Clinical delineation,sex differences,and genotype–phenotype correlation in pathogenic KDM6A variants causing X-linked Kabuki syndrome type 2

PurposeThe variant spectrum and the phenotype of X-linked Kabuki syndrome type 2 (KS2) are poorly understood.MethodsGenetic and clinical details of new and published individuals with pathogenic KDM6A variants were compiled and analyzed.ResultsSixty-one distinct pathogenic KDM6A variants (50 truncating, 11 missense) from 80 patients (34 males, 46 females) were identified. Missense variants clustered in the TRP 2, 3, 7 and Jmj-C domains. Truncating variants were significantly more likely to be de novo. Thirteen individuals had maternally inherited variants and one had a paternally inherited variant. Neonatal feeding difficulties, hypoglycemia, postnatal growth retardation, poor weight gain, motor delay, intellectual disability (ID), microcephaly, congenital heart anomalies, palate defects, renal malformations, strabismus, hearing loss, recurrent infections, hyperinsulinism, seizures, joint hypermobility, and gastroesophageal reflux were frequent clinical findings. Facial features of over a third of patients were not typical for KS. Males were significantly more likely to be born prematurely, have shorter stature, and severe developmental delay/ID.ConclusionWe expand the KDM6A variant spectrum and delineate the KS2 phenotype. We demonstrate that the variability of the KS2 phenotypic depends on sex and the variant type. We also highlight the overlaps and differences between the phenotypes of KS2 and KS1.     

CDKN2A testing and genetic counseling promote reductions in objectively measured sun exposure one year later

PurposeThis study investigated whether genetic counseling and test reporting for the highly penetrant CDKN2A melanoma predisposition gene promoted decreases in sun exposure.MethodsA prospective, nonequivalent control group design compared unaffected participants (N = 128, M_age = 35.24, 52% men) from (1) families known to carry a CDKN2A pathogenic variant, who received counseling about management recommendations and a positive or negative genetic test result and (2) no-test control families known not to carry a CDKN2A pathogenic variant, who received equivalent counseling based on their comparable family history. Changes in daily ultraviolet radiation (UVR) exposure (J/m²), skin pigmentation (melanin index), and sunburns between baseline and one year following counseling were compared among carriers (n = 32), noncarriers (n = 46), and no-test control participants (n = 50).ResultsBoth carriers and no-test control participants exhibited a decrease one year later in daily UVR dose (B = −0.52, −0.33, p < 0.01). Only carriers exhibited a significant decrease in skin pigmentation at the wrist one year later (B = −0.11, p < 0.001), and both carriers and no-test control participants reported fewer sunburns than noncarriers (p < 0.05). Facial pigmentation did not change for any group. Noncarriers did not change on any measure of UVR exposure.ConclusionsThese findings support the clinical utility of disclosing CDKN2A test results and providing risk management education to high-risk individuals.     

KDM1A inactivation causes hereditary food-dependent Cushing syndrome

PurposeThis study aimed to investigate the genetic cause of food-dependent Cushing syndrome (FDCS) observed in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) and adrenal ectopic expression of the glucose-dependent insulinotropic polypeptide receptor. Germline ARMC5 alterations have been reported in about 25% of PBMAH index cases but are absent in patients with FDCS.MethodsA multiomics analysis of PBMAH tissues from 36 patients treated by adrenalectomy was performed (RNA sequencing, single-nucleotide variant array, methylome, miRNome, exome sequencing).ResultsThe integrative analysis revealed 3 molecular groups with different clinical features, namely G1, comprising 16 patients with ARMC5 inactivating variants; G2, comprising 6 patients with FDCS with glucose-dependent insulinotropic polypeptide receptor ectopic expression; and G3, comprising 14 patients with a less severe phenotype. Exome sequencing revealed germline truncating variants of KDM1A in 5 G2 patients, constantly associated with a somatic loss of the KDM1A wild-type allele on 1p, leading to a loss of KDM1A expression both at messenger RNA and protein levels (P = 1.2 × 10^–12 and P < .01, respectively). Subsequently, KDM1A pathogenic variants were identified in 4 of 4 additional index cases with FDCS.ConclusionKDM1A inactivation explains about 90% of FDCS PBMAH. Genetic screening for ARMC5 and KDM1A can now be offered for most PBMAH operated patients and their families, opening the way to earlier diagnosis and improved management.     

