Phenotype expansion of heterozygous FOXC1 pathogenic variants toward involvement of congenital anomalies of the kidneys and urinary tract (CAKUT)

PurposeCongenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in childhood and adolescence. We aim to identify novel monogenic causes of CAKUT.MethodsExome sequencing was performed in 550 CAKUT-affected families.ResultsWe discovered seven FOXC1 heterozygous likely pathogenic variants within eight CAKUT families. These variants are either never reported, or present in <5 alleles in the gnomAD database with ~141,456 controls. FOXC1 is a causal gene for Axenfeld–Rieger syndrome type 3 and anterior segment dysgenesis 3. Pathogenic variants in FOXC1 have not been detected in patients with CAKUT yet. Interestingly, mouse models for Foxc1 show severe CAKUT phenotypes with incomplete penetrance and variable expressivity. The FOXC1 variants are enriched in the CAKUT cohort compared with the control. Genotype–phenotype correlations showed that Axenfeld–Rieger syndrome or anterior segment dysgenesis can be caused by both truncating and missense pathogenic variants, and the missense variants are located at the forkhead domain. In contrast, for CAKUT, there is no truncating pathogenic variant, and all variants except one are located outside the forkhead domain.ConclusionWe thereby expanded the phenotype of FOXC1 pathogenic variants toward involvement of CAKUT, which can potentially be explained by allelism.     

Assessment of incidental findings in 232 whole-exome sequences from the Baylor–Hopkins Center for Mendelian Genomics

PurposeIn March 2013 the American College of Medical Genetics and Genomics published a list of 56 genes with the recommendation that pathogenic and likely pathogenic variants detected incidentally by clinical sequencing be reported to patients. As an initial step in determining the practical consequences of this recommendation in the research setting, we searched for variants in these genes in 232 whole-exome sequences from the Baylor-Hopkins Center for Mendelian Genomics.MethodsWe identified rare, nonsynonymous, and splicing single-nucleotide variants and insertions/deletions and assessed variant classification using the Human Gene Mutation, Emory, and ClinVar databases. We analyzed the burden of mutation in each of the 56 genes and determined which variants should be reported to patients.ResultsOur filtering resulted in 249 distinct variants, with a mean of 1.69 variants per individual. Half of these were novel missense mutations not classified by any of the three reference databases. Of 101 variants listed in the Human Gene Mutation Database, 48 were also in ClinVar and 3 were also in Emory; half of these shared variants were classified discordantly between databases. Some genes consistently had greater variation than others. In total, 0.86% of individuals had a reportable incidental variant.ConclusionThese observations demonstrate some current challenges of assessing phenotypic consequences of incidental variants for counseling patients.     

Sequence variants in malignant hyperthermia genes in Iceland: classification and actionable findings in a population database

Malignant hyperthermia (MH) susceptibility is a rare life-threatening disorder that occurs upon exposure to a triggering agent. MH is commonly due to protein-altering variants in RYR1 and CACNA1S. The American College of Medical Genetics and Genomics recommends that when pathogenic and likely pathogenic variants in RYR1 and CACNA1S are incidentally found, they should be reported to the carriers. The detection of actionable variants allows the avoidance of exposure to triggering agents during anesthesia. First, we report a 10-year-old Icelandic proband with a suspected MH event, harboring a heterozygous missense variant NM_000540.2:c.6710G>A r.(6710g>a) p.(Cys2237Tyr) in the RYR1 gene that is likely pathogenic. The variant is private to four individuals within a three-generation family and absent from 62,240 whole-genome sequenced (WGS) Icelanders. Haplotype sharing and WGS revealed that the variant occurred as a somatic mosaicism also present in germline of the proband’s paternal grandmother. Second, using a set of 62,240 Icelanders with WGS, we assessed the carrier frequency of actionable pathogenic and likely pathogenic variants in RYR1 and CACNA1S. We observed 13 actionable variants in RYR1, based on ClinVar classifications, carried by 43 Icelanders, and no actionable variant in CACNA1S. One in 1450 Icelanders carries an actionable variant for MH. Extensive sequencing allows for better classification and precise dating of variants, and WGS of a large fraction of the population has led to incidental findings of actionable MH genotypes.Subject terms: Disease genetics, Genetics research     

Genome sequencing identifies multiple deleterious variants in autism patients with more severe phenotypes

