Journal: Brain

Article Title: Combined genomics and proteomics unveils elusive variants and vast aetiologic heterogeneity in dystonia

doi: 10.1093/brain/awaf059

Figure Lengend Snippet: Linearly growing number of dystonia-associated genes in the WES cohort . ( A ) Recruitment sites in the study with inclusion of normally underrepresented patient groups and the associated overall diagnostic rates achieved by WES. Geographical areas with underserved dystonia populations and new recruitment foci are highlighted in green. ( B ) Cumulative dystonia patient ascertainment for WES over time, with the number of index patients analysed at the time cut-offs of the study in 2019 –2021 –2024 represented by the size of each point; a detailed description of the recruitment process is provided elsewhere. The number of identified disease genes increased with increasing cohort size with no signs of plateauing. The diagnostic yield was relatively stable at ∼19%–22%. Disease genes identified by WES in the entire cohort over a decade are as follows [alphabetical order; 141/205 (68.8%) found in a single family only; for details, see ]: AARS1 , ACTB , ADAR , ADCY5 , AFG3L2 , ALS2 , ANK2 , ANO3 , AOPEP , ARHGEF9 , ARSA , ASXL3 , ATL1 , ATM , ATP1A3 , ATP2B2 , ATP5F1A , ATP5F1B , ATP5MC3 , ATP7B , ATP8A2 , AUTS2 , BCL11B , BRAF , BRPF1 , C19orf12 , CACNA1A , CACNA1E , CAMK4 , CAMTA1 , CASK , CD40LG , CHD3 , CHD4 , CHD8 , CNTNAP1 , COQ8A , CP , CSDE1 , CTNNB1 , CUL3 , CUX1 , CWF19L1 , DCAF17 , DDC , DHCR24 , DHDDS , DLG4 , DLL1 , DNAJC6 , DNM1L , DNMT1 , EBF3 , ECHS1 , EEF1A2 , EFTUD2 , EIF2AK2 , EIF4A2 , ERCC4 , ERCC8 , FA2H , FBXO31 , FGF14 , FITM2 , FOXG1 , FOXP2 , FRMD5 , FRYL , FTL , GABBR2 , GABRA1 , GAD1 , GCH1 , GJA1 , GJC2 , GNAL , GNAO1 , GNB1 , GRIA2 , GRIA3 , GRID2 , GRIN1 , GRIN2A , HECW2 , HEXA , HIBCH , IFIH1 , IMPDH2 , INTS11 , IRF2BPL , KCNA2 , KCNB1 , KCNJ10 , KCNMA1 , KCTD17 , KIF1A , KIF5A , KMT2B , LIG4 , LRRK2 , MAG , MATR3 , MECP2 , MECR , MED23 , MICU1 , MMAA , MORC2 , MRE11 , MSL3 , NAA15 , NARS2 , NAV3 , NEFL , NFIX , NGLY1 , NKX2-1 , NPC1 , NR4A2 , NUP54 , OPA1 , PAK1 , PANK2 , PARK7 , PCDH12 , PDE10A , PDHA1 , PINK1 , PLA2G6 , PNKD , PNPLA6 , POGZ , POLG , POLR1A , POLR3A , PPP2R5D , PPT1 , PRKCG , PRKN , PRRT2 , PSEN1 , PTS , PURA , RALA , RARB , RERE , RHOBTB2 , SATB1 , SCN2A , SCO2 , SCP2 , SERAC1 , SETX , SGCE , SHANK3 , SHQ1 , SLC16A2 , SLC19A3 , SLC20A2 , SLC2A1 , SLC6A1 , SLC6A3 , SLC9A6 , SNAP25 , SNX14 , SON , SOX2 , SOX6 , SPAST , SPG11 , SPG7 , SPR , SPTBN1 , SRRM2 , SUCLG1 , SUOX , SYNE1 , TBC1D24 , TBCD , TBX1 , TCF20 , TECPR2 , TFE3 , TH , THAP1 , TMEM240 , TOR1A , TTPA , TUBB4A , UBE3A , UBTF , VLDLR , VPS16 , WAC , WARS2 , WASHC5 , WDR45 , WDR73 , WFS1 , YY1 , ZC4H2 , ZEB2 , ZMYND11 , ZNF142 , ZNF335 . ( C ) Pedigrees for three families (black fill, individual with dystonia; grey fill, individual with neurodevelopmental phenotype without dystonia; index patients indicated with arrows) with ANK2 heterozygous predicted loss-of-function (pLoF) variants, and the positions of the variants mapped to the ANK2 protein sequence; ANK2 pLoF mutations previously reported in autism and other NDDs are shown in the protein schematic for comparison ( bottom ). Patient ANK2-GM-A was identified via GeneMatcher. The dystonia-related ANK2 variants p.Glu346* and p.Thr1269Hisfs*19 were absent from gnomAD v4.1.0; p.Arg1427* was present in a single gnomAD-v.4.1.0 subject, as seen for an increasing number of NDD-causing variants associated with variable expressivity ; p.Arg1427* has also been identified in independent clinically affected individuals (listed as ‘likely pathogenic’ in ClinVar, ID: 3338732). Immunoblotting performed on fibroblasts from one WES-cohort individual (M-WES-S143) and three controls (C1–C3) showed significantly reduced ANK2 expression in the patient, compatible with the described haploinsufficiency mechanism of the disorder. Blots are representative of three biological replicates (see also ); in bar plots for quantification, results are shown as mean ± standard deviation represented by error bars (statistical significance determined by Student’s t -test). +/− = monoallelic variant carrier; +/+ = homozygous reference allele; ANK2 , NM_001148.6. ( D ) Burden testing to demonstrate significant enrichment of heterozygous CHD3 pLoF variants in adult-onset isolated dystonia. Differences in carrier rates of rare (MAF < 0.0005) pLoF variants (defined as nonsense, frameshift and splice-site alterations) between all individuals with late adulthood-onset isolated dystonia in the WES cohort ( n = 303) and controls from gnomAD-v.2.1.1 [non-Finish European (NFE) subset, n = 64 603] were determined according to established methods (Test Rare vAriants with Public Data/TRAPD approach). , A flow chart of the analysis strategy is shown. Quantile-quantile plots for the study of NDD-associated genes ( n = 1615; SysNDD database ) and all CCDS genes ( n = 20 000) highlight a single significant signal for CHD3 ( P < 3.1 × 10 −5 and P < 2.5 × 10 −6 , respectively, indicated with dashed horizontal lines; Fisher’s exact test; genomic inflation factor λ is provided). CCDS = consensus coding sequence; gnomAD = Genome Aggregation Database; MAF = minor allele frequency; NDD = neurodevelopmental disorder; WES = whole-exome sequencing; WGS = whole-genome sequencing.

Article Snippet: Total protein lysates processed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were probed with the following primary antibodies: ANK2 (Santa Cruz, sc-12718, 1:1000), GLS (GeneTex, GTX131263, 1:5000), GAPDH (Sigma, G8795-25UL, 1:20 000) and β-tubulin (abeomics, 11-13002, 1:5000).

Techniques: Diagnostic Assay, Sequencing, Comparison, Western Blot, Expressing, Standard Deviation, Variant Assay, Isolation