Population data improves variant interpretation in autosomal dominant polycystic kidney disease

PurposeAutosomal dominant polycystic kidney disease (ADPKD) is a common adult-onset monogenic disorder, with prevalence of 1/1000. Population databases including ExAC have improved pathogenic variant prioritization in many diseases. Due to pseudogene homology of PKD1, the predominant ADPKD disease gene, and the variable disease severity and age of onset, we aimed to investigate the utility of ExAC for variant assessment in ADPKD.MethodsWe assessed coverage and variant quality in the ExAC cohort and combined allele frequency and age data from the ExAC database (n = 60,706) with curated variants from 2000 ADPKD pedigrees (ADPKD Mutation Database).ResultsSeventy-six percent of PKD1 and PKD2 were sequenced adequately for variant discovery and variant quality was high in ExAC. In ExAC, we identified 25 truncating and 393 previously reported disease-causing variants in PKD1 and PKD2, 6.9-fold higher than expected. Fifty-four different variants, previously classified as disease-causing, were observed in ≥5 participants in ExAC.ConclusionOur study demonstrates that many previously implicated disease-causing variants are too common, challenging their pathogenicity, or penetrance. The presence of protein-truncating variants in older participants in ExAC demonstrates the complexity of variant classification and highlights need for further study of prevalence and penetrance of this common monogenic disease.     

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.     

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.     

Molecular genetic study of 59 Chinese Oculocutaneous albinism families

Oculocutaneous albinism is an autosomal recessive disorder characterized by either a complete lack of or reduction in melanin biosynthesis in the skin, hair, and eyes. The aim of the present study was to identify the molecular basis for 59 Chinese OCA families. In this study, compound heterozygous or homozygous pathogenic variants were found in 53 families, 4 families possessed only one heterozygous variant, and the pathogenic variants of 2 families remain undiscovered by using Sanger sequencing, whole exome sequencing and multiplex ligation-dependent probe amplification. We have identified a total of 55 variants including 21 novel variants in TYR, OCA2, SLC45A2, SLC24A5, and HPS1. The 21 novel variants include 11 missense changes, 4 nonsense changes, 2 splice site changes, 1 frameshift and 3 gross deletions. Forty-six variants including 14 novel variants were segregated with the phenotype in 37 families. We conducted RT-PCR of the novel splicing site variant (c.399-14G > A) of HPS1 and verified that the variant would result in the inclusion of 12 bp of intronic material in exon 6 of HPS1. The results of platelet whole mount electron microscopy further confirmed the diagnosis of HPS1. These novel variants identified in our study expand the mutational spectrum of the disease, which contributes to prenatal diagnosis and genetic counselling.     

Prevalence of mutations in a panel of breast cancer susceptibility genes in BRCA1/2-negative patients with early-onset breast cancer

PurposeClinical testing for germ-line variation in multiple cancer susceptibility genes is available using massively parallel sequencing. Limited information is available for pretest genetic counseling regarding the spectrum of mutations and variants of uncertain significance in defined patient populations.MethodsWe performed massively parallel sequencing using targeted capture of 22 cancer susceptibility genes in 278 BRCA1/2-negative patients with early-onset breast cancer (diagnosed at younger than 40 years of age).ResultsThirty-one patients (11%) were found to have at least one deleterious or likely deleterious variant. Seven patients (2.5% overall) were found to have deleterious or likely deleterious variants in genes for which clinical guidelines exist for management, namely TP53 (4), CDKN2A (1), MSH2 (1), and MUTYH (double heterozygote). Twenty-four patients (8.6%) had deleterious or likely deleterious variants in a cancer susceptibility gene for which clinical guidelines are lacking, such as CHEK2 and ATM. Fifty-four patients (19%) had at least one variant of uncertain significance, and six patients were heterozygous for a variant in MUTYH.ConclusionThese data demonstrate that massively parallel sequencing identifies reportable variants in known cancer susceptibility genes in more than 30% of patients with early-onset breast cancer. However, only few patients (2.5%) have definitively actionable mutations given current clinical management guidelines.Genet Med17 8, 630–638.     

BLOC1S5 pathogenic variants cause a new type of Hermansky–Pudlak syndrome

PurposeHermansky–Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, excessive bleeding, and often additional symptoms. Variants in ten different genes have been involved in HPS. However, some patients lack variants in these genes. We aimed to identify new genes involved in nonsyndromic or syndromic forms of albinism.MethodsTwo hundred thirty albinism patients lacking a molecular diagnosis of albinism were screened for pathogenic variants in candidate genes with known links to pigmentation or HPS pathophysiology.ResultsWe identified two unrelated patients with distinct homozygous variants of the BLOC1S5 gene. Patients had mild oculocutaneous albinism, moderate bleeding diathesis, platelet aggregation deficit, and a dramatically decreased number of platelet dense granules, all signs compatible with HPS. Functional tests performed on platelets of one patient displayed an absence of the obligate multisubunit complex BLOC-1, showing that the variant disrupts BLOC1S5 function and impairs BLOC-1 assembly. Expression of the patient-derived BLOC1S5 deletion in nonpigmented murine Bloc1s5^-/- melan-mu melanocytes failed to rescue pigmentation, the assembly of a functional BLOC-1 complex, and melanosome cargo trafficking, unlike the wild-type allele.ConclusionMutation of BLOC1S5 is disease-causing, and we propose that BLOC1S5 is the gene for a new form of Hermansky–Pudlak syndrome, HPS-11.     

