gene-condition pairs and are prospectively asserting on gene-condition pairs under assessment, having completed over 170 adult and 188 pediatric gene-condition pairs. CONCLUSION: The AWG expanded its framework to provide actionability assertions to enhance the clinical value of their resources and increase their utility as decision aids regarding return of secondary findings.

Defining and Reducing Variant Classification Disparities.

Background: Multiplexed Assays of Variant Effects (MAVEs) can test all possible single variants in a gene of interest. The resulting saturation-style data may help resolve variant classification disparities between populations, especially for variants of uncertain significance (VUS). Methods: We analyzed clinical significance classifications in 213,663 individuals of European-like genetic ancestry versus 206,975 individuals of non-European-like genetic ancestry from All of Us and the Genome Aggregation Database. Then, we incorporated clinically calibrated MAVE data into the Clinical Genome Resource's Variant Curation Expert Panel rules to automate VUS reclassification for BRCA1, TP53, and PTEN . Results: Using two orthogonal statistical approaches, we show a higher prevalence ( p ≤5.95e-06) of VUS in individuals of non-European-like genetic ancestry across all medical specialties assessed in all three databases. Further, in the non-European-like genetic ancestry group, higher rates of Benign or Likely Benign and variants with no clinical designation ( p ≤2.5e-05) were found across many medical specialties, whereas Pathogenic or Likely Pathogenic assignments were higher in individuals of European-like genetic ancestry ( p ≤2.5e-05). Using MAVE data, we reclassified VUS in individuals of non-European-like genetic ancestry at a significantly higher rate in comparison to reclassified VUS from European-like genetic ancestry ( p =9.1e-03) effectively compensating for the VUS disparity. Further, essential code analysis showed equitable impact of MAVE evidence codes but inequitable impact of allele frequency ( p =7.47e-06) and computational predictor ( p =6.92e-05) evidence codes for individuals of non-European-like genetic ancestry. Conclusions: Generation of saturation-style MAVE data should be a priority to reduce VUS disparities and produce equitable training data for future computational predictors.

Human embryonic genetic mosaicism and its effects on development and disease.

Nearly every mammalian cell division is accompanied by a mutational event that becomes fixed in a daughter cell. When carried forward to additional cell progeny, a clone of variant cells can emerge. As a result, mammals are complex mosaics of clones that are genetically distinct from one another. Recent high-throughput sequencing studies have revealed that mosaicism is common, clone sizes often increase with age and specific variants can affect tissue function and disease development. Variants that are acquired during early embryogenesis are shared by multiple cell types and can affect numerous tissues. Within tissues, variant clones compete, which can result in their expansion or elimination. Embryonic mosaicism has clinical implications for genetic disease severity and transmission but is likely an under-recognized phenomenon. To better understand its implications for mosaic individuals, it is essential to leverage research tools that can elucidate the mechanisms by which expanded embryonic variants influence development and disease.

Multilocus pathogenic variants contribute to intrafamilial clinical heterogeneity: a retrospective study of sibling pairs with neurodevelopmental disorders.

BACKGROUND: Multilocus pathogenic variants (MPVs) are genetic changes that affect multiple gene loci or regions of the genome, collectively leading to multiple molecular diagnoses. MPVs may also contribute to intrafamilial phenotypic variability between affected individuals within a nuclear family. In this study, we aim to gain further insights into the influence of MPVs on a disease manifestation in individual research subjects and explore the complexities of the human genome within a familial context. METHODS: We conducted a systematic reanalysis of exome sequencing data and runs of homozygosity (ROH) regions of 47 sibling pairs previously diagnosed with various neurodevelopmental disorders (NDD). RESULTS: We found siblings with MPVs driven by long ROH regions in 8.5% of families (4/47). The patients with MPVs exhibited significantly higher FROH values (p-value = 1.4e-2) and larger total ROH length (p-value = 1.8e-2). Long ROH regions mainly contribute to this pattern; the siblings with MPVs have a larger total size of long ROH regions than their siblings in all families (p-value = 6.9e-3). Whereas the short ROH regions in the siblings with MPVs are lower in total size compared to their sibling pairs with single locus pathogenic variants (p-value = 0.029), and there are no statistically significant differences in medium ROH regions between sibling pairs (p-value = 0.52). CONCLUSION: This study sheds light on the significance of considering MPVs in families with affected sibling pairs and the role of ROH as an adjuvant tool in explaining clinical variability within families. Identifying individuals carrying MPVs may have implications for disease management, identification of possible disease risks to different family members, genetic counseling and exploring personalized treatment approaches.

Core planar cell polarity genes VANGL1 and VANGL2 in predisposition to congenital vertebral malformations.

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß-catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss-of-function and dominant-negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock-in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment-dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder.

NODAL variants are associated with a continuum of laterality defects from simple D-transposition of the great arteries to heterotaxy.

