CMMC: Nürnberg, Peter | Hussain, Muhammad Sajid

Introduction

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.

Our Aims

To investigate the effects of C1orf131 mutations on nucleolar and mitotic pathways
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

Previous Work

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

Table 1: Progress in identifying novel disease-associated genes for neurodevelopmental disorders in recent years
No	Disorder	Gene	Consequences of the identified mutations
1	MCPH	CEP135	Centrosomal dysfunctions, multiple and fragmented centrosomes, disorganized microtubules.
2	MCPH	CDK6	Association with centrosome, depletion causes supernumerary centrosomes, disorganized microtubules.
3	MCPH	KIF14	Midbody protein, mutation triggered loss of CRIK (citron rho-interacting kinase) at the midbody that consequently impaired cytokinesis and led to apoptosis.
4	MCPH	NUP37	Component of outer rings of the nuclear pore complex (NPC), mutation altered the amount and localization of other NPCs — NUP160 and NUP133 — and decreased the number of nuclear pores.
5	MPD	PLK4	Mutation activates a novel cryptic splice acceptor site and introduces a premature termination codon. It impaired centriole biogenesis and resulted in a monopolar configuration, centriole loss and defective spindle formation.
6	FS	CKAP2L	First genetic cause of Filippi syndrome, mutation caused disorganized spindle microtubules, multipolar configurations and defects in chromosome segregation.
7	ARID	C1orf131	New ARID gene encoding a novel nucleolar protein, its depletion causes a distortion of the nucleoli.

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).

Project Related Publications

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.
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.
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.
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.
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.

Publications 2020

Balogh E, Chandler JC, Varga M, Tahoun M, Menyhard DK, Schay G, Goncalves T, Hamar R, Legradi R, Szekeres A, Gribouval O, Kleta R, Stanescu H, Bockenhauer D, Kerti A, Williams H, Kinsler V, Di WL, Curtis D, Kolatsi-Joannou M, Hammid H, Szocs A, Perczel K, Maka E, Toldi G, Sava F, Arrondel C, Kardos M, Fintha A, Hossain A, D'Arco F, Kaliakatsos M, Koeglmeier J, Mifsud W, Moosajee M, Faro A, Javorszky E, Rudas G, Saied MH, Marzouk S, Kelen K, Gotze J, Reusz G, Tulassay T, Dragon F, Mollet G, Motameny S, Thiele H, Dorval G, Nurnberg P, Perczel A, Szabo AJ, Long DA, Tomita K, Antignac C, Waters AM, and Tory K (2020). Pseudouridylation defect due to DKC1 and NOP10 mutations causes nephrotic syndrome with cataracts, hearing impairment, and enterocolitis. Proc Natl Acad Sci U S A 117, 15137-47.

Bamborschke D, Daimaguler HS, Hahn A, Hussain MS, Nurnberg P, and Cirak S (2020a). Mutation in CEP135 causing primary microcephaly and subcortical heterotopia. American journal of medical genetics Part A 10.1002/ajmg.a.61762.

Bamborschke D, Ozdemir O, Kreutzer M, Motameny S, Thiele H, Kribs A, Dotsch J, Altmuller J, Nurnberg P, and Cirak S (2020b). Ultra-rapid emergency genomic diagnosis of Donahue syndrome in a preterm infant within 17 hours. American journal of medical genetics Part A 10.1002/ajmg.a.61917.

Budde BS, Aly MA, Mohamed MR, Bress A, Altmuller J, Motameny S, Kawalia A, Thiele H, Konrad K, Becker C, Toliat MR, Nurnberg G, Sayed EAF, Mohamed ES, Pfister M, and Nurnberg P (2020). Comprehensive molecular analysis of 61 Egyptian families with hereditary nonsyndromic hearing loss. Clin Genet 10.1111/cge.13754.

Ding C, Scicluna BP, Stroo I, Yang J, Roelofs JJ, de Boer OJ, de Vos AF, Nurnberg P, Revenko AS, Crosby J, Van't Veer C, and van der Poll T (2020). Prekallikrein inhibits innate immune signaling in the lung and impairs host defense during pneumosepsis in mice. J Pathol 250, 95-106.

Erger F, Norling D, Borchert D, Leenen E, Habbig S, Wiesener MS, Bartram MP, Wenzel A, Becker C, Toliat MR, Nurnberg P, Beck BB, and Altmuller J (2020). cfNOMe - A single assay for comprehensive epigenetic analyses of cell-free DNA. Genome Med 12, 54.