PurposeTo maximize the discovery of potentially pathogenic variants to better understand the diagnostic utility of genome sequencing (GS) and to assess how the presence of multiple risk events might affect the phenotypic severity in autism spectrum disorders (ASD).MethodsGS was applied to 180 simplex and multiplex ASD families (578 individuals, 213 patients) with exome sequencing and array comparative genomic hybridization further applied to a subset for validation and cross-platform comparisons.ResultsWe found that 40.8% of patients carried variants with evidence of disease risk, including a de novo frameshift variant in NR4A2 and two de novo missense variants in SYNCRIP, while 21.1% carried clinically relevant pathogenic or likely pathogenic variants. Patients with more than one risk variant (9.9%) were more severely affected with respect to cognitive ability compared with patients with a single or no-risk variant. We observed no instance among the 27 multiplex families where a pathogenic or likely pathogenic variant was transmitted to all affected members in the family.ConclusionThe study demonstrates the diagnostic utility of GS, especially for multiple risk variants that contribute to the phenotypic severity, shows the genetic heterogeneity in multiplex families, and provides evidence for new genes for follow up.     

Variant recurrence in neurodevelopmental disorders: the use of publicly available genomic data identifies clinically relevant pathogenic missense variants

PurposeNext-generation sequencing has revealed the major impact of de novo variants (DNVs) in developmental disorders (DD) such as intellectual disability, autism, and epilepsy. However, a substantial fraction of these predicted pathogenic DNVs remains challenging to distinguish from background DNVs, notably the missense variants acting via nonhaploinsufficient mechanisms on specific amino acid residues. We hypothesized that the detection of the same missense variation in at least two unrelated individuals presenting with a similar phenotype could be a powerful approach to reveal novel pathogenic variants.MethodsWe looked for variations independently present in both our database of >1200 solo exomes and in denovo-db, a large, publicly available collection of de novo variants identified in patients with DD.ResultsThis approach identified 30 variants with strong evidence of pathogenicity, including variants already classified as pathogenic or probably pathogenic by our team, and also several new variants of interest in known OMIM genes or in novel genes. We identified FEM1B and GNAI2 as good candidate genes for syndromic intellectual disability and confirmed the implication of ACTL6B in a neurodevelopmental disorder.ConclusionAnnotation of local variants with denovo-db can highlight missense variants with high potential for pathogenicity, both facilitating the time-consuming reanalysis process and allowing novel DD gene discoveries.     

eyeVarP: A computational framework for the identification of pathogenic variants specific to eye disease

PurposeDisease-specific pathogenic variant prediction tools that differentiate pathogenic variants from benign have been improved through disease specificity recently. However, they have not been evaluated on disease-specific pathogenic variants compared with other diseases, which would help to prioritize disease-specific variants from several genes or novel genes. Thus, we hypothesize that features of pathogenic variants alone would provide a better model.MethodsWe developed an eye disease–specific variant prioritization tool (eyeVarP), which applied the random forest algorithm to the data set of pathogenic variants of eye diseases and other diseases. We also developed the VarP tool and generalized pipeline to filter missense and insertion-deletion variants and predict their pathogenicity from exome or genome sequencing data, thus we provide a complete computational procedure.ResultseyeVarP outperformed pan disease–specific tools in identifying eye disease–specific pathogenic variants under the top 10. VarP outperformed 12 pathogenicity prediction tools with an accuracy of 95% in correctly identifying the pathogenicity of missense and insertion-deletion variants. The complete pipeline would help to develop disease-specific tools for other genetic disorders.ConclusioneyeVarP performs better in identifying eye disease–specific pathogenic variants using pathogenic variant features and gene features. Implementing such complete computational procedure would significantly improve the clinical variant interpretation for specific diseases.     

Loss-of-function and missense variants in NSD2 cause decreased methylation activity and are associated with a distinct developmental phenotype

PurposeDespite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf–Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood.MethodsWe collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro.ResultsThe core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2’s folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants.ConclusionNSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch–Steindl syndrome after the delineators of this phenotype.     

Genome sequencing as a first-line genetic test in familial dilated cardiomyopathy

PurposeWe evaluated genome sequencing (GS) as an alternative to multigene panel sequencing (PS) for genetic testing in dilated cardiomyopathy (DCM).MethodsForty-two patients with familial DCM underwent PS and GS, and detection rates of rare single-nucleotide variants and small insertions/deletions in panel genes were compared. Loss-of-function variants in 406 cardiac-enriched genes were evaluated, and an assessment of structural variation was performed.ResultsGS provided broader and more uniform coverage than PS, with high concordance for rare variant detection in panel genes. GS identified all PS-identified pathogenic or likely pathogenic variants as well as two additional likely pathogenic variants: one was missed by PS due to low coverage, the other was a known disease-causing variant in a gene not included on the panel. No loss-of-function variants in the extended gene set met clinical criteria for pathogenicity. One BAG3 structural variant was classified as pathogenic.ConclusionOur data support the use of GS for genetic testing in DCM, with high variant detection accuracy and a capacity to identify structural variants. GS provides an opportunity to go beyond suites of established disease genes, but the incremental yield of clinically actionable variants is limited by a paucity of genetic and functional evidence for DCM association.     

