Molecular Genetics of Distal Hereditary Motor Neuropathies: Modelling the DHMN1 Complex Insertion

Abstract

Distal hereditary motor neuropathies (dHMN) are a group of progressive diseases with length-dependent axonal degeneration primarily affecting the lower motor neurons of the peripheral nervous system. This causes denervation of distal muscles resulting in atrophy and paresis affecting the lower limbs leading to chronic disability. Using whole genome sequencing we recently identified a large chromosomal insertion within the DHMN1 locus on chromosome 7q34-q36.2 (DHMN1; OMIM %182960) in a large Australian family (F-54) (Drew et al. Hum Genet. 2016). The complex insertion DNA re-arrangement involves a 1.35 Mb region of chromosome 7q36.3 that has been duplicated and inserted into the DHMN1 disease locus in the reverse orientation. The genomic structure of genes within the DHMN1 locus, are not disrupted by the insertion re-arrangement. Furthermore, previous genetic analysis of F-54 excluded all protein-coding point mutations and copy number variations within the locus. Structural variation represents a major source of variability in the human genome that can potentially contribute to disease. Our discovery represents a new disease mechanism for dHMN. Structural variations such as the DHMN1 insertion could act by several mechanisms including gene dosage changes, disrupting the local gene regulatory environment or by altering chromatin organisation. For this project the hypothesis is that the DHMN1 complex insertion will dysregulate one or more genes within or adjacent to the DHMN1 linkage region leading to the subsequent axonal degeneration of lower motor neurons. The overall aim is to further characterise the DHMN1 insertion and determine the effect of the large genomic re-arrangement on gene regulation. This will involve: 1) developing and establishing a spinal motor neuron (sMN) model for DHMN1 using induced pluripotent stem cell (iPSC) technologies, 2) Examining DHMN1 iPSC-derived sMN for axonal degeneration and abnormal pathology and function, 3) Examining both the local and global gene expression in patient and control sMN using NanoString and RNA-seq technologies. In this PhD, the development of a neuronal model of DHMN1 was dependent on having appropriate protocols in place for the differentiation of sMN from iPSC lines reprogrammed from patient fibroblasts. Following a period of training in iPSC culture and neuronal differentiation techniques at the University of Miami, I facilitated the transition of iPSC and differentiation protocols into the Northcott Neuroscience Laboratory at the ANZAC Research Institute. To address the aims of this project, it was important that pure sMN populations were generated in high quantity with a short turn-around time. This was not feasible with the original protocols transitioned into the laboratory from Miami. A review of the literature identified several key elements lacking from the initial transitioned protocols that could address issues of yield, purity and flexibility. Therefore, a protocol was implemented that incorporated: 1) a 3-D culture step; 2) small molecule, chemical purification of heterogeneous neural cell mixtures and 3) a flexible workflow allowing for the of cryopreservation and biobanking of intermediate cells (motor neuron progenitors, MNP) that could be cultured into terminally differentiated sMN. Using this protocol, we were able to rapidly produce highly pure populations of mature sMN from iPSC in high quantities that displayed canonical molecular and morphological features of sMN. This protocol was therefore instrumental to the development of a neuronal model of DHMN1. The flexibility in neuronal culturing (both 2D and 3D formats) allowed the development of a 3D neuronal platform (neuospheres) for the examination of neuronal pathology during this project. This led to the identification of an abnormal delayed axonal outgrowth phenotype for the DHMN1 neurospheres. The phenotype was only observed within the first 36 hours during the development and extension of axonal processes, but was not observed after 10 days of culturing. This growth phenotype could not be observed using the 2D culture format due to the formation of dense overlapping networks of neuronal processes. Targeted and global transcriptome profiling approaches were used to examine DHMN1 sMN for changes in gene expression associated with gene dysregulation caused by the complex DHMN1 insertion. A custom designed NanoString nCounter gene expression assay was used to target 63 genes within a 3 Mb interval on either side of the DHMN1 complex insertion breakpoints. This analysis revealed local gene dysregulation occurring due to the DHMN1 complex insertion with the identification nine dysregulated candidate genes (MNX1/HB9, CRYGN, SHH, BLACE, TMEM176B, LINC00244, UBE3C, GALNTL5, GIMAP4). Similarly, global transcriptome profiling with RNA-seq also revealed the local gene dysregulation with the identification of several dysregulated isoforms of genes within the DHMN1 locus and the complex insertion sequence (MNX1/HB9, LMBR1, CASP2, ZYX, EZH2, TMEM176B, FASTK, AGAP3, EPHB6, ZNF398, ZNF212, ACTR3B). Furthermore, both the RNA-seq and NanoString assay revealed several dysregulated genes in DHMN1 sMN that have key roles in motor neuron axonal growth and guidance. Together, these approaches were helpful in identifying several potential DHMN1 candidate genes, as well as bringing to light changes in potential downstream pathways that may result from primary mutation. Although further work is needed to identify the gene causing DHMN1, the generation of a patient derived iPSC sMN model was essential for this project to investigate disease-relevant tissues containing the DHMN1 mutation background. The results of this project highlight the DHMN1 model of sMN is an ideal naturally occurring paradigm for understanding the role of SV in IPN pathogenesis and will be a fundamental resource to develop future therapies for DHMN1.