Quantitative Genomics Medicine Laboratories (qGenomics)

ACTB encodes β-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, β-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic β-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, β-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development. ACTB encodes β-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, β-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic β-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, β-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development. Developmental disorders (DDs) are thought to affect 2%–5% of individuals and are genetically heterogeneous.1Deciphering Developmental Disorders StudyPrevalence and architecture of de novo mutations in developmental disorders.Nature. 2017; 542: 433-438Crossref PubMed Scopus (771) Google Scholar They range from isolated internal organ malformations and intellectual disability to complex syndromic presentations. In developed economies, congenital malformations are one of the leading causes of death among children and account for almost 25% of neonatal deaths.2Khokha M.K. Mitchell L.E. Wallingford J.B. An opportunity to address the genetic causes of birth defects.Pediatr. Res. 2017; 81: 282-285Crossref PubMed Scopus (6) Google Scholar DDs constitute a large proportion of the life-long global health burden in terms of medical expenditure, hospitalizations, and mortality.2Khokha M.K. Mitchell L.E. Wallingford J.B. An opportunity to address the genetic causes of birth defects.Pediatr. Res. 2017; 81: 282-285Crossref PubMed Scopus (6) Google Scholar Accurate diagnosis and better mechanistic understanding are key to improving medical management. Rare copy-number variations associated with human DDs can provide insights into single-gene conditions and their molecular mechanisms.3Cooper G.M. Coe B.P. Girirajan S. Rosenfeld J.A. Vu T.H. Baker C. Williams C. Stalker H. Hamid R. Hannig V. et al.A copy number variation morbidity map of developmental delay.Nat. Genet. 2011; 43: 838-846Crossref PubMed Scopus (934) Google Scholar, 4Coe B.P. Witherspoon K. Rosenfeld J.A. van Bon B.W. Vulto-van Silfhout A.T. Bosco P. Friend K.L. Baker C. Buono S. Vissers L.E. et al.Refining analyses of copy number variation identifies specific genes associated with developmental delay.Nat. Genet. 2014; 46: 1063-1071Crossref PubMed Scopus (391) Google Scholar, 5Yagi H. Furutani Y. Hamada H. Sasaki T. Asakawa S. Minoshima S. Ichida F. Joo K. Kimura M. Imamura S. et al.Role of TBX1 in human del22q11.2 syndrome.Lancet. 2003; 362: 1366-1373Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 6Banka S. Cain S.A. Carim S. Daly S.B. Urquhart J.E. Erdem G. Harris J. Bottomley M. Donnai D. Kerr B. et al.Leri’s pleonosteosis, a congenital rheumatic disease, results from microduplication at 8q22.1 encompassing GDF6 and SDC2 and provides insight into systemic sclerosis pathogenesis.Ann. Rheum. Dis. 2015; 74: 1249-1256Crossref PubMed Scopus (19) Google Scholar, 7Kasher P.R. Schertz K.E. Thomas M. Jackson A. Annunziata S. Ballesta-Martinez M.J. Campeau P.M. Clayton P.E. Eaton J.L. Granata T. et al.Small 6q16.1 deletions encompassing POU3F2 cause susceptibility to obesity and variable developmental delay with intellectual disability.Am. J. Hum. Genet. 2016; 98: 363-372Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar From more than 15,000 individuals who underwent clinical array comparative genomic hybridization for suspected genetic DDs at our center, we identified five individuals from four families with 7p22.1 deletions (Figure 1A; Table S1) and an overlapping phenotype (families I–IV in Table 1; Figure 2A). ACTB [MIM: 102630] was the only gene common to all four deletions, leading us to hypothesize that ACTB haploinsufficiency leads to a distinct clinical syndrome. We ascertained 26 additional individuals from 19 families with likely or definitely pathogenic 7p22.1 deletions that were <3Mb and encompassed ACTB (Figure 1B; Table S1). Next, we interrogated data from 4,293 trios in the Deciphering Developmental Disorders study1Deciphering Developmental Disorders StudyPrevalence and architecture of de novo mutations in developmental disorders.Nature. 2017; 542: 433-438Crossref PubMed Scopus (771) Google Scholar for de novo nonsense or frameshift variants in all known protein-coding genes on chromosome 7p22.1. We identified two ACTB point mutations, c.1097dupG; p.Ser368LeufsTer13 and c.1117A>T; p.Lys373Ter, in two children (NM_001101.3; ENST00000331789) (Figures 1C; Table S1). Finally, we identified another individual with a c.329delT; p.Leu110ArgfsTer10 ACTB point mutation in the CAUSES Study by using the analytical pipeline described previously.8Tarailo-Graovac M. Shyr C. Ross C.J. Horvath G.A. Salvarinova R. Ye X.C. Zhang L.H. Bhavsar A.P. Lee J.J. Drögemöller B.I. et al.Exome sequencing and the management of neurometabolic disorders.N. Engl. J. Med. 2016; 374: 2246-2255Crossref PubMed Scopus (199) Google Scholar The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national), and proper informed consent was obtained. Multiple lines of evidence establish ACTB loss-of-function mutations as a cause of a pleiotropic clinical syndrome. First, of the genes on human chromosomal region 7p22.1, ACTB is the only one with a high-probability loss-of-function intolerance (pLI) score9Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. O’Donnell-Luria A.H. Ware J.S. Hill A.J. Cummings B.B. et al.Exome Aggregation ConsortiumAnalysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (6586) Google Scholar and low residual variation intolerance score (RVIS)10Petrovski S. Wang Q. Heinzen E.L. Allen A.S. Goldstein D.B. Genic intolerance to functional variation and the interpretation of personal genomes.PLoS Genet. 2013; 9: e1003709Crossref PubMed Scopus (643) Google Scholar as well as a low haploinsufficiency index (HI)11Huang N. Lee I. Marcotte E.M. Hurles M.E. Characterising and predicting haploinsufficiency in the human genome.PLoS Genet. 2010; 6: e1001154Crossref PubMed Scopus (448) Google Scholar (Table S2). Second, it was the only gene deleted within the minimum critical region in both the discovery and validation cohorts of individuals with 7p22.1 deletions. Importantly, we also identified three individual with ACTB point mutations that, like the deletions, are expected to produce a heterozygous null ACTB genotype. Third, mutations, including all point mutations, were proven to have arisen de novo in 12 individuals. Biological parentage was proven in the three individuals who were identified via exome sequencing. In all multiplex families, the deletions segregated with the phenotype. Fourth, the striking phenotypic convergence in a large cohort ascertained on the basis of genotyping followed by reverse phenotyping12de Goede C. Yue W.W. Yan G. Ariyaratnam S. Chandler K.E. Downes L. Khan N. Mohan M. Lowe M. Banka S. Role of reverse phenotyping in interpretation of next generation sequencing data and a review of INPP5E related disorders.Eur. J. Paediatr. Neurol. 2016; 20: 286-295Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar rules out possibility of a chance association. Collectively, in this cohort of 33 individuals from 25 unrelated families we observed a high frequency of developmental delay, apparent intellectual disability, internal organ malformations (affecting heart, kidneys, spine, and palate, among others), growth retardation, and facial dysmorphism (interrupted eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) (Figure 2A; Table 1).Table 1Clinical Features of Individuals with Deletions, Stop-Gained, or Frameshift Mutations Involving ACTBCase IDInheritanceGenderAge (Years)Prenatal and Neonatal HistoryPN Growth RetardationMicrocephalyMotor DelaySpeech DelayDD/ IDBehavioral, Psychiatric, and Neurological FeaturesMalformations and Physical AnomaliesAdditional CommentsDiscovery CohortIDNM4SGA and feeding difficultiesYNYYmodpossible absence and focal seizuresVSD with tortuous aortic arch, horseshoe kidney, cryptorchidism, BL inguinal hernia, deep sacral dimple and BL single palmar creasesearly-onset hypothyroidism, limitation of joint mobility and cutis marmorataIIDNM7SGA and feeding difficultiesYNYYmildsociable, empathetic, hand flapping tendency and attention deficitRt pelvic kidney, Rt inguinal hernia and scoliosis.GOR, asthma and allergies.IIIDNM7SGA, polycythaemia, jaundice and hypoglycaemia. Congenital CMV infectionYYNYMildattention deficit, echolalia and tantrums.inguinal hernia, cryptorchidism, proximally placed second toes and microcornea.perineal and scalp abscesses, recurrent chest and ear infections, allergies and nephrotic syndrome.IVaMatF32SGAYNYYmodempathetic personality.scoliosisglaucoma, asthma, and eczemaIVbUF68UUUUUmildUhorseshoe kidney with multiple cystshiatus herniaValidation CohortVDNF13SGA and feeding difficultiesYNYYmodsociable personality, mild ventriculomegaly, and multifocal small T2 hyperintensitites in the cerebral white matterVSD, PDA, BL 5th finger clinodactyly, BL 2-3 & Lt 3-4 toe syndactylyNVIUM20NYNNYmildstress intolerance.short and broad uvula, broad halluces, short distal phalanx of finger and toes, small nails, and 5th finger clinodactyly.frequent otisis media, GH deficiency and limitation of joint mobilityVIIDNF12SGA, hypotonia and feeding difficultiesYYYYmodemotional problems and hypotoniatricuspid valve dysplasia, 2-3-4 fingers and 2-3 toes syndactylyBL severe SNHL and dorsal hypertrichosisVIIIDNM7SGA, hypotonia and feeding difficultiesYNYYsevThin CC, septum pellucidum cyst, megacisterna magna, mild ventricular dilation and subependymal heterotopiaBL CLAP, VSD, Lt extra nipple, hypospadias, UL cryptorchidism and sacral dimplecutis marmorata; additional de novo 1.65 Mb loss 7:6243891-7889083IXDNM6hypotonia and feeding difficultiesYYYYmodcortical and subcortical atrophyatrial septal defect and BL inguinal herniaGORXDNF0 (fetus)antenatal ultrasound: cleft lip and palate, septum pellucidum agenesisNANANANANAabsent septum pellucidum and hydrocephalushorseshoe kidney and non-midline CLAPNXIDNM6hypotonia and feeding difficultiesNNYYmodsociable personality, ASD, hypotonia, possible seizures and periventricular heterotopiasBL absent thumbs, bowed radii, shortened forearms, chordee and parameatal cystGOR and hypermetropia. et K. S. M. T. N. de A. M. J. S. et ACTB associated with developmental delay, short and J. Med. Genet. 