A gene-centric strategy for identifying disease-causing rare variants in dilated cardiomyopathy

PurposeWe evaluated strategies for identifying disease-causing variants in genetic testing for dilated cardiomyopathy (DCM).MethodsCardiomyopathy gene panel testing was performed in 532 DCM patients and 527 healthy control subjects. Rare variants in 41 genes were stratified using variant-level and gene-level characteristics.ResultsA majority of DCM cases and controls carried rare protein-altering cardiomyopathy gene variants. Variant-level characteristics alone had limited discriminative value. Differentiation between groups was substantially improved by addition of gene-level information that incorporated ranking of genes based on literature evidence for disease association. The odds of DCM were increased to nearly 9-fold for truncating variants or high-impact missense variants in the subset of 14 genes that had the strongest biological links to DCM (P <0.0001). For some of these genes, DCM-associated variants appeared to be clustered in key protein functional domains. Multiple rare variants were present in many family probands, however, there was generally only one “driver” pathogenic variant that cosegregated with disease.ConclusionRare variants in cardiomyopathy genes can be effectively stratified by combining variant-level and gene-level information. Prioritization of genes based on their a priori likelihood of disease causation is a key factor in identifying clinically actionable variants in cardiac genetic testing.     

The ACMG SF v3.0 gene list increases returnable variant detection by 22% when compared with v2.0 in the ClinSeq cohort

PurposeThe American College of Medical Genetics and Genomics (ACMG) recommends the return of pathogenic and likely pathogenic (P/LP) secondary findings from exome and genome sequencing. The latest version (ACMG secondary finding [SF] v3.0) includes 14 additional genes. We interrogated the ClinSeq cohort for variants in these genes to determine the additional yield in unselected individuals.MethodsExome data from 1473 individuals (60% White, 34% African American or Black, 6% other) were analyzed. We restricted our analyses to coding variants; +1,+2,–1, and –2 splice site variants; and the pathogenic GAA variant, NM_000152.5:c.-32-13T>G. Variants were assessed with slightly modified ACMG/Association of Molecular Pathology guidelines.ResultsA total of 25 P/LP variants were identified. In total, 7 individuals had P/LP variants in genes recommended for return of heterozygous variants, namely HNF1A (1), PALB2 (3), TMEM127 (1), and TTN (2). In total, 4 individuals had a homozygous variant in a gene recommended for biallelic variant return, namely HFE, NM_000410.3(HFE):c.845G>A p.Cys282Tyr. A total of 17 P/LP variants were identified in the heterozygous state in genes recommended only for biallelic variant reporting and were not returned. The frequency of returnable P/LP variants did not significantly differ by race.ConclusionUsing the ACMG SF v3.0, the returnable P/LP variant frequency increased in the ClinSeq cohort by 22%, from 3.4% (n = 50, ACMG SF v2.0) to 4.1% (n = 61, ACMG SF v3.0).     

FOXG1 syndrome: genotype–phenotype association in 83 patients with FOXG1 variants

PurposeThe study aimed at widening the clinical and genetic spectrum and assessing genotype–phenotype associations in FOXG1 syndrome due to FOXG1 variants.MethodsWe compiled 30 new and 53 reported patients with a heterozygous pathogenic or likely pathogenic variant in FOXG1. We grouped patients according to type and location of the variant. Statistical analysis of molecular and clinical data was performed using Fisher’s exact test and a nonparametric multivariate test.ResultsAmong the 30 new patients, we identified 19 novel FOXG1 variants. Among the total group of 83 patients, there were 54 variants: 20 frameshift (37%), 17 missense (31%), 15 nonsense (28%), and 2 in-frame variants (4%). Frameshift and nonsense variants are distributed over all FOXG1 protein domains; missense variants cluster within the conserved forkhead domain. We found a higher phenotypic variability than previously described. Genotype–phenotype association revealed significant differences in psychomotor development and neurological features between FOXG1 genotype groups. More severe phenotypes were associated with truncating FOXG1 variants in the N-terminal domain and the forkhead domain (except conserved site 1) and milder phenotypes with missense variants in the forkhead conserved site 1.ConclusionsThese data may serve for improved interpretation of new FOXG1 sequence variants and well-founded genetic counseling.     