BACKGROUND: NODAL signaling plays a critical role in embryonic patterning and heart development in vertebrates. Genetic variants resulting in perturbations of the TGF-ß/NODAL signaling pathway have reproducibly been shown to cause laterality defects in humans. To further explore this association and improve genetic diagnosis, the study aims to identify and characterize a broader range of NODAL variants in a large number of individuals with laterality defects. METHODS: We re-analyzed a cohort of 321 proband-only exomes of individuals with clinically diagnosed laterality congenital heart disease (CHD) using family-based, rare variant genomic analyses. To this cohort we added 12 affected subjects with known NODAL variants and CHD from institutional research and clinical cohorts to investigate an allelic series. For those with candidate contributory variants, variant allele confirmation and segregation analysis were studied by Sanger sequencing in available family members. Array comparative genomic hybridization and droplet digital PCR were utilized for copy number variants (CNV) validation and characterization. We performed Human Phenotype Ontology (HPO)-based quantitative phenotypic analyses to dissect allele-specific phenotypic differences. RESULTS: Missense, nonsense, splice site, indels, and/or structural variants of NODAL were identified as potential causes of heterotaxy and other laterality defects in 33 CHD cases. We describe a recurrent complex indel variant for which the nucleic acid secondary structure predictions implicate secondary structure mutagenesis as a possible mechanism for formation. We identified two CNV deletion alleles spanning NODAL in two unrelated CHD cases. Furthermore, 17 CHD individuals were found (16/17 with known Hispanic ancestry) to have the c.778G > A:p.G260R NODAL missense variant which we propose reclassification from variant of uncertain significance (VUS) to likely pathogenic. Quantitative HPO-based analyses of the observed clinical phenotype for all cases with p.G260R variation, including heterozygous, homozygous, and compound heterozygous cases, reveal clustering of individuals with biallelic variation. This finding provides evidence for a genotypic-phenotypic correlation and an allele-specific gene dosage model. CONCLUSION: Our data further support a role for rare deleterious variants in NODAL as a cause for sporadic human laterality defects, expand the repertoire of observed anatomical complexity of potential cardiovascular anomalies, and implicate an allele specific gene dosage model.

Variants in ZFX are associated with an X-linked neurodevelopmental disorder with recurrent facial gestalt.

Pathogenic variants in multiple genes on the X chromosome have been implicated in syndromic and non-syndromic intellectual disability disorders. ZFX on Xp22.11 encodes a transcription factor that has been linked to diverse processes including oncogenesis and development, but germline variants have not been characterized in association with disease. Here, we present clinical and molecular characterization of 18 individuals with germline ZFX variants. Exome or genome sequencing revealed 11 variants in 18 subjects (14 males and 4 females) from 16 unrelated families. Four missense variants were identified in 11 subjects, with seven truncation variants in the remaining individuals. Clinical findings included developmental delay/intellectual disability, behavioral abnormalities, hypotonia, and congenital anomalies. Overlapping and recurrent facial features were identified in all subjects, including thickening and medial broadening of eyebrows, variations in the shape of the face, external eye abnormalities, smooth and/or long philtrum, and ear abnormalities. Hyperparathyroidism was found in four families with missense variants, and enrichment of different tumor types was observed. In molecular studies, DNA-binding domain variants elicited differential expression of a small set of target genes relative to wild-type ZFX in cultured cells, suggesting a gain or loss of transcriptional activity. Additionally, a zebrafish model of ZFX loss displayed an altered behavioral phenotype, providing additional evidence for the functional significance of ZFX. Our clinical and experimental data support that variants in ZFX are associated with an X-linked intellectual disability syndrome characterized by a recurrent facial gestalt, neurocognitive and behavioral abnormalities, and an increased risk for congenital anomalies and hyperparathyroidism.

Atypical Diabetes: What Have We Learned and What Does the Future Hold?

As our understanding of the pathophysiology of diabetes evolves, we increasingly recognize that many patients may have a form of diabetes that does not neatly fit with a diagnosis of either type 1 or type 2 diabetes. The discovery and description of these forms of "atypical diabetes" have led to major contributions to our collective understanding of the basic biology that drives insulin secretion, insulin resistance, and islet autoimmunity. These discoveries now pave the way to a better classification of diabetes based on distinct endotypes. In this review, we highlight the key biological and clinical insights that can be gained from studying known forms of atypical diabetes. Additionally, we provide a framework for identification of patients with atypical diabetes based on their clinical, metabolic, and molecular features. Helpful clinical and genetic resources for evaluating patients suspected of having atypical diabetes are provided. Therefore, appreciating the various endotypes associated with atypical diabetes will enhance diagnostic accuracy and facilitate targeted treatment decisions.

Expanding the phenotype of PPP1R21-related neurodevelopmental disorder.