2016; PubMed Scopus Google Scholar personality, attention deficit, and seizures in clinodactyly, short thumbs, finger finger and 2-3 toe reduced and BL and Lt aortic and feeding and Rt single finger and toe of Lt scalp in et K. S. M. T. N. de A. M. J. S. et ACTB associated with developmental delay, short and J. Med. Genet. 2016; PubMed Scopus Google Scholar loss and and BL inguinal and recurrent and feeding attention and and and of and finger septal and of horseshoe kidney, and and Rt at 26 and hypotonia and feeding deficit, and cortical have BL SNHL and additional in et K. S. M. T. N. de A. M. J. S. et ACTB associated with developmental delay, short and J. Med. Genet. 2016; PubMed Scopus Google Scholar & in et K. S. M. T. N. de A. M. J. S. et ACTB associated with developmental delay, short and J. Med. Genet. 2016; PubMed Scopus Google Scholar of & and additional and feeding personality, ASD, hypotonia and cortical congenital hernia, finger and and BL and and renal hernia, high palate, and and overlapping toes, short and fingers and in both of congenital on and septal defect and distal joint of 5th BL mild in deficiency to heterozygous pathogenic mutations in additional and the clinical of individuals in the discovery or validation cohort and point mutations ACTB spectrum CC, CLAP, cleft lip and developmental de growth intellectual or not PDA, small for sensorineural VSD, ventricular septal and in a the clinical of individuals in the discovery or validation cohort and point mutations ACTB spectrum CC, CLAP, cleft lip and developmental de growth intellectual or not PDA, small for sensorineural VSD, ventricular septal and We and ACTB mRNA expression in lymphoblastic cell lines and levels in affected were than in control (Figure have been in Y. cell migration, and the 2011; PubMed Scopus Google Scholar and in derived from one individual with a 7p22.1 K. S. M. T. N. de A. M. J. S. et ACTB associated with developmental delay, short and J. Med. Genet. 2016; PubMed Scopus Google Scholar Next, we β-actin expression in a is this is to the of human β-actin was prominent in affected by the cortical and in the of the and the and in the of the heart (Figures it was not in all cell from within an β-actin is for a number of cytoplasmic as of cell shape and Y. cell migration, and the 2011; PubMed Scopus Google Scholar, L. R. C. J. and in cell 2014; PubMed Scopus Google Scholar, E.M. and 2015; PubMed Scopus Google Scholar We an and the amount of β-actin in the cytoplasmic protein of affected individual but consistent in β-actin expression were observed in affected (Figure affected fibroblasts were more than was in the cell (Figure S1). was also in comparison to that of control fibroblasts (Figure Importantly, of ACTB in control fibroblasts (Figure a in (Figure and (Figure that the in with a 7p22.1 were to ACTB In the β-actin gene cell and N. P. of 2010; Scopus Google Scholar, N. P. roles for 2015; PubMed Scopus Google Scholar in to the cytoplasmic the protein showed a reduced amount of β-actin in derived from affected individuals (Figure with β-actin with levels of and cell M. P. K. E. D. C.J. de P. by 2016; PubMed Scopus Google Scholar we decreased in affected (Figures and We expression of cell-cycle genes by using on derived from two and two from affected individuals (Table In from affected we increased expression of [MIM: a key gene that encodes for to from into the (Figure K. M. in levels the cell the of a PubMed Scopus Google Scholar is consistent with the that β-actin leads to in the C. F. P.R. of fibroblasts in in to is on but not on 2003; PubMed Scopus Google Scholar the expression levels of several and genes were in from affected individuals than in control (Figure chromosome 7p22.1 deletions have been described in a small number of affected individuals ACTB not been proven to the gene responsible for the L. R. C. J. and in cell 2014; PubMed Scopus Google Scholar We have described affected families, that the syndrome by loss-of-function ACTB mutations have been in the phenotype to in the of loss of additional genes or or to genetic or two point mutations in are to and one in is to cause protein it is that the phenotype of individuals is to that of individuals with ACTB deletions as to [MIM: J.B. van Bon B.W. A. C. S. et novo mutations in the genes ACTB and cause Baraitser-Winter Genet. PubMed Scopus Google Scholar that the of β-actin an in human development. individuals in our cohort developmental delay and apparent intellectual disability. In several was empathetic, or was in individuals. were to have or disorders. are a of and are critical for and M. The can shape consequences on and complex 2016; PubMed Scopus Google Scholar, of the 2015; PubMed Scopus Google Scholar the developmental and phenotype of affected individuals. individuals who were with were to have including in two individuals (Table is consistent with prominent expression of β-actin in cortical of and our of in in from affected individuals. cortical cerebral a a septum pellucidum cyst, megacisterna magna, ventricular and were in a of and one individual been in with an ACTB V. G. S. M. A. R. J. G. et al.A mutation of that alters is associated with developmental malformations, deafness, and J. Hum. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar Congenital as ventricular and septal tortuous arch, valve dysplasia, and ventricular were observed in out of individuals who were by or (Table renal were in out of 19 individuals renal or were four individuals with horseshoe kidneys, and one with renal pelvic kidney, and kidney (Table and were also in individuals. who renal malformations, a high frequency of as inguinal hypospadias, and and were also in and four Collectively, our data show that internal organ malformations, in the heart and kidneys, are more in individuals with loss-of-function ACTB mutations than in the in congenital heart The 2016; PubMed Scopus Google Scholar, A. A. J. B. J. T. et Developmental Disorders genetic for syndromic and congenital heart identified by exome Genet. 2016; PubMed Scopus Google Scholar, A. A. M. S. C. Study of congenital renal malformations by an of in 12 J. Med. Genet. PubMed Scopus Google Scholar, G. R. G.M. the of horseshoe kidney from data at a single 2003; Full Text Full Text PDF PubMed Scopus Google Scholar of β-actin in is by our of this protein in the heart and kidney in in from affected we showed altered migration, and proliferation, are all key developmental growth were not for all affected we growth in individuals. related to in cell growth and proliferation, we also observed in the affected N. P. of 2010; Scopus Google Scholar The individuals described have overlapping dysmorphism with wavy eyebrows, dense eyelashes, a wide nose, a wide mouth, and a prominent is distinct from the typical facial dysmorphism of we overlapping as a wide and in both of our individuals were to have or coloboma, are in A. N. J. M. B. J. H. D. N. et of the spectrum in J. Hum. Genet. 2015; PubMed Scopus Google Scholar, J. Baraitser-Winter Genet. 2017; PubMed Scopus Google Scholar that ACTB loss-of-function mutations cause a specific syndrome distinct from BRWS. The facial characteristics of individual described those in several as the in syndrome [MIM: and S. R. E. S. N. M.J. Kerr B. H. et genetically is in review and analyses of mutation and phenotypic J. Hum. Genet. 20: PubMed Scopus Google Scholar, S. D. V. E. E. S. Kerr B. S. D. et mutations and a clinical and molecular review of the syndrome Genet. 2015; PubMed Scopus Google Scholar and the typical lip and large in syndrome [MIM: β-actin is a of as and N. P. roles for 2015; PubMed Scopus Google Scholar and mutations in cause syndrome [MIM: J. C. M.J. J. D. J.S. et in cause J. Hum. Genet. Full Text Full Text PDF PubMed Scopus Google Scholar and syndrome [MIM: E. Y. R. C. M. Y. et in complex gene cause Genet. PubMed Scopus Google Scholar we decreased amounts of β-actin, with gene in of affected individuals. The of β-actin been the of M.J. The of 2014; PubMed Scopus Google Scholar, P. of in chromatin-remodeling 2014; Full Text Full Text PDF PubMed Scopus Google Scholar and our results the of in human development. In we have in 33 a pleiotropic by haploinsufficiency of encodes for the abundant cytoplasmic β-actin. we have of the consequences of reduced of ACTB in studies to we are to the the from loss-of-function ACTB the ACTB missense mutations with also The of of individuals with and ACTB loss-of-function mutations suggest that the of is not as of but also from loss-of-function or are that mutations in the gene can have genetic and can in M. Yue W.W. K. J. K. et Developmental Disorders missense mutations in developmental disorders with J. Hum. Genet. 2017; Full Text Full Text PDF PubMed Scopus Google Scholar missense [MIM: the only mutations also in J.B. van Bon B.W. A. C. S. et novo mutations in the genes ACTB and cause Baraitser-Winter Genet. PubMed Scopus Google Scholar we not distinct phenotype that to deletions not the score for is the that heterozygous loss-of-function mutations in this gene are a cause of an human overlapping but distinct roles for the two and our studies suggest that a critically reduced amount of β-actin alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development. We are to all individual and their families for in the We the of the and and and We are to the and for the number is by a is by and the of is by a is a clinical of the for We for to of the The Deciphering Developmental Disorders by the number a the and the of and the number The in this are those of the and not those of the or the of The by the and by the of The the of the for the of the have of to with and and

