Abstract
Motor, sensory, and integrative activities of the brain are coordinated by a series of midline-bridging neuronal commissures whose development is tightly regulated. Here we report a new human syndrome in which these commissures are widely disrupted, thus causing clinical manifestations of horizontal gaze palsy, scoliosis, and intellectual disability. Affected individuals were found to possess biallelic loss-of-function mutations in the gene encoding the axon-guidance receptor 'deleted in colorectal carcinoma' (DCC), which has been implicated in congenital mirror movements when it is mutated in the heterozygous state but whose biallelic loss-of-function human phenotype has not been reported. Structural MRI and diffusion tractography demonstrated broad disorganization of white-matter tracts throughout the human central nervous system (CNS), including loss of all commissural tracts at multiple levels of the neuraxis. Combined with data from animal models, these findings show that DCC is a master regulator of midline crossing and development of white-matter projections throughout the human CNS.
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Acknowledgements
We thank A. Rozzo, J. Partlow, B. Barry, and R. Hill for logistical and administrative assistance; C. Carruthers, research assistant in FNNDSC, for creating and compressing the DTT movie files; and H. Somhegyi for expert illustrations. We thank the individuals and their families reported on herein for their participation in this research. This research was supported in part by the Repository Core for Neurological Disorders, Department of Neurology, Boston Children's Hospital, and the IDDRC (NIH P30HD018655). S.S.J. is supported by the National Medical Research Council, Singapore, and the Singhealth-Duke NUS Paediatric Academic Programme Nurturing Clinician Scientist Scheme. A.M.D. is supported by the National Institute of General Medical Sciences (T32GM07753) and a National Institutes of Health Ruth L. Kirschstein National Research Service Award (5T32 GM007226-39). E.C.E.'s contributions to this work were supported by NEI R01EY12498, IDDRC grant P30 HD018655, and the Manton Center for Orphan Disease Research. C.A.W. is supported by grants from the National Institute of Mental Health (R01MH083565), the National Institute of Neurological Disorders and Stroke (R01NS032457 and R01NS035129), the Simons Foundation, and the Manton Center for Orphan Disease Research. C.A.W. and E.C.E. are supported as Investigators of the Howard Hughes Medical Institute. T.W.Y.'s contributions to this work were supported by grants from the National Institute of Mental Health (R01MH083565), the Simons Foundation, and the Nancy Lurie Marks Foundation.
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S.S.J., E.C.E., C.A.W., and T.W.Y. designed experiments. K.S.-A. and K.M. performed the homozygosity and CNV analysis. S.S.J., A.M.D., A.-T.N.L., and S.S. performed the ddPCR, junction PCR, and RT–PCR. W.-M.C. performed Sanger sequencing of family 2. M.P., M.D., and P.E.G. performed the DTI and tractography analyses. M.R.D., N.J.C., M.G., Z.A.Z., M.A.D., A.A.J., K.A.-A., and T.M.B. performed the phenotypic assessment of the patients. A.J.B., C.A.W., M.D., M.P., C.D.R., I.A.A., P.E.G., E.C.E., and T.W.Y. reviewed the MRI imaging studies. W.W. performed the phenotypic assessment of patient 3, and E.S.L. performed the in situ hybridization analyses. S.S.J., E.C.E., C.A.W., and T.W.Y. wrote the manuscript. All coauthors reviewed and approved the final version of the submitted manuscript.
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Integrated supplementary information
Supplementary Figure 1 Additional clinicoradiographic features of DCC−/− syndrome.
(a) Horizontal gaze palsy as evidenced by failure of horizontal movements of the eye, with preservation of upward and downward gaze. (b-g) Agenesis of the corpus callosum. Axial and sagittal images showing ACC and variable interhemispheric cyst in Family 1: II:5 (b) and Family 1: II:7 (c). Axial view: control (d) vs. patient (Family 1: II:7) (f). Coronal view: control (e) vs. patient (Family 1: II:7) (g).
Supplementary Figure 2 Homozygosity mapping.
(a) Homozygosity analysis of the two affected individuals of Family 1 identified multiple regions of homozygosity across the genome. Observed homozygosity for the two siblings (II:5 and II:7) was 3.9% and 4.9% of the genome. (b) The observation is consistent with first and second cousin marriage (expectation 6.25% and 1.56%, respectively, as illustrated in the bottom figure; however, the distributions are broad, and there is not enough information to distinguish between the two). Note: only homozygous regions with 2 cM or longer are shown.