Continuous Bayesian variant interpretation accounts for incomplete penetrance among Mendelian cardiac channelopathies

PurposeThe congenital Long QT Syndrome (LQTS) and Brugada Syndrome (BrS) are Mendelian autosomal dominant diseases that frequently precipitate fatal cardiac arrhythmias. Incomplete penetrance is a barrier to clinical management of heterozygotes harboring variants in the major implicated disease genes KCNQ1, KCNH2, and SCN5A. We apply and evaluate a Bayesian penetrance estimation strategy that accounts for this phenomenon.MethodsWe generated Bayesian penetrance models for KCNQ1-LQT1 and SCN5A-LQT3 using variant-specific features and clinical data from the literature, international arrhythmia genetic centers, and population controls. We analyzed the distribution of posterior penetrance estimates across 4 genotype-phenotype relationships and compared continuous estimates with ClinVar annotations. Posterior estimates were mapped onto protein structure.ResultsBayesian penetrance estimates of KCNQ1-LQT1 and SCN5A-LQT3 are empirically equivalent to 10 and 5 clinically phenotype heterozygotes, respectively. Posterior penetrance estimates were bimodal for KCNQ1-LQT1 and KCNH2-LQT2, with a higher fraction of missense variants with high penetrance among KCNQ1 variants. There was a wide distribution of variant penetrance estimates among identical ClinVar categories. Structural mapping revealed heterogeneity among “hot spot” regions and featured high penetrance estimates for KCNQ1 variants in contact with calmodulin and the S6 domain.ConclusionsBayesian penetrance estimates provide a continuous framework for variant interpretation.     

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.     

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.     

Exploring concordance and discordance for return of incidental findings from clinical sequencing

PurposeThe aim of this study was to explore specific conditions and types of genetic variants that specialists in genetics recommend should be returned as incidental findings in clinical sequencing.MethodsSixteen specialists in clinical genetics and/or molecular medicine selected variants in 99 common conditions to return to the ordering physician if discovered incidentally through whole-genome sequencing. For most conditions, the specialists independently considered three molecular scenarios for both adults and minor children: a known pathogenic mutation, a truncating variant presumed pathogenic (where other truncating variants are known to be pathogenic), and a missense variant predicted in silico to be pathogenic.ResultsOn average, for adults and children, respectively, each specialist selected 83.5 and 79.0 conditions or genes of 99 in the known pathogenic mutation categories, 57.0 and 53.5 of 72 in the truncating variant categories, and 33.4 and 29.7 of 72 in the missense variant categories. Concordance in favor of disclosure within the adult/known pathogenic mutation category was 100% for 21 conditions or genes and 80% or higher for 64 conditions or genes.ConclusionSpecialists were highly concordant for the return of findings for 64 conditions or genes if discovered incidentally during whole-exome sequencing or whole-genome sequencing.Genet Med 2012:14(4):405–410     

Identification of novel loci for pediatric cholestatic liver disease defined by KIF12, PPM1F,USP53, LSR,and WDR83OS pathogenic variants

PurposeGenetic testing in pediatric cholestasis can be very informative but genetic causes have not been fully characterized.MethodsExome sequencing and positional mapping in seven families with cholestatic liver disease and negative clinical testing for known disease genes.ResultsKIF12, which encodes a microtubule motor protein with a tentative role in cell polarity, was found to harbor three homozygous likely deleterious variants in three families with sclerosing cholangitis. KIF12 expression is dependent on HNF-1β, deficiency which is known to cause bile duct dysmorphogenesis associated with loss of KIF12 expression. In another extended family, we mapped an apparently novel syndrome of sclerosing cholangitis, short stature, hypothyroidism, and abnormal tongue pigmentation in two cousins to a homozygous variant in PPM1F (POPX2), a regulator of kinesin-mediated ciliary transport. In the fifth family, a syndrome of normal gamma glutamyltransferase (GGT) cholestasis and hearing loss was found to segregate with a homozygous truncating variant in USP53, which encodes an interactor with TJP2. In the sixth family, we mapped a novel syndrome of transient neonatal cholestasis, intellectual disability, and short stature to a homozygous variant in LSR, an important regulator of liver development. In the last family of three affected siblings, a novel syndrome of intractable itching, hypercholanemia, short stature, and intellectual disability was mapped to a single locus that contains a homozygous truncating variant in WDR83OS (C19orf56), known to interact with ATP13A2 and BSEP.ConclusionOur results expand the genetic heterogeneity of pediatric cholestatic liver disease and highlight the vulnerability of bile homeostasis to a wide range of molecular perturbations.     

