995V Poster Online - Virtual Posters
Wednesday April 06, 4:00 PM - 7:00 PM

Authors:
Yan Huang ^1,2; Mengqi Ma ^1,2; Xiao Mao ^3,4; Davut Pehlivan ^1,5,6; Oguz Kanca ^1,2; Gulsen Akay ¹; Tadahiro Mitani ¹; Shenzhao Lu ^1,2; Sukru Candan ⁷; Bo Xiao ⁸; James Lupski ^1,6; Hugo Bellen ^1,2

Affiliations:
1) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA ; 2) Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas, USA ; 3) National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China; 4) Department of Medical Genetics, Maternal and Child Health Hospital of Hunan Province, Changsha, Hunan, China; 5) Division of Neurology and Developmental Neuroscience, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA ; 6) Texas Children’s Hospital, Houston, Texas, USA ; 7) Medical Genetics Section, Balikesir Ataturk Public Hospital, Balikesir, Turkey; 8) Neurology Department, Xiangya Hospital, Central South University, Changsha, Hunan, China

Keywords:
b. neural disorder; a. axon guidance

The Roundabout (Robo) receptors present on growth cones of neurons induce axon repulsion in response to the extracellular ligand Slit. The Robo family of proteins controls midline crossing of commissural neurons during development in vertebrate and invertebrate model organisms. Mono- and bi-allelic loss-of-function (LoF) of human ROBO1 (MIM: 602430) has been associated with a breath of phenotypes, including neurodevelopmental defects such as strabismus, pituitary defects, intellectual impairment, as well as non-neuronal defects in heart and kidney, with reduced penetrance and highly variable expression. We identified two novel ROBO1 variants associated with distinct phenotypes. In family #1, we identified a biallelic missense (p.S1522L) variant in three affected siblings with a recessive trait isolated nystagmus. In family #2, we identified a de novo p.D422G variant in the proband who presented with a severe early-onset epileptic encephalopathy. To assess the perturbance to biological homeostasis for these variants, we performed functional assays in Drosophila. We generated a null allele of robo1 by inserting a CRIMIC T2A-GAL4 in an intron. To our surprise, transheterozygous robo1 null mutation (T2A-Gal4/Df) leads to reduced viability but not lethality. In contrast, overexpression of either human ROBO1 or fly robo1 is toxic but also reduces viability. Note that human ROBO1 is not able to replace fly robo1 when driven by the Gal4 insertion. The fly cDNA driven by the Gal4 rescues the midline crossing at 18^oC. However, the p.D413G variant in the fly cDNA fails to rescue midline crossing suggesting that it is a LoF allele. The recessive ROBO1 variant cannot be tested as the p.S1522 is not conserved in fly robo1.
We therefore turned to gain-of-function (GoF) assays using the T2A-GAL4 to drive the human reference cDNA which leads to toxicity. ROBO1 p.S1522L is less toxic than the reference human cDNA with respect to viability and midline crossing. In contrast, the dominant p.D413G variant leads to a highly aberrant protein distribution of fly Robo1 and creates novel defects in several assays. This suggests that it is a neomorphic allele. In summary, our studies expand the phenotypic spectrum associated with ROBO1 variant alleles and assesses the potential nature of the variants.