Increased frequency of FBN1 truncating and splicing variants in Marfan syndrome patients with aortic events

PurposeMarfan syndrome is a systemic disorder that typically involves FBN1 mutations and cardiovascular manifestations. We investigated FBN1 genotype–phenotype correlations with aortic events (aortic dissection and prophylactic aortic surgery) in patients with Marfan syndrome.MethodsGenotype and phenotype information from probands (n = 179) with an FBN1 pathogenic or likely pathogenic variant were assessed.ResultsA higher frequency of truncating or splicing FBN1 variants was observed in Ghent criteria–positive patients with an aortic event (n = 34) as compared with all other probands (n = 145) without a reported aortic event (79 vs. 39%; P < 0.0001), as well as Ghent criteria–positive probands (n = 54) without an aortic event (79 vs. 48%; P = 0.0039). Most probands with an early aortic event had a truncating or splicing variant (100% (n = 12) and 95% (n = 21) of patients younger than 30 and 40 years old, respectively). Aortic events occurred at a younger median age in patients with truncating/splicing variants (29 years) as compared with those with missense variants (51 years). A trend toward a higher frequency of truncating/splicing variants in patients with aortic dissection (n = 21) versus prophylactic surgery (n = 13) (85.7 vs. 69.3%; not significant) was observed.ConclusionThese aortic event– and age-associated findings may have important implications for the management of Marfan syndrome patients with FBN1 truncating and splicing variants.     

Genotype-phenotype correlations in RHOBTB2-associated neurodevelopmental disorders

PurposeMissense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy with early-onset seizures and severe intellectual disability.MethodsBy international collaboration, we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders. By western blotting, we investigated the consequences of missense variants in vitro.ResultsIn accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe developmental and epileptic encephalopathy in 16 individuals. Now, we also identified de novo missense variants in the GTPase domain in 6 individuals with apparently more variable neurodevelopmental phenotypes with or without epilepsy. In contrast to variants in the BTB domain region, variants in the GTPase domain do not impair proteasomal degradation of RHOBTB2 in vitro, indicating different functional consequences. Furthermore, we observed biallelic splice-site and truncating variants in 9 families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well.ConclusionBy identifying genotype-phenotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying biallelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2-related phenotypes, including both autosomal dominant and recessive neurodevelopmental disorders.     

Genetic heterogeneity of heritable ectopic mineralization disorders in a large international cohort

PurposeHeritable ectopic mineralization disorders comprise a group of conditions with a broad range of clinical manifestations in nonskeletal connective tissues. We report the genetic findings from a large international cohort of 478 patients afflicted with ectopic mineralization.MethodsSequence variations were identified using a next-generation sequencing panel consisting of 29 genes reported in association with ectopic mineralization. The pathogenicity of select splicing and missense variants was analyzed in experimental systems in vitro and in vivo.ResultsA total of 872 variants of unknown significance as well as likely pathogenic and pathogenic variants were disclosed in 25 genes. A total of 159 distinct variants were identified in 425 patients in ABCC6, the gene responsible for pseudoxanthoma elasticum, a heritable multisystem ectopic mineralization disorder. The interpretation of variant pathogenicity relying on bioinformatic predictions did not provide a consensus. Our in vitro and in vivo functional assessment of 14 ABCC6 variants highlighted this dilemma and provided unambiguous interpretations to their pathogenicity.ConclusionThe results expand the ABCC6 variant repertoire, shed new light on the genetic heterogeneity of heritable ectopic mineralization disorders, and provide evidence that functional characterization in appropriate experimental systems is necessary to determine the pathogenicity of genetic variants.     

The mitochondrial seryl-tRNA synthetase SARS2 modifies onset in spastic paraplegia type 4

PurposeHereditary spastic paraplegia type 4 is extremely variable in age at onset; the same variant can cause onset at birth or in the eighth decade. We recently discovered that missense variants in SPAST, which influences microtubule dynamics, are associated with earlier onset and more severe disease than truncating variants, but even within the early and late-onset groups there remained significant differences in onset. Given the rarity of the condition, we adapted an extreme phenotype approach to identify genetic modifiers of onset.MethodsWe performed a genome-wide association study on 134 patients bearing truncating pathogenic variants in SPAST, divided into early- and late-onset groups (aged ≤15 and ≥45 years, respectively). A replication cohort of 419 included patients carrying either truncating or missense variants. Finally, age at onset was analyzed in the merged cohort (N = 553).ResultsWe found 1 signal associated with earlier age at onset (rs10775533, P = 8.73E-6) in 2 independent cohorts and in the merged cohort (N = 553, Mantel–Cox test, P < .0001). Western blotting in lymphocytes of 20 patients showed that this locus tends to upregulate SARS2 expression in earlier-onset patients.ConclusionSARS2 overexpression lowers the age of onset in hereditary spastic paraplegia type 4. Lowering SARS2 or improving mitochondrial function could thus present viable approaches to therapy.     