Dominant negative effects of SCN5A missense variants

PurposeUp to 30% of patients with Brugada syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A encoding for the protein Na_V1.5. Recent studies suggested that Na_V1.5 can dimerize, and some variants exert dominant negative effects. In this study, we sought to explore the generality of missense variant Na_V1.5 dominant negative effects and their clinical severity.MethodsWe identified 35 LoF variants (<10% of wild type [WT] peak current) and 15 partial LoF variants (10%-50% of WT peak current) that we assessed for dominant negative effects. SCN5A variants were studied in HEK293T cells, alone or in heterozygous coexpression with WT SCN5A using automated patch clamp. To assess the clinical risk, we compared the prevalence of dominant negative vs putative haploinsufficient (frameshift, splice, or nonsense) variants in a BrS consortium and the Genome Aggregation Database population database.ResultsIn heterozygous expression with WT, 32 of 35 LoF and 6 of 15 partial LoF variants showed reduction to <75% of WT-alone peak current, showing a dominant negative effect. Individuals with dominant negative LoF variants had an elevated disease burden compared with the individuals with putative haploinsufficient variants (2.7-fold enrichment in BrS cases, P = .019).ConclusionMost SCN5A missense LoF variants exert a dominant negative effect. This class of variant confers an especially high burden of BrS.     

Enhancing rare variant interpretation in inherited arrhythmias through quantitative analysis of consortium disease cohorts and population controls

PurposeStringent variant interpretation guidelines can lead to high rates of variants of uncertain significance (VUS) for genetically heterogeneous disease like long QT syndrome (LQTS) and Brugada syndrome (BrS). Quantitative and disease-specific customization of American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines can address this false negative rate.MethodsWe compared rare variant frequencies from 1847 LQTS (KCNQ1/KCNH2/SCN5A) and 3335 BrS (SCN5A) cases from the International LQTS/BrS Genetics Consortia to population-specific gnomAD data and developed disease-specific criteria for ACMG/AMP evidence classes—rarity (PM2/BS1 rules) and case enrichment of individual (PS4) and domain-specific (PM1) variants.ResultsRare SCN5A variant prevalence differed between European (20.8%) and Japanese (8.9%) BrS patients (p = 5.7 × 10⁻¹⁸) and diagnosis with spontaneous (28.7%) versus induced (15.8%) Brugada type 1 electrocardiogram (ECG) (p = 1.3 × 10⁻¹³). Ion channel transmembrane regions and specific N-terminus (KCNH2) and C-terminus (KCNQ1/KCNH2) domains were characterized by high enrichment of case variants and >95% probability of pathogenicity. Applying the customized rules, 17.4% of European BrS and 74.8% of European LQTS cases had (likely) pathogenic variants, compared with estimated diagnostic yields (case excess over gnomAD) of 19.2%/82.1%, reducing VUS prevalence to close to background rare variant frequency.ConclusionLarge case–control data sets enable quantitative implementation of ACMG/AMP guidelines and increased sensitivity for inherited arrhythmia genetic testing.     

Targeted gene sequencing in 6994 individuals with neurodevelopmental disorder with epilepsy

PurposeWe aimed to gain insight into frequencies of genetic variants in genes implicated in neurodevelopmental disorder with epilepsy (NDD+E) by investigating large cohorts of patients in a diagnostic setting.MethodsWe analyzed variants in NDD+E using epilepsy gene panel sequencing performed between 2013 and 2017 by two large diagnostic companies. We compared variant frequencies in 6994 panels with another 8588 recently published panels as well as exome-wide de novo variants in 1942 individuals with NDD+E and 10,937 controls.ResultsGenes with highest frequencies of ultrarare variants in NDD+E comprised SCN1A, KCNQ2, SCN2A, CDKL5, SCN8A, and STXBP1, concordant with the two other epilepsy cohorts we investigated. In only 46% of the analyzed 262 dominant and X-linked panel genes ultrarare variants in patients were reported. Among genes with contradictory evidence of association with epilepsy, CACNB4, CLCN2, EFHC1, GABRD, MAGI2, and SRPX2 showed equal frequencies in cases and controls.ConclusionWe show that improvement of panel design increased diagnostic yield over time, but panels still display genes with low or no diagnostic yield. With our data, we hope to improve current diagnostic NDD+E panel design and provide a resource of ultrarare variants in individuals with NDD+E to the community.