Supplementary Figure 3 Homozygosity mapping and copy-number analysis.
Analysis identified a block of homozygosity on chromosome 18, bounded by SNP markers rs10502546 and rs9950483 and included 139 UCSC genes (homozygous SNPs are shown in red or blue, heterozygous SNPs are shown in green, and SNPs for which no genotype could be assigned are shown in white). Copy number analysis (using Variant Explorer) identified a rare homozygous deletion (orange box) on chromosome 18q21.2 near exon 1 of DCC, and a common non-genic homozygous deletion on chromosome 18q12.3.
Supplementary Figure 4 Junction PCR.
(a) Digital droplet PCR using probes located within intron 1 of DCC (chr18: 49869550 (hg19) and chr18: 49871307 (hg19)) confirmed the homozygous deletion in both affected siblings (II:5 and II:7 from Family 1) and showed that their unaffected mother (I:2) was heterozygous for the deletion. (b) Schematic of the DCC gene with the predicted deletion near exon 1 (grey boxes). SNPs in blue indicate where copy number 2 was detected on the SNP array, and SNPs in green indicate where copy number 0 was detected on the SNP array, indicative of maximum and minimum boundaries of the predicted deletion, respectively. Nested PCR primers spanning the predicted deletion are indicated by F →, R ←. (c) Amplification with primers F5+R2 and F6+R2 yielded PCR products containing the deletion breakpoint in both the affected siblings (II:5 and II:7) and their mother (I:2), but not from an unrelated control individual. (d) Amplification with primers F2+R0 and F1+R0 yielded PCR products in the control sample, but non-specific bands in the affected individual II:7, confirming that the region can be amplified in the control sample.
Supplementary Figure 5 RT–PCR.
(a) Schematic representation of primers (forward →, reverse ←) used in DCC RT-PCR experiments to show selective loss of all products containing exon 1 in the affected individual II:7 (from Family 1) but not in the control. (b) RT-PCR on RNA demonstrated loss of all products containing exon 1 in the affected individual but not in the control. (c) qPCR on RNA demonstrated non-expression of exon 1 the in affected individual. The homozygous deletion identified in two affected individuals from Family 1 partially deletes exon 1 of the predominant DCC isoform that is expressed in normal adult and fetal brain, X76132 (Refseq mRNA NM_005215.3).
Supplementary Figure 6 Diffusion tensor tractography.
(a) Whole brain diffusion tensor tractography (DTT) fiber tract colored direction map of Family 2 (DCC) in comparison to an age and gender matched control individual and a subject with isolated agenesis of corpus callosum (ACC) (red, left right orientation; green, anterior posterior orientation; blue, superior inferior orientation), showing anterior, posterior coronal view, left and right sagittal views. (b) Whole brain fiber tract-fractional anisotropy (red/yellow scalar map) of control, DCC and ACC subject (yellow = 0 or low anisotropy, red = 1 or high anisotropy), showing anterior, posterior coronal view, left and right sagittal views.
Supplementary Figure 7 Diffusion tensor tractography.
(a,b) Diffusion Tensor Imaging (DTI) of the corpus callosum, obtained by placing a region of interest on the four midline sagittal slices; left and right hemisphere contribution to the tracts were visualized (two left hemisphere slices, two right hemisphere slices) and pseudocolored yellow and blue. A mixture of yellow and blue fibers, representing the left and right hemisphere tract reconstructions, and the presence of red fibers indicate a well-defined interhemispheric crossing in the control. In individual II:1 from Family 2, there appears to be no midline region of interest corresponding to the corpus callosum. Instead, in each hemisphere, a distinct longitudinal tract known as the Probst bundle can be visualized; each hemisphere seems to have a separate connection to the frontal, motor strip, and occipital areas, but these fibers do not extend into the contralateral hemisphere. Solid white line: genu of corpus callosum. Dashed white line: splenium of corpus callosum. Dashed orange line: anterior commissure.
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Jamuar, S., Schmitz-Abe, K., D'Gama, A. et al. Biallelic mutations in human DCC cause developmental split-brain syndrome. Nat Genet 49, 606–612 (2017). https://doi.org/10.1038/ng.3804
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DOI: https://doi.org/10.1038/ng.3804
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