BACKGROUND: Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders with high heritability. Recent findings support a highly heterogeneous and complex genetic etiology including rare de novo and inherited mutations or chromosomal rearrangements as well as double or multiple hits. METHODS: We performed whole-exome sequencing (WES) and blood cell transcriptome by RNAseq in a subset of male patients with idiopathic ASD (n = 36) in order to identify causative genes, transcriptomic alterations, and susceptibility variants. RESULTS: We detected likely monogenic causes in seven cases: five de novo (SCN2A, MED13L, KCNV1, CUL3, and PTEN) and two inherited X-linked variants (MAOA and CDKL5). Transcriptomic analyses allowed the identification of intronic causative mutations missed by the usual filtering of WES and revealed functional consequences of some rare mutations. These included aberrant transcripts (PTEN, POLR3C), deregulated expression in 1.7% of mutated genes (that is, SEMA6B, MECP2, ANK3, CREBBP), allele-specific expression (FUS, MTOR, TAF1C), and non-sense-mediated decay (RIT1, ALG9). The analysis of rare inherited variants showed enrichment in relevant pathways such as the PI3K-Akt signaling and the axon guidance. CONCLUSIONS: Integrative analysis of WES and blood RNAseq data has proven to be an efficient strategy to identify likely monogenic forms of ASD (19% in our cohort), as well as additional rare inherited mutations that can contribute to ASD risk in a multifactorial manner. Blood transcriptomic data, besides validating 88% of expressed variants, allowed the identification of missed intronic mutations and revealed functional correlations of genetic variants, including changes in splicing, expression levels, and allelic expression.

