Peter Nürnberg | Muhammad Sajid Hussain – C 12

In this project, we are studying C1orf131 that we identified as a novel gene involved in intellectual disability. The protein product plays a crucial role in maintaining the structural integrity of the nucleolus. It seems to be essential for a normal brain development and function. A better understanding of the nucleolar and mitotic function of C1orf131 will shed light on the cellular processes disrupted in C1orf131-mutated individuals. Data obtained from this study will give us an unprecedented opportunity to gain new insights into the gene regulatory networks required to form an optimally functional brain.

Intellectual disability (ID) is characterized by substantial limitations in both intellectual functioning and adaptive behavior. Its worldwide prevalence is estimated to range from 1% to 3%, which casts a serious socio-economic burden on societies. Based on the inheritance patterns, ID is classified into X-linked, autosomal recessive (ARID) and dominant. We studied a Pakistani family featuring ARID and identified linkage regions with a maximum possible LOD score of 2.4 on chromosomes 1, 2, 15 and 21. Whole-exome sequencing (WES) of two affected members revealed a missense mutation (NM_152379.3;c.112G>A; p.Asp38Asn) in C1orf131 located in the linkage region on chromosome 1. The mutated residue is highly conserved and its substitution is predicted to be pathogenic by several in silico tools. Virtually nothing has been reported about C1orf131 or its protein product. Our preliminary data suggest that it is a novel nucleolar component that relocates to the chromosomal periphery during mitosis. Depletion of the protein as observed in both patient fibroblasts or cells treated with siRNA resulted in distorted nucleoli. Intriguingly, pulldown data also showed that it interacts with several nucleolar and centrosomal proteins.

Here we propose to explore the potential involvement of the C1orf131 protein in nucleolar functions and cell cycle progression and to study their impairment. We also aim to investigate its role in neurogenesis by generating single-cell RNA sequencing (scRNA-seq) data of cerebral organoids, generated from patient-derived fibroblasts via induced pluripotent stem cells (iPSC). These data will allow us to dissect the progenitor/neuronal cell ratio and progenitor-to-neuron lineage relationships. Assuming high relevance for their etiology, we are confident to elucidate yet unknown mechanisms underlying ARID and microcephaly.

1. To investigate the effects of C1orf131 mutations on nucleolar and mitotic pathways  
2. To study the role of C1orf131 for a normal brain function with the help of
   • Transcriptional profiling by scRNA-seq of human cerebral organoids derived from C1orf131-mutated cellI
   • In vivo knockdown by in utero electroporation in mouse embryonic brain

The main focus of our research is to unravel genetic causes of neurodevelopmental disorders and to explore the consequences of the identified mutations. In the last decade, we have provided new insights into the genetic etiology of families afflicted with primary microcephaly (MCPH), microcephalic primordial dwarfism (MPD) and Filippi syndrome (FS) using state-of-the-art genetic, biochemical and cell-biological methods (Table 1). Somatic cells of a patient from a Pakistani MCPH family with a homozygous truncating frame shift mutation (c.970delC, p.Gln324Serfs*2) of CEP135 showed characteristic features of aberrant centrosomes like multiple or fragmented centrosomes along with disorganized microtubules (Figure 1A). We also found a homozygous nonsense mutation (NM_017668.2; c.658C>T, p.Arg220*) of NDE1 in an

MCPH patient of Yemeni origin. Primary fibroblasts of the patient showed an impaired localization pattern of the mutant protein (NudE) in form of aggregates within the nucleus as well as faint staining at the centrosomes as detected by ϒ-tubulin (Figure 1B). Investigation of CENPJ-mutated cells(NM_018451.4: c.18delC, p.Ser7Profs*2) resulted in the impairment of centriole (Figure 1C, left panel) and centrosome (Figure 1C, right panel) duplication, which depicts the role of CENP-J in centriole duplication (Figure 1C). One of our most recent findings is the identification of novel MCPH gene named KIAA0408 which encodes a novel centrosomal protein (Figure 1D).

1. Braun DA, Lovric S, Schapiro D, Schneider R, Marquez J, Asif M, Hussain MS, Daga A, Widmeier E, Rao J, Ashraf S, Tan W, Lusk CP, Kolb A, Jobst-Schwan T, Schmidt JM, Hoogstraten CA, Eddy K, Kitzler TM, Shril S, Moawia A, Schrage K, Khayyat AIA, Lawson JA1, Gee HY, Warejko JK, Hermle T, Majmundar AJ, Hugo H, Budde B, Motameny S, Altmüller J, Noegel AA, Fathy HM, Gale DP, Waseem SS, Khan A, Kerecuk L, Hashmi S, Mohebbi N, Ettenger R, Serdaroğlu E, Alhasan KA, Hashem M, Goncalves S, Ariceta G, Ubetagoyena M, Antonin W, Baig SM, Alkuraya FS, Shen Q, Xu H, Antignac C, Lifton RP, Mane S, Nürnberg P, Khokha MK, Hildebrandt F. Mutations in multiple components of the nuclear pore complex cause nephrotic syndrome. J Clin Invest. (2018); 128: 4313-4328.
2. Moawia A, Shaheen R, Rasool S, Waseem SS, Ewida N, Budde B, Kawalia A, Motameny S, Khan K, Fatima A, Jameel M, Ullah F, Akram T, Ali Z, Abdullah U, Irshad S, Höhne W, Noegel AA, Al-Owain M, Hörtnagel K, Stöbe P, Baig SM, Nürnberg P,** Alkuraya FS, Hahn A, Hussain MS. Mutations of KIF14 cause primary microcephaly by impairing cytokinesis. Ann Neurol. (2017); 82(4): 562-577. **Joint senior author.
3. Hussain MS, Battaglia, A. Szczepanski, S. Kaygusuz, E. Toliat, M.R. Sakakibara, S. Altmüller, J. Thiele, H. Nürnberg, G. Moosa, S. Yigit G, Beleggia F, Tinschert S, Clayton-Smith J, Vasudevan P, Urquhart JE, Donnai D, Fryer A, Percin F, Brancati F, Dobbie A, Smigiel R, Gillessen-Kaesbach G, Wollnik B, Noegel AA, Newman WG, Nürnberg P. Mutations in CKAP2L, the Human Ortholog of the Mouse Radmis Gene, Cause Filippi Syndrome. Am. J. Hum. Genet. (2014); 95: 622-632.
4. Martin, C. Ahmad, I. Klingseisen, A. Hussain, MS*. Bicknell, LS. Leitch, A. Nürnberg, G. Toliat, MR. Murray, JE. Hunt, D. Khan F, Ali Z, Tinschert S, Ding J, Keith C, Harley ME, Heyn P, Müller R, Hoffmann I, Cormier-Daire V, Dollfus H, Dupuis L, Bashamboo A, McElreavey K, Kariminejad A, Mendoza-Londono R, Moore AT, Saggar A, Schlechter C, Weleber R, Thiele H, Altmüller J, Höhne W, Hurles ME, Noegel AA, Baig SM, Nürnberg P,** Jackson AP. Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy. Nature Genetics, (2014); 46: 1283-1292. *Joint first author, **Joint senior author.
5. Hussain M.S, Baig S.M, Neumann S, Nürnberg G, Farooq M, Ahmad I, Wajid M, Alef T, Hennies H-C, Technau M, Altmüller J, Frommolt P, Thiele H, Noegel A.A, Nürnberg P. A Truncating Mutation of CEP135 is associated with primary microcephaly and disturbed centrosomal function. Am. J. Hum. Genet. (2012); 90: 871–878.