Accurate assignment of disease liability to genetic variants using only population data

PurposeThe growing size of public variant repositories prompted us to test the accuracy of pathogenicity prediction of DNA variants using population data alone.MethodsUnder the a priori assumption that the ratio of the prevalence of variants in healthy population vs that in affected populations form 2 distinct distributions (pathogenic and benign), we used a Bayesian method to assign probability to a variant belonging to either distribution.ResultsThe approach, termed Bayesian prevalence ratio (BayPR), accurately parsed 300 of 313 expertly curated CFTR variants: 284 of 296 pathogenic/likely pathogenic variants in 1 distribution and 16 of 17 benign/likely benign variants in another. BayPR produced an area under the receiver operating characteristic curve of 0.99 for 103 functionally confirmed missense CFTR variants, which is equal to or exceeds 10 commonly used algorithms (area under the receiver operating characteristic curve range = 0.54-0.99). Application of BayPR to expertly curated variants in 8 genes associated with 7 Mendelian conditions led to the assignment of a disease-causing probability of ≥80% to 1350 of 1374 (98.3%) pathogenic/likely pathogenic variants and of ≤20% to 22 of 23 (95.7%) benign/likely benign variants.ConclusionIrrespective of the variant type or functional effect, the BayPR approach provides probabilities of pathogenicity for DNA variants responsible for Mendelian disorders using only the variant counts in affected and unaffected population samples.     

Targeted Next‐Generation Sequencing Analysis of 1,000 Individuals with Intellectual Disability

To identify genetic causes of intellectual disability (ID), we screened a cohort of 986 individuals with moderate to severe ID for variants in 565 known or candidate ID‐associated genes using targeted next‐generation sequencing. Likely pathogenic rare variants were found in ～11% of the cases (113 variants in 107/986 individuals: ～8% of the individuals had a likely pathogenic loss‐of‐function [LoF] variant, whereas ～3% had a known pathogenic missense variant). Variants in SETD5, ATRX, CUL4B, MECP2, and ARID1B were the most common causes of ID. This study assessed the value of sequencing a cohort of probands to provide a molecular diagnosis of ID, without the availability of DNA from both parents for de novo sequence analysis. This modeling is clinically relevant as 28% of all UK families with dependent children are single parent households. In conclusion, to diagnose patients with ID in the absence of parental DNA, we recommend investigation of all LoF variants in known genes that cause ID and assessment of a limited list of proven pathogenic missense variants in these genes. This will provide 11% additional diagnostic yield beyond the 10%–15% yield from array CGH alone.     

Genotype-phenotype associations in a large PTEN Hamartoma Tumor Syndrome (PHTS) patient cohort

BackgroundPathogenic PTEN germline variants cause PTEN Hamartoma Tumor Syndrome (PHTS), a rare disease with a variable genotype and phenotype. Knowledge about these spectra and genotype-phenotype associations could help diagnostics and potentially lead to personalized care. Therefore, we assessed the PHTS genotype and phenotype spectrum in a large cohort study.MethodsInformation was collected of 510 index patients with pathogenic or likely pathogenic (LP/P) PTEN variants (n = 467) or variants of uncertain significance. Genotype-phenotype associations were assessed using logistic regression analyses adjusted for sex and age.ResultsAt time of genetic testing, the majority of children (n = 229) had macrocephaly (81%) or developmental delay (DD, 61%), and about half of the adults (n = 238) had cancer (51%), macrocephaly (61%), or cutaneous pathology (49%). Across PTEN, 268 LP/P variants were identified, with exon 5 as hotspot. Missense variants (n = 161) were mainly located in the phosphatase domain (PD, 90%) and truncating variants (n = 306) across all domains. A trend towards 2 times more often truncating variants was observed in adults (OR = 2.3, 95%CI = 1.5–3.4) and patients with cutaneous pathology (OR = 1.6, 95%CI = 1.1–2.5) or benign thyroid pathology (OR = 2.0, 95%CI = 1.1–3.5), with trends up to 2–4 times more variants in PD. Whereas patients with DD (OR = 0.5, 95%CI = 0.3–0.9) or macrocephaly (OR = 0.6, 95%CI = 0.4–0.9) had about 2 times less often truncating variants compared to missense variants. In DD patients these missense variants were often located in domain C2.ConclusionThe PHTS phenotypic diversity may partly be explained by the PTEN variant coding effect and the combination of coding effect and domain. PHTS patients with early-onset disease often had missense variants, and those with later-onset disease often truncating variants.