Autoinflammatory diseases (AIDs) were first described as clinical disorders characterized by recurrent episodes of seemingly unprovoked sterile inflammation. In the past few years, the identification of novel AIDs expanded their phenotypes toward more complex clinical pictures associating vasculopathy, autoimmunity, or immunodeficiency. Herein, we describe two unrelated patients suffering since the neonatal period from a complex disease mainly characterized by severe sterile inflammation, recurrent bacterial infections, and marked humoral immunodeficiency. Whole-exome sequencing detected a novel, de novo heterozygous PLCG2 variant in each patient (p.Ala708Pro and p.Leu845_Leu848del). A clear enhanced PLCγ2 activity for both variants was demonstrated by both ex vivo calcium responses of the patient's B cells to IgM stimulation and in vitro assessment of PLC activity. These data supported the autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) diagnosis in both patients. Immunological evaluation revealed a severe decrease of immunoglobulins and B cells, especially class-switched memory B cells, with normal T and NK cell counts. Analysis of bone marrow of one patient revealed a reduced immature B cell fraction compared with controls. Additional investigations showed that both PLCG2 variants activate the NLRP3-inflammasome through the alternative pathway instead of the canonical pathway. Collectively, the evidences here shown expand APLAID diversity toward more severe phenotypes than previously reported including dominantly inherited agammaglobulinemia, add novel data about its genetic basis, and implicate the alternative NLRP3-inflammasome activation pathway in the basis of sterile inflammation.

