ACTB Loss-of-Function Mutations Result in a Pleiotropic Developmental Disorder

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. 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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

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