Hauke J, Harter P, Ernst C, Burges A, Schmidt S, Reuss A, Borde J, De Gregorio N, Dietrich D, El-Balat A, Kayali M, Gevensleben H, Hilpert F, Altmuller J, Heimbach A, Meier W, Schoemig-Markiefka B, Thiele H, Kimmig R, Nurnberg P, Kast K, Richters L, Sehouli J, Schmutzler RK, and Hahnen E (2020). Sensitivity and specificity of loss of heterozygosity analysis for the classification of rare germline variants in BRCA1/2: results of the observational AGO-TR1 study (NCT02222883). Journal of medical genetics 10.1136/jmedgenet-2020-107353.

Karsak M, Glebov K, Scheffold M, Bajaj T, Kawalia A, Karaca I, Rading S, Kornhuber J, Peters O, Diez-Fairen M, Frolich L, Hull M, Wiltfang J, Scherer M, Riedel-Heller S, Schneider A, Heneka MT, Fliessbach K, Sharaf A, Thiele H, Lennarz M, Jessen F, Maier W, Kubisch C, Ignatova Z, Nurnberg P, Pastor P, Walter J, and Ramirez A (2020). A rare heterozygous TREM2 coding variant identified in familial clustering of dementia affects an intrinsically disordered protein region and function of TREM2. Hum Mutat 41, 169-81.

Lal D, May P, Perez-Palma E, Samocha KE, Kosmicki JA, Robinson EB, Moller RS, Krause R, Nurnberg P, Weckhuysen S, De Jonghe P, Guerrini R, Niestroj LM, Du J, Marini C, Euro E-RESC, Ware JS, Kurki M, Gormley P, Tang S, Wu S, Biskup S, Poduri A, Neubauer BA, Koeleman BPC, Helbig KL, Weber YG, Helbig I, Majithia AR, Palotie A, and Daly MJ (2020). Gene family information facilitates variant interpretation and identification of disease-associated genes in neurodevelopmental disorders. Genome Med 12, 28.

Lessel I, Chen MJ, Luttgen S, Arndt F, Fuchs S, Meien S, Thiele H, Jones JR, Shaw BR, Crossman DK, Nurnberg P, Korf BR, Kubisch C, and Lessel D (2020). Two novel cases further expand the phenotype of TOR1AIP1-associated nuclear envelopathies. Hum Genet 139, 483-98.

Niestroj LM, Perez-Palma E, Howrigan DP, Zhou Y, Cheng F, Saarentaus E, Nurnberg P, Stevelink R, Daly MJ, Palotie A, Lal D, and Epi C (2020). Epilepsy subtype-specific copy number burden observed in a genome-wide study of 17 458 subjects. Brain 143, 2106-18.

Patil P, Cieslak A, Bernhart SH, Toprak UH, Wagener R, Lopez C, Wiehle L, Bens S, Altmuller J, Franitza M, Scholz I, Jayne S, Ahearne MJ, Scheffold A, Jebaraj BMC, Schneider C, Costa D, Braun T, Schrader A, Campo E, Dyer MJS, Nurnberg P, Durig J, Johansson P, Bottcher S, Schlesner M, Herling M, Stilgenbauer S, Macintyre E, and Siebert R (2020). Reconstruction of rearranged T-cell receptor loci by whole genome and transcriptome sequencing gives insights into the initial steps of T-cell prolymphocytic leukemia. Genes Chromosomes Cancer 59, 261-7.

Pergande M, Motameny S, Ozdemir O, Kreutzer M, Wang H, Daimaguler HS, Becker K, Karakaya M, Ehrhardt H, Elcioglu N, Ostojic S, Chao CM, Kawalia A, Duman O, Koy A, Hahn A, Reimann J, Schoner K, Schanzer A, Westhoff JH, Schwaibold EMC, Cossee M, Imbert-Bouteille M, von Pein H, Haliloglu G, Topaloglu H, Altmuller J, Nurnberg P, Thiele H, Heller R, and Cirak S (2020b). The genomic and clinical landscape of fetal akinesia. Genet Med 22, 511-23.

Pohl-Rescigno E, Hauke J, Loibl S, Mobus V, Denkert C, Fasching PA, Kayali M, Ernst C, Weber-Lassalle N, Hanusch C, Tesch H, Muller V, Altmuller J, Thiele H, Untch M, Lubbe K, Nurnberg P, Rhiem K, Furlanetto J, Lederer B, Jackisch C, Nekljudova V, Schmutzler RK, Schneeweiss A, and Hahnen E (2020). Association of Germline Variant Status With Therapy Response in High-risk Early-Stage Breast Cancer: A Secondary Analysis of the GeparOcto Randomized Clinical Trial. JAMA Oncol 10.1001/jamaoncol.2020.0007.

Rasool S, Baig JM, Moawia A, Ahmad I, Iqbal M, Waseem SS, Asif M, Abdullah U, Makhdoom EUH, Kaygusuz E, Zakaria M, Ramzan S, Haque SU, Mir A, Anjum I, Fiaz