Background: The identification of expression quantitative trait methylation (eQTMs), defined as associations between DNA methylation levels and gene expression, might help the biological interpretation of epigenome-wide association studies (EWAS). We aimed to identify autosomal cis eQTMs in children's blood, using data from 832 children of the Human Early Life Exposome (HELIX) project. Methods: Blood DNA methylation and gene expression were measured with the Illumina 450K and the Affymetrix HTA v2 arrays, respectively. The relationship between methylation levels and expression of nearby genes (1 Mb window centered at the transcription start site, TSS) was assessed by fitting 13.6 M linear regressions adjusting for sex, age, cohort, and blood cell composition. Results: We identified 39,749 blood autosomal cis eQTMs, representing 21,966 unique CpGs (eCpGs, 5.7% of total CpGs) and 8,886 unique transcript clusters (eGenes, 15.3% of total transcript clusters, equivalent to genes). In 87.9% of these cis eQTMs, the eCpG was located at <250 kb from eGene's TSS; and 58.8% of all eQTMs showed an inverse relationship between the methylation and expression levels. Only around half of the autosomal cis-eQTMs eGenes could be captured through annotation of the eCpG to the closest gene. eCpGs had less measurement error and were enriched for active blood regulatory regions and for CpGs reported to be associated with environmental exposures or phenotypic traits. In 40.4% of the eQTMs, the CpG and the eGene were both associated with at least one genetic variant. The overlap of autosomal cis eQTMs in children's blood with those described in adults was small (13.8%), and age-shared cis eQTMs tended to be proximal to the TSS and enriched for genetic variants. Conclusions: This catalogue of autosomal cis eQTMs in children's blood can help the biological interpretation of EWAS findings and is publicly available at https://helixomics.isglobal.org/ and at Dryad (doi:10.5061/dryad.fxpnvx0t0). Funding: The study has received funding from the European Community's Seventh Framework Programme (FP7/2007-206) under grant agreement no 308333 (HELIX project); the H2020-EU.3.1.2. - Preventing Disease Programme under grant agreement no 874583 (ATHLETE project); from the European Union's Horizon 2020 research and innovation programme under grant agreement no 733206 (LIFECYCLE project), and from the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL and Instituto de Salud Carlos III) under the grant agreement no AC18/00006 (NutriPROGRAM project). The genotyping was supported by the projects PI17/01225 and PI17/01935, funded by the Instituto de Salud Carlos III and co-funded by European Union (ERDF, "A way to make Europe") and the Centro Nacional de Genotipado-CEGEN (PRB2-ISCIII). BiB received core infrastructure funding from the Wellcome Trust (WT101597MA) and a joint grant from the UK Medical Research Council (MRC) and Economic and Social Science Research Council (ESRC) (MR/N024397/1). INMA data collections were supported by grants from the Instituto de Salud Carlos III, CIBERESP, and the Generalitat de Catalunya-CIRIT. KANC was funded by the grant of the Lithuanian Agency for Science Innovation and Technology (6-04-2014_31V-66). The Norwegian Mother, Father and Child Cohort Study is supported by the Norwegian Ministry of Health and Care Services and the Ministry of Education and Research. The Rhea project was financially supported by European projects (EU FP6-2003-Food-3-NewGeneris, EU FP6. STREP Hiwate, EU FP7 ENV.2007.1.2.2.2. Project No 211250 Escape, EU FP7-2008-ENV-1.2.1.4 Envirogenomarkers, EU FP7-HEALTH-2009- single stage CHICOS, EU FP7 ENV.2008.1.2.1.6. Proposal No 226285 ENRIECO, EU- FP7- HEALTH-2012 Proposal No 308333 HELIX), and the Greek Ministry of Health (Program of Prevention of obesity and neurodevelopmental disorders in preschool children, in Heraklion district, Crete, Greece: 2011-2014; "Rhea Plus": Primary Prevention Program of Environmental Risk Factors for Reproductive Health, and Child Health: 2012-15). We acknowledge support from the Spanish Ministry of Science and Innovation through the "Centro de Excelencia Severo Ochoa 2019-2023" Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. MV-U and CR-A were supported by a FI fellowship from the Catalan Government (FI-DGR 2015 and #016FI_B 00272). MC received funding from Instituto Carlos III (Ministry of Economy and Competitiveness) (CD12/00563 and MS16/00128).

BACKGROUND: Understanding the genetic contribution to phenotype variation of human groups is necessary to elucidate differences in disease predisposition and response to pharmaceutical treatments in different human populations. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the genome-wide profile of structural variation on pooled samples from the three populations studied in the HapMap project by comparative genome hybridization (CGH) in different array platforms. We have identified and experimentally validated 33 genomic loci that show significant copy number differences from one population to the other. Interestingly, we found an enrichment of genes related to environment adaptation (immune response, lipid metabolism and extracellular space) within these regions and the study of expression data revealed that more than half of the copy number variants (CNVs) translate into gene-expression differences among populations, suggesting that they could have functional consequences. In addition, the identification of single nucleotide polymorphisms (SNPs) that are in linkage disequilibrium with the copy number alleles allowed us to detect evidences of population differentiation and recent selection at the nucleotide variation level. CONCLUSIONS: Overall, our results provide a comprehensive view of relevant copy number changes that might play a role in phenotypic differences among major human populations, and generate a list of interesting candidates for future studies.

Obesity is a multifactorial disorder with high heritability (50-75%), which is probably higher in early-onset and severe cases. Although rare monogenic forms and several genes and regions of susceptibility, including copy number variants (CNVs), have been described, the genetic causes underlying the disease still remain largely unknown. We searched for rare CNVs (>100kb in size, altering genes and present in <1/2000 population controls) in 157 Spanish children with non-syndromic early-onset obesity (EOO: body mass index >3 standard deviations above the mean at <3 years of age) using SNP array molecular karyotypes. We then performed case control studies (480 EOO cases/480 non-obese controls) with the validated CNVs and rare sequence variants (RSVs) detected by targeted resequencing of selected CNV genes (n = 14), and also studied the inheritance patterns in available first-degree relatives. A higher burden of gain-type CNVs was detected in EOO cases versus controls (OR = 1.71, p-value = 0.0358). In addition to a gain of the NPY gene in a familial case with EOO and attention deficit hyperactivity disorder, likely pathogenic CNVs included gains of glutamate receptors (GRIK1, GRM7) and the X-linked gastrin-peptide receptor (GRPR), all inherited from obese parents. Putatively functional RSVs absent in controls were also identified in EOO cases at NPY, GRIK1 and GRPR. A patient with a heterozygous deletion disrupting two contiguous and related genes, SLCO4C1 and SLCO6A1, also had a missense RSV at SLCO4C1 on the other allele, suggestive of a recessive model. The genes identified showed a clear enrichment of shared co-expression partners with known genes strongly related to obesity, reinforcing their role in the pathophysiology of the disease. Our data reveal a higher burden of rare CNVs and RSVs in several related genes in patients with EOO compared to controls, and implicate NPY, GRPR, two glutamate receptors and SLCO4C1 in highly penetrant forms of familial obesity.

BACKGROUND: Congenital malformations are present in approximately 2-3% of liveborn babies and 20% of stillborn fetuses. The mechanisms underlying the majority of sporadic and isolated congenital malformations are poorly understood, although it is hypothesized that the accumulation of rare genetic, genomic and epigenetic variants converge to deregulate developmental networks. METHODOLOGY/PRINCIPAL FINDINGS: We selected samples from 95 fetuses with congenital malformations not ascribed to a specific syndrome (68 with isolated malformations, 27 with multiple malformations). Karyotyping and Multiplex Ligation-dependent Probe Amplification (MLPA) discarded recurrent genomic and cytogenetic rearrangements. DNA extracted from the affected tissue (46%) or from lung or liver (54%) was analyzed by molecular karyotyping. Validations and inheritance were obtained by MLPA. We identified 22 rare copy number variants (CNV) [>100 kb, either absent (n = 7) or very uncommon (n = 15, <1/2,000) in the control population] in 20/95 fetuses with congenital malformations (21%), including 11 deletions and 11 duplications. One of the 9 tested rearrangements was de novo while the remaining were inherited from a healthy parent. The highest frequency was observed in fetuses with heart hypoplasia (8/17, 62.5%), with two events previously related with the phenotype. Double events hitting candidate genes were detected in two samples with brain malformations. Globally, the burden of deletions was significantly higher in fetuses with malformations compared to controls. CONCLUSIONS/SIGNIFICANCE: Our data reveal a significant contribution of rare deletion-type CNV, mostly inherited but also de novo, to human congenital malformations, especially heart hypoplasia, and reinforce the hypothesis of a multifactorial etiology in most cases.

Chronic lymphocytic leukemia (CLL) is a common disease with highly variable clinical course. Several recurrent chromosomal alterations are associated with prognosis and may guide risk-adapted therapy. We have developed a targeted genome-wide array to provide a robust tool for ascertaining abnormalities in CLL and to overcome limitations of the 4-marker fluorescence in situ hybridization (FISH). DNA from 180 CLL patients were hybridized to the qChip®Hemo array with a high density of probes covering commonly altered loci in CLL (11q22-q23, 13q14, and 17p13), nine focal regions (2p15-p16.1, 2p24.3, 2q13, 2q36.3-q37.1, 3p21.31, 8q24.21, 9p21.3, 10q24.32, and 18q21.32-q21.33) and two larger regions (6q14.1-q22.31 and 7q31.33-q33). Overall, 86% of the cases presented copy number alterations (CNA) by array. There was a high concordance of array findings with FISH (84% sensitivity, 100% specificity); all discrepancies corresponded to subclonal alterations detected only by FISH. A chromothripsis-like pattern was detected in eight cases. Three showed concomitant shattered 5p with gain of TERT along with isochromosome 17q. Presence of 11q loss was associated with shorter time to first treatment (P = 0.003), whereas 17p loss, increased genomic complexity, and chromothripsis were associated with shorter overall survival (P < 0.001, P = 0.001, and P = 0.02, respectively). In conclusion, we have validated a targeted array for the diagnosis of CLL that accurately detects, in a single experiment, all relevant CNAs, genomic complexity, chromothripsis, copy number neutral loss of heterozygosity, and CNAs not covered by the FISH panel. This test may be used as a practical tool to stratify CLL patients for routine diagnostics or clinical trials.

Progression of non-alcoholic fatty liver disease (NAFLD) in the context of metabolic syndrome (MetS) is only partially explored due to the lack of preclinical models. In order to study the alterations in hepatic metabolism that accompany this condition, we developed a model of MetS accompanied by the onset of steatohepatitis (NASH) by challenging golden hamsters with a high-fat diet low in vitamin E and selenium (HFD), since combined deficiency results in hepatic necroinflammation in rodents. Metabolomics and transcriptomics integrated analyses of livers revealed an unexpected accumulation of hepatic S-Adenosylmethionine (SAM) when compared with healthy livers likely due to diminished methylation reactions and repression of GNMT. SAM plays a key role in the maintenance of cellular homeostasis and cell cycle control. In agreement, analysis of over-represented transcription factors revealed a central role of c-myc and c-Jun pathways accompanied by negative correlations between SAM concentration, MYC expression and AMPK phosphorylation. These findings point to a drift of cell cycle control toward senescence in livers of HFD animals, which could explain the onset of NASH in this model. In contrast, hamsters with NAFLD induced by a conventional high-fat diet did not show SAM accumulation, suggesting a key role of selenium and vitamin E in SAM homeostasis. In conclusion, our results suggest that progression of NAFLD in the context of MetS can take place even in a situation of hepatic SAM excess and that selenium and vitamin E status might be considered in current therapies against NASH based on SAM supplementation.

