# Novel Phenotypic Elements of Type IV Collagenopathy Revealed by the _Drosophila_ Model
## 1 Introduction
Sequencing revealed three close glycine mutations in the highly conserved exons 24 and 25 of the _COL4A1_ gene, affecting the integrin-binding site within the COL4A1 protein. Within the three families recapitulating the key features of HANAC, three different heterozygous missense mutations in the _COL4A1_ gene were demonstrated [34]. In a Spanish family with tortuosity of the retinal arteries, retinal hemorrhage was identified [35]. Interestingly, the lesion was not associated with muscle cramps, renal or brain anomalies, although the p.G510R mutation was previously detected in a French family with HANAC syndrome [34]. The different apparent expressivity of the associated trait was explained by environmental factors or genetic modifiers that may influence the phenotypic manifestation and the extent of organ involvement in _COL4A1_-related disease. In a study on the susceptibility to intracerebral hemorrhage, in two of 96 unrelated patients with adult-onset hemorrhagic stroke, the p.P352L substitution at the highly conserved Y-position of a gly-X-Y repeat and the p.R538G replacement were identified within the _COL4A1_ gene [36]. Heterozygosity for a missense mutation in the _COL4A1_ gene was identified in a boy with schizencephaly, renovascular hypertension, retinal arteriosclerosis and alveolar hemorrhage [37; 38]. In 183 mostly pediatric patients with cerebral hemorrhage or porencephaly, 21 _COL4A1_ and three _COL4A2_ mutations were identified at a 40% sporadic mutation rate [39]. Diagnosis in some patients with _COL4A1_ mutations were consistent with Walker-Warburg Syndrome or muscle-eye-brain disease, a distinct form of congenital muscular dystrophy [40]. Mouse _Col4a1_ mutants recapitulate human disease. An allelic series of induced dominant mouse mutants was identified and showed ocular anterior segment defects or Axenfeld-Rieger anomaly, vacuolar cataracts, corneal opacities, iris defects, iris or corneal adhesions, bupthalmos, optic nerve cupping, retinal detachment and developed renal glomerulopathy [41]. Similar ophthalmic defects were noted in the _Col4a1_ splice site of mutant C57 BL6/J mice. When C57 BL6/J mice were crossed with 129/SvEvTac and CAST/Eij inbred strains, the F1 progeny were phenotypically almost indistinguishable from the wild-type. Genetic mapping identified a co-segregating dominant locus on chromosome 1, that likely contains the suppressor and modifier genes [42]. _Col4a1C498V/C498V_ homozygous mice showed severe muscular dystrophy, including muscle mass decrease, fiber atrophy, centronuclear fibers, fibrosis, focal perivascular inflammation and intramuscular hemorrhaging [43]. Variants closer to the C-terminus tended to result in increased intracellular levels of the Col4a1 protein, whereas those closer to the N-terminus tended to accumulate lower amounts of intracellular proteins with unchanged clinical severity [44]. The renal manifestation in mouse models of HANAC syndrome include albuminuria, hematuria, glomerular cysts and delays in glomerogenesis and podocyte differentiation [45]. In _col4a1_ fly mutants, we recorded severe myopathy, reduced concentration of COL4A1 protein [24], chronic inflammation, intestinal dysfunction [25], detachment of the cells from the BM by electron microscopy [26], compromised excretory system [27], heavy membrane peroxidation in epithelial cells of the Malpighian tubules [28] and the onset of muscular dystrophy in all mutants [29]. Temperature-sensitive _col4a1_ alleles in _Drosophila_ were generated by ethyl-methanesulfonate mutagenesis, resulting in G to A transitions in all alleles, in turn leading to glycine substitutions by Glu, Asp and Ser [29]. The phenotypic changes appeared in adult flies following 14 days of incubation at 29 \({}^{\circ}\)C and were not reversible, the flies did not recover at 20 \({}^{\circ}\)C following a shift in heat. We established the complementation map of the _col4a1-col4a2_ locus with the aid of 34 mutants in all combinations in 561 independent crosses.