RAD51 haploinsufficiency causes congenital mirror movements in humans

Abstract

Congenital mirror movements (CMM) are characterized by involuntary movements of one side of the body that mirror intentional movements on the opposite side. CMM reflect dysfunctions and structural abnormalities of the motor network and are mainly inherited in an autosomal-dominant fashion. Recently, heterozygous mutations in DCC, the gene encoding the receptor for netrin 1 and involved in the guidance of developing axons toward the midline, have been identified but CMM are genetically heterogeneous. By combining genome-wide linkage analysis and exome sequencing, we identified heterozygous mutations introducing premature termination codons in RAD51 in two families with CMM. RAD51 mRNA was significantly downregulated in individuals with CMM resulting from the degradation of the mutated mRNA by nonsense-mediated decay. RAD51 was specifically present in the developing mouse cortex and, more particularly, in a subpopulation of corticospinal axons at the pyramidal decussation. The identification of mutations in RAD51, known for its key role in the repair of DNA double-strand breaks through homologous recombination, in individuals with CMM reveals a totally unexpected role of RAD51 in neurodevelopment. These findings open a new field of investigation for researchers attempting to unravel the molecular pathways underlying bimanual motor control in humans.

Figure 1

Identification of RAD51 Mutations in Two Independent Families (A) LOD score plot for genome-wide linkage analysis in Family A, revealing a single locus with a maximal multipoint LOD score value on chromosome arm 15q (Z = +2.4). Twenty-six family members (7 symptomatic individuals, 3 obligate carriers, 12 at-risk asymptomatic relatives, and 4 spouses) were genotyped by linkage-24 microarrays (Illumina). Multipoint LOD scores were calculated with Merlin 1.0 (affected-only analysis, autosomal-dominant trait, disease allele frequency of 0.00001, penetrance of 80%, null phenocopy rate). All regions with LOD scores >−2 other than that on chromosome 15 were further analyzed with microsatellite markers and excluded on the basis of the absence of a common haplotype in all affected family members. (B) Refinement of the chromosome 15 interval with eight microsatellite markers (D15S1010, D15S102, D15S221, D15S129, D15S514, D15S659, D15S119, D15S982) showing a common haplotype segregating in all affected family members. Multipoint LOD scores were recalculated from the microsatellite markers in the chromosome 15 interval via Allegro 1.0 with the same parameters as those previously used (Zmax = +2.7). (C) Confirmation of the c.760C>T (p.Arg254^∗) mutation in RAD51 by Sanger sequencing in Family A. (D) The coding region of RAD51 was amplified with 11 primer pairs (sequences available on request) in the index cases of Families B (from Germany) and C (from UK). The c.855dupA (p.Pro286Thrfs^∗37) mutation in RAD51 was identified in Family B (pedigree).

Figure 2

Downregulation of RAD51 mRNA in Lymphoblasts from Affected Individuals and Degradation of the Mutated mRNA by Nonsense-Mediated Decay In parallel with the whole-exome analysis, we used transcriptomic analysis to identify the gene responsible for MM in Family A, postulating that the mutation might decrease the mRNA expression in lymphoblasts from affected individuals compared to healthy spouses from the same family. Total RNA extracted from lymphoblasts of four affected individuals and three spouses were hybridized on lllumina HumanHT12 beadchips. Expression profiles were extracted and normalized with Bead studio software (Illumina). Normalized expression data were log2 transformed. The 131 genes expressed on bead chips from the 223 candidate genes on chromosome 15 were included for further analysis. Two complementary statistical analyses by two independent investigators were performed to identify genes differentially expressed between the groups. Results of classic class comparison analysis are presented in Table 2. (A) Gene clustering approach with the pattern discovery tool of GeneATWork software (IBM Research). The filtering criteria for a gene's inclusion in a pattern are a maximum deviation of 0.05 and a p value of 0.001. The best patterns classifying the “phenotype” and “control” groups were retained. This approach distinguished the affected individuals from the controls on the basis of four genes underexpressed in the affected subjects. Among these four genes (CATSPER2P1, CAPN3, RAD51, and PLA2G4B), only two (RAD51 and CAPN3) were expressed in the brain. (B) RAD51 was the only gene lying at the intersection of the gene lists obtained with the two statistical analyses. (C) Expression data obtained for RAD51 on HumanHT12 beadchips. (D) Lymphoblastic cells from three affected individuals and three asymptomatic spouses from Family A were treated overnight with 10 μg/ml emetin to inhibit nonsense-mediated decay (NMD). Total RNA was extracted with the QIAGEN RNeasy Mini kit (Invitrogen) and reverse-transcribed with the SuperScript III First-Strand Kit (Invitrogen). RAD51 cDNA was amplified and sequenced with specific primers located in exons 7 (Forward) and 10 (Reverse). Chromatograms for one affected individual and one spouse, showing lower levels of mutated mRNA compared to WT mRNA in untreated cells and a comparable expression levels of both mRNA in cells pretreated with emetin, are shown.

Figure 3

Comparative Expression and Localization of RAD51 and DCC in Developing Mouse Cortex (A) Quantification of RAD51 and DCC expression in mouse cerebral cortex sampled in quadruplicate at several stages of development (E12, E15, E18, P1, P7, P15, 1 month, and 3 months) by real-time PCR. Quantification of each sample was carried out with the QIAGEN QuantiTect primer assays for DCC and RAD51. PPIA and PGK1 were used as control genes. Each sample was run in triplicate on a Lightcycler-1536 apparatus (Roche). Forty-five two-step cycles (15 s at 95°C and 30 s at 60°C) were performed. Analysis was performed with qbase Plus software (Biogazelle). (B–G) Sagittal sections of the neocortex of E12 mouse embryos, fixed overnight in paraformaldehyde 4%, and immunolabeled with anti-RAD51 (1/50, sc-6862, Santa Cruz Biotechnology, Santa Cruz, CA) (B–E) or anti-DCC (1/100, sc-6535, Santa Cruz) (F and G) and counterstained with DAPI (C, E, G). Confocal plane (D, E). Scale bar represents 220 μm (B, C, F, G) or 15 μm (D, E).

Figure 4

Comparative Localization of RAD51 and DCC in Mouse Brain at Postnatal Stages (A–H) Cortical coronal section of a newborn (P0) mouse triple immunostained with anti-TBR1 (1/500, Millipore, Molsheim, France) to label the subplate and layer VI, anti-CTIP2 (1/500, Abcam, Cambridge, UK) to label layer V, and either anti-RAD51 (1/50, sc-6862, Santa Cruz) (A, B) or anti-DCC (1/100, sc-6535, Santa Cruz) (C, D), or immunolabeled only with anti-RAD51 (E, F) or anti-DCC (G, H) and counterstained with DAPI (F, H). (I and J) Coronal section of a P2 mouse at the pyramidal decussation, immunostained with anti-RAD51 (I, J) and anti-PKCγ (1/100, sc211, Santa-Cruz) to label the corticospinal tract (J). (I′ and J′) Enlargements of (I) and (J); arrows point to the same area. Scale bar represents 120 μm (A–D), 30 μm (E–H), 250 μm (I, J), or 100 μm (I′, J′).