Abstract Accurate annotations of genes and their transcripts is a foundation of genomics, but no annotation technique presently combines throughput and accuracy. As a result, reference gene collections remain incomplete: many gene models are fragmentary, while thousands more remain uncatalogued–particularly for long noncoding RNAs (lncRNAs). To accelerate lncRNA annotation, the GENCODE consortium has developed RNA Capture Long Seq (CLS), combining targeted RNA capture with third-generation long-read sequencing. We present an experimental re-annotation of the GENCODE intergenic lncRNA population in matched human and mouse tissues, resulting in novel transcript models for 3574 / 561 gene loci, respectively. CLS approximately doubles the annotated complexity of targeted loci, outperforming existing short-read techniques. Full-length transcript models produced by CLS enable us to definitively characterize the genomic features of lncRNAs, including promoter- and gene-structure, and protein-coding potential. Thus CLS removes a longstanding bottleneck of transcriptome annotation, generating manual-quality full-length transcript models at high-throughput scales. Abbreviations bp base pair FL full length nt nucleotide ROI read of insert, i.e. PacBio read SJ splice junction SMRT single-molecule real-time TM transcript model

BACKGROUND: The human chromosome 8p23.1 region contains a 3.8-4.5 Mb segment which can be found in different orientations (defined as genomic inversion) among individuals. The identification of single nucleotide polymorphisms (SNPs) tightly linked to the genomic orientation of a given region should be useful to indirectly evaluate the genotypes of large genomic orientations in the individuals. RESULTS: We have identified 16 SNPs, which are in linkage disequilibrium (LD) with the 8p23.1 inversion as detected by fluorescent in situ hybridization (FISH). The variability of the 8p23.1 orientation in 150 HapMap samples was predicted using this set of SNPs and was verified by FISH in a subset of samples. Four genes (NEIL2, MSRA, CTSB and BLK) were found differentially expressed (p<0.0005) according to the orientation of the 8p23.1 region. Finally, we have found variable levels of mosaicism for the orientation of the 8p23.1 as determined by FISH. CONCLUSION: By means of dense SNP genotyping of the region, haplotype-based computational analyses and FISH experiments we could infer and verify the orientation status of alleles in the 8p23.1 region by detecting two short haplotype stretches at both ends of the inverted region, which are likely the relic of the chromosome in which the original inversion occurred. Moreover, an impact of 8p23.1 inversion on gene expression levels cannot be ruled out, since four genes from this region have statistically significant different expression levels depending on the inversion status. FISH results in lymphoblastoid cell lines suggest the presence of mosaicism regarding the 8p23.1 inversion.

BACKGROUND: Distal motor neuropathies with a genetic origin have a heterogeneous clinical presentation with overlapping features affecting distal nerves and including spinal muscular atrophies and amyotrophic lateral sclerosis. This indicates that their genetic background is heterogeneous. PATIENT AND METHODS: In this work, we have identified and characterized the genetic and molecular base of a patient with a distal sensorimotor neuropathy of unknown origin. For this study, we performed whole-exome sequencing, molecular modelling, cloning and expression of mutant gene, and biochemical and cell biology analysis of the mutant protein. RESULTS: A novel homozygous recessive mutation in the human VRK1 gene, coding for a chromatin kinase, causing a substitution (c.637T > C; p.Tyr213His) in exon 8, was detected in a patient presenting since childhood a progressive distal sensorimotor neuropathy and spinal muscular atrophy syndrome, with normal intellectual development. Molecular modelling predicted this mutant VRK1 has altered the kinase activation loop by disrupting its interaction with the C-terminal regulatory region. The p.Y213H mutant protein has a reduced kinase activity with different substrates, including histones H3 and H2AX, proteins involved in DNA damage responses, such as p53 and 53BP1, and coilin, the scaffold for Cajal bodies. The mutant VRK1(Y213H) protein is unable to rescue the formation of Cajal bodies assembled on coilin, in the absence of wild-type VRK1. CONCLUSION: The VRK1(Y213H) mutant protein alters the activation loop, impairs the kinase activity of VRK1 causing a functional insufficiency that impairs the formation of Cajal bodies assembled on coilin, a protein that regulates SMN1 and Cajal body formation.

The combined analysis of haplotype panels with phenotype clinical cohorts is a common approach to explore the genetic architecture of human diseases. However, genetic studies are mainly based on single nucleotide variants (SNVs) and small insertions and deletions (indels). Here, we contribute to fill this gap by generating a dense haplotype map focused on the identification, characterization, and phasing of structural variants (SVs). By integrating multiple variant identification methods and Logistic Regression Models (LRMs), we present a catalogue of 35 431 441 variants, including 89 178 SVs (≥50 bp), 30 325 064 SNVs and 5 017 199 indels, across 785 Illumina high coverage (30x) whole-genomes from the Iberian GCAT Cohort, containing a median of 3.52M SNVs, 606 336 indels and 6393 SVs per individual. The haplotype panel is able to impute up to 14 360 728 SNVs/indels and 23 179 SVs, showing a 2.7-fold increase for SVs compared with available genetic variation panels. The value of this panel for SVs analysis is shown through an imputed rare Alu element located in a new locus associated with Mononeuritis of lower limb, a rare neuromuscular disease. This study represents the first deep characterization of genetic variation within the Iberian population and the first operational haplotype panel to systematically include the SVs into genome-wide genetic studies.

Intellectual disability (ID) has a prevalence of 1-3% and aproximately 30-50% of ID cases have a genetic cause. Development of next-generation sequencing has shown a high diagnostic potential. The aim of this work was to evaluate the diagnostic yield of clinical exome sequencing in 188 ID patients and the economic impact of its introduction in clinical practice. An analysis of diagnostic yield according to the different clinical variables was performed in order to establish an efficient diagnostic protocol for ID patients. Diagnostic yield of clinical exome sequencing was significant (34%) supporting its utility in diagnosis of ID patients. Wide genetic heterogeneity and predominance of autosomal dominant de novo variants in ID patients were observed. Time to diagnosis was shortened and diagnostic study costs decreased by 62% after implementation of clinical exome sequencing. No association was found between any of the variables analyzed and a higher diagnostic yield; added to the fact that many of the diagnoses weren't clinically detectable, the reduction of time to diagnosis and the economic savings with respect to classical diagnostic studies, strengthen the clinical and economical convenience of early implementation of clinical exome sequencing in the diagnostic workup of ID patients in clinical practice.

Predicting the therapeutic response to ocular hypotensive drugs is crucial for the clinical treatment and management of glaucoma. Our aim was to identify a possible genetic contribution to the response to current pharmacological treatments of choice in a white Mediterranean population with primary open-angle glaucoma (POAG) or ocular hypertension (OH). We conducted a prospective, controlled, randomized, partial crossover study that included 151 patients of both genders, aged 18 years and older, diagnosed with and requiring pharmacological treatment for POAG or OH in one or both eyes. We sought to identify copy number variants (CNVs) associated with differences in pharmacological response, using a DNA pooling strategy of carefully phenotyped treatment responders and non-responders, treated for a minimum of 6 weeks with a beta-blocker (timolol maleate) and/or prostaglandin analog (latanoprost). Diurnal intraocular pressure reduction and comparative genome wide CNVs were analyzed. Our finding that copy number alleles of an intronic portion of the MLIP gene is a predictor of pharmacological response to beta blockers and prostaglandin analogs could be used as a biomarker to guide first-tier POAG and OH treatment. Our finding improves understanding of the genetic factors modulating pharmacological response in POAG and OH, and represents an important contribution to the establishment of a personalized approach to the treatment of glaucoma.

In order to report clinically actionable incidental findings in genetic testing, the American College of Medical Genetics and Genomics (ACMG) recommended the evaluation of variants in 59 genes associated with highly penetrant mutations. However, there is a lack of epidemiological data on medically actionable rare variants in these genes in Arab populations. We used whole genome sequencing data from 6045 participants from the Qatar Genome Programme and integrated it with phenotypic data collected by the Qatar Biobank. We identified novel putative pathogenic variants in the 59 ACMG genes by filtering previously unrecorded variants based on computational prediction of pathogenicity, variant rarity and segregation evidence. We assessed the phenotypic associations of candidate variants in genes linked to cardiovascular diseases. Finally, we used a zebrafish knockdown and synthetic human mRNA co-injection assay to functionally characterize two of these novel variants. We assessed the zebrafish cardiac function in terms of heart rate, rhythm and hemodynamics, as well as the heart structure. We identified 52 492 novel variants, which have not been reported in global and disease-specific databases. A total of 74 novel variants were selected with potentially pathogenic effect. We prioritized two novel cardiovascular variants, DSP c.1841A > G (p.Asp614Gly) and LMNA c.326 T > G (p.Val109Gly) for functional characterization. Our results showed that both variants resulted in abnormal zebrafish heart rate, rhythm and structure. This study highlights medically actionable variants that are specific to the Middle Eastern Qatari population.

Hereditary hearing loss (HHL) is a common genetic disorder accounting for at least 60% of pre-lingual deafness in children, of which 70% is inherited in an autosomal recessive pattern. The long tradition of consanguinity among the Qatari population has increased the prevalence of HHL, which negatively impacts the quality of life. Here, we functionally validated the pathogenicity of the c.178G>C, p.E60Q mutation in the MYO6 gene, which was detected previously in a Qatari HHL family, using cellular and animal models. In vitro analysis was conducted in HeLa cells transiently transfected with plasmids carrying MYO6WT or MYO6p.E60Q, and a zebrafish model was generated to characterize the in vivo phenotype. Cells transfected with MYO6WT showed higher expression of MYO6 in the plasma membrane and increased ATPase activity. Modeling the human MYO6 variants in zebrafish resulted in severe otic defects. At 72 h post-injection, MYO6p.E60Q embryos demonstrated alterations in the sizes of the saccule and utricle. Additionally, zebrafish with MYO6p.E60Q displayed super-coiled and bent hair bundles in otic hair cells when compared to control and MYO6WT embryos. In conclusion, our cellular and animal models add support to the in silico prediction that the p.E60Q missense variant is pathogenic and damaging to the protein. Since the c.178G>C MYO6 variant has a 0.5% allele frequency in the Qatari population, about 400 times higher than in other populations, it could contribute to explaining the high prevalence of hearing impairment in Qatar.

ABSTRACT The combined analysis of haplotype panels with phenotype clinical cohorts is a common approach to explore the genetic architecture of human diseases. However, genetic studies are mainly based on single nucleotide variants (SNVs) and small insertions and deletions (indels). Here, we contribute to fill this gap by generating a dense haplotype map focused on the identification, characterization and phasing of structural variants (SVs). By integrating multiple variant identification methods and Logistic Regression models, we present a catalogue of 35,431,441 variants, including 89,178 SVs (≥50bp), 30,325,064 SNVs and 5,017,199 indels, across 785 Illumina high coverage (30X) whole-genomes from the Iberian GCAT Cohort, containing 3.52M SNVs, 606,336 indels and 6,393 SVs in median per individual. The haplotype panel is able to impute up to 14,360,728 SNVs/indels and 23,179 SVs, showing a 2.7-fold increase for SVs compared with available genetic variation panels. The value of this panel for SVs analysis is shown through an imputed rare Alu element located in a new locus associated with mononeuritis of lower limb, a rare neuromuscular disease. This study represents the first deep characterization of genetic variation within the Iberian population and the first operational haplotype panel to systematically include the SVs into genome-wide genetic studies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Functional annotation and interpretation of genetic variants are a critical step in genetic diagnosis, as it may lead to personalized therapeutic options and genetic counseling. While the number of confirmed pathogenic genetic variants in an individual is relatively low, the number of variants of uncertain significance (VOUS) can be considerably higher, increasing the number of potential carriers of genetic disorders. Thus, reducing uncertainty and assessing the real effect of VOUS are crucial for clinical and medical genetics. In this study, we evaluated the efficacy of genetic screening technologies in accurately predicting pathogenic variants and their corresponding disease prevalence in a cohort of over 6000 healthy individuals involved in assisted reproduction programs. Using data from 305 genes associated with recessive disorders, we determined the frequency of carriers of pathogenic variants and VOUS in our dataset and compared the predicted prevalence based on this information with reported population prevalence data. The higher predicted prevalence in some disorders when considering VOUS suggests a mostly benign effect.