Bi-allelic loss-of-function variants in BCAS3 cause a syndromic neurodevelopmental disorder

BCAS3 microtubule-associated cell migration factor (BCAS3) is a large, highly conserved cytoskeletal protein previously proposed to be critical in angiogenesis and implicated human embryogenesis tumorigenesis. Here, we established loss-of-function variants as causative for neurodevelopmental disorder. We report 15 individuals from eight unrelated families with germline bi-allelic BCAS3. All probands share global developmental delay accompanied by pyramidal tract involvement, microcephaly, short stature, strabismus, dysmorphic facial features, seizures. The phenotype less severe compared the Bcas3 knockout mouse model cannot explained angiogenic defects alone. Consistent being alleles, observed absence of probands’ primary fibroblasts. By comparing transcriptomic proteomic data based on fibroblasts those model, identified similar dysregulated pathways resulting over-representation analysis, while dysregulation some key interactors could not confirmed. 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Craigen W.J. al.Undiagnosed Diseases NetworkDe Novo ATPase Module MORC2 Cause Neurodevelopmental Disorder Growth Retardation Variable Craniofacial Dysmorphism.Am. 107: 352-363Abstract (17) dermal maintained 37°C, 5% CO2, 100% relative humidity fibroblast medium consisting Dulbecco’s modified Eagle’s (DMEM) (Merck) supplemented 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) culture flasks. Cells split trypsinization passaged into flasks seeded defined density Oris assay. Fibroblast investigated plate (Platypus Technologies). Control (four lines) (two isolated 6 × 104 per 96-well manufacturer’s guidelines (5 wells line). preincubation step 24 h, stoppers removed incubated 30 h. Afterward, stained ActinRed 555 (Sigma) instructions imaged Operetta High Content Imaging System (Perkin Elmer). Using ImageJ, calculated covered area well. Upon reaching confluence, washed cold PBS, scraped off centrifuged 800 g 5 min, frozen ?80°C. Pellets lysed RIPA buffer containing protease inhibitors (Roche) 45 min rotator 4°C. Cell debris pelleted 15,800 4°C min. concentration determined Pierce BCA Protein Assay Kit instructions. 10 ?g eluted 5× pink 95°C. Samples separated polyacrylamide gels transferred onto Hypond-P polyvinylidene difluoride (PVDF) membrane (Merck). Membranes blocked milk TBS-T overnight antibodies against rabbit ?-BCAS3 (1:1,000, Bethyl Laboratories), ?-CDC42 (1:5,000, Abcam), ?-Vimentin Signaling Technologies), ?-GAPDH (1:20,000, Meridian Life Sciences) Western Blocking Reagent 4°C, followed washes incubation HRP-conjugated secondary (Jackson ImmunoResearch) 1 h room temperature. Proteins visualized Immobilon chemiluminescent HRP substrate For extraction, lines (F3-II.1 F4-II.1) control (CO-1, female, years old CO-2, male, 22 old) cultivated. independently grown each line (biological replicates, 4 3 samples) QIAGEN RNeasy Mini Kit. quality assessed Agilent 2100 Bioanalyzer Nano (Agilent Technologies, Santa Clara, CA, United States). samples had integrity numbers (RIN > 9). NEBNext Ultra II Directional Prep (New Biolabs, Ipswich, MA, States) ng total input library, generated poly(A)+-selected libraries manual. sequenced Illumina NovaSeq 6000 platform paired-end mode 2 51 bp reads depth approximately million clusters each. same individual, design aimed minimize introduction technical batch effects chosen. raw (RNA-seq) FASTQ files ReadQC (v.2019_11) identify potential cycles low average base distribution bias. Reads preprocessed SeqPurge aligned STAR (v.2.7.3a), allowing spliced read alignment reference (build GRCh37). Alignment MappingQC (ngs-bits v.2019_11) visually inspected Broad Integrative Genome Viewer (v.2.7.0). On basis Ensembl annotation (GRCh37, release 97), counts all subread (v.2.0.0). retain only least count (cpm) half normalized them trimmed mean M values (TMM) procedure, leaving >13,000 determining differential pairwise comparisons samples. edgeR (v.3.26.8) negative binomial distributions genewise testing generalized linear models. proband, triplicate profiles corresponding both sets lines. addition, (Table S2). Gene changes expressed log2-fold group baseline. Significant adjusted p value (false discovery rate [FDR]) than 0.01 absolute change reported. liquid chromatography-mass spectrometry (nanoLC-MS/MS) replicates label-free quantification [LFQ] intensity [pat]_1, 2, LFQ pat_4, 5, Table S4) (LFQ [ctrl]_1, 3). extracts purified SDS-PAGE (Invitrogen). Coomassie-stained gel pieces excised in-gel digested trypsin described previously.16Borchert Dieterich Krug Schütz W. 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Crosses up 25°C 29°C kept incubators 12-h day-night cycle. following utilized this study: UAS-RNAi rudhira, UAS-rudhira-RNAi-a (Bloomington Stock [BDSC] #51691, RNAi inserted 3rd chromosome) UAS-rudhira-RNAi-b (Vienna Resource [VDRC ID dna9673]); GAL4 driver elav-Gal4 (BDSC #8765, BDSC #5144), D42-Gal4 #8816), Appl-Gal4 (from Aaron Voigt, Department Neurology RWTH Aachen University); rudhira-RNAi-a #36304, TRiP background attP40 chromosome); UAS-GFP-RNAi #9331, GFP 2nd second line. To generate transgenic line, diluted construct received VDRC (1 ?L ?50–100 ng/?L stock) adding TE mixed thoroughly. DH5?-competent transformed mixture. Agar plates ampicillin plated 37°C. Individual colonies picked inoculated plasmid purification. Purified sent Bestgene (BestGene, embryo microinjection. unravel disorder, F1, F2, F3 (Figure 1) WES. molecular these families. WES next screened variants, heterozygous, variants. Independently, families, predicted identified, namely, stop-gain c.73C>T (p.Gln25?) 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Mutat. 36: 928-930Crossref (624) matched responsible GENESIS database, Solve-RD Munich (EVAdB), (BH-CMG) 100KGP, GeneDx, Queen Square additional strengthen evidence genotype-phenotype association. way, able five total, studied 1A), six latter anticipated given historical reports consanguinity majority very (minor [MAF] 0.01% gnomAD). residues (phyloP 100-way 7.49 9.48) silico prediction scores (CADD 26.4 24.1) 1B, S1). F8-II.1, combined trio led intragenic spanning exons 13 23 (chr17: g.60921177_61232193del [c.994?3230_2426?136134del]) maternally near splice-site bases (c.2074+4_2074+7delAGTA). genomes father sequencing. Breakpoint locations microhomology Alu-LINE (long interspersed nuclear element) rearrangement27Song Beck C.R. Du Coban-Akdemir Gu Breman A.M. Stankiewicz Ira Lupski J.R. Predicting susceptible instability Alu/Alu-mediated rearrangements.Genome 1228-1242Crossref (35) mechanism harboring presented characteristic core (GDD), stature 1, Figure 2A). At time examination, 19 months age. GDD (ID) (F3-II.2 too young formally diagnosed ID). Ten minimal vocabulary (between words), four never learned speak. motor involvement hyperreflexia spasticity lower limbs (15/15), dystonic dyskinetic movements (5/15). 14 achieved ability walk, although milestone significantly delayed. Regression abilities starting ages older probands. Seizures 7 probands—two febrile convulsions tonic tonic-clonic Only one pharmaco-resistant epilepsy.Table 1Clinical findings variantsF1-II.1F1-II.2F2-II.2F2-II.3F2-II.7F3-II.1F3-II.2F4-II.1F5-II.1F5-II.2F6-II.1F6-II.2F7-II.1F7-II.2F8-II.2F9-II.1cDNA NM_001099432.3)c.73C>T, homc.73C>T, homc.726T>G , homc.1457C>G, homc.[1700C>T]; [1729G>A]c.2227C>T, homc.2227C>T, homc.1133delT, homc.530delT, homc.2074+4_2074+7delAGTA; intragentic