PPP1R21 encodes for a conserved protein that is involved in endosomal maturation. Biallelic pathogenic variants in PPP1R21 have been associated with a syndromic neurodevelopmental disorder from studying 13 affected individuals. In this report, we present 11 additional individuals from nine unrelated families and their clinical, radiological, and molecular findings. We identified eight different variants in PPP1R21, of which six were novel variants. Global developmental delay and hypotonia are neurological features that were observed in all individuals. There is also a similar pattern of dysmorphic features with coarse faces as a gestalt observed in several individuals. Common findings in 75% of individuals with available brain imaging include delays in myelination, wavy outline of the bodies of the lateral ventricles, and slight prominence of the bodies of the lateral ventricles. PPP1R21-related neurodevelopmental disorder is associated with a consistent phenotype and should be considered in highly consanguineous individuals presenting with developmental delay/intellectual disability along with coarse facial features.

Decoding complex inherited phenotypes in rare disorders: the DECIPHERD initiative for rare undiagnosed diseases in Chile.

Rare diseases affect millions of people worldwide, and most have a genetic etiology. The incorporation of next-generation sequencing into clinical settings, particularly exome and genome sequencing, has resulted in an unprecedented improvement in diagnosis and discovery in the past decade. Nevertheless, these tools are unavailable in many countries, increasing health care gaps between high- and low-and-middle-income countries and prolonging the "diagnostic odyssey" for patients. To advance genomic diagnoses in a setting of limited genomic resources, we developed DECIPHERD, an undiagnosed diseases program in Chile. DECIPHERD was implemented in two phases: training and local development. The training phase relied on international collaboration with Baylor College of Medicine, and the local development was structured as a hybrid model, where clinical and bioinformatics analysis were performed in-house and sequencing outsourced abroad, due to lack of high-throughput equipment in Chile. We describe the implementation process and findings of the first 103 patients. They had heterogeneous phenotypes, including congenital anomalies, intellectual disabilities and/or immune system dysfunction. Patients underwent clinical exome or research exome sequencing, as solo cases or with parents using a trio design. We identified pathogenic, likely pathogenic or variants of unknown significance in genes related to the patients´ phenotypes in 47 (45.6%) of them. Half were de novo informative variants, and half of the identified variants have not been previously reported in public databases. DECIPHERD ended the diagnostic odyssey for many participants. This hybrid strategy may be useful for settings of similarly limited genomic resources and lead to discoveries in understudied populations.

RNA methyltransferase SPOUT1/CENP-32 links mitotic spindle organization with the neurodevelopmental disorder SpADMiSS.

SPOUT1/CENP-32 encodes a putative SPOUT RNA methyltransferase previously identified as a mitotic chromosome associated protein. SPOUT1/CENP-32 depletion leads to centrosome detachment from the spindle poles and chromosome misalignment. Aided by gene matching platforms, we identified 24 individuals with neurodevelopmental delays from 18 families with bi-allelic variants in SPOUT1/CENP-32 detected by exome/genome sequencing. Zebrafish spout1/cenp-32 mutants showed reduction in larval head size with concomitant apoptosis likely associated with altered cell cycle progression. In vivo complementation assays in zebrafish indicated that SPOUT1/CENP-32 missense variants identified in humans are pathogenic. Crystal structure analysis of SPOUT1/CENP-32 revealed that most disease-associated missense variants mapped to the catalytic domain. Additionally, SPOUT1/CENP-32 recurrent missense variants had reduced methyltransferase activity in vitro and compromised centrosome tethering to the spindle poles in human cells. Thus, SPOUT1/CENP-32 pathogenic variants cause an autosomal recessive neurodevelopmental disorder: SpADMiSS ( SPOUT1 Associated Development delay Microcephaly Seizures Short stature) underpinned by mitotic spindle organization defects and consequent chromosome segregation errors.

Phenotypic heterogeneity associated with KIF21A: Two new cases and review of the literature.

Our understanding of genetic and phenotypic heterogeneity associated with the clinical spectrum of rare diseases continues to expand. Thorough phenotypic descriptions and model organism functional studies are valuable tools in dissecting the biology of the disease process. Kinesin genes are well known to be associated with specific disease phenotypes and a subset of kinesin genes, including KIF21A, have been associated with more than one disease. Here we report two patients with KIF21A variants identified by exome sequencing; one with biallelic variants, supporting a novel KIF21A related syndrome with recessive inheritance and the second report of this condition, and another with a heterozygous de novo variant allele representing a phenotypic expansion of the condition described to date. We provide detailed phenotypic information on both families, including a novel neuropathology finding of neuroaxonal dystrophy associated with biallelic variants in KIF21A. Additionally, we studied the dominant variant in Saccharomyces cerevisiae to assess variant pathogenicity and found that this variant appears to impair protein function. KIF21A associated disease has mounting evidence for phenotypic heterogeneity; further patients and study of an allelic series are required to define the phenotypic spectrum and further explore the molecular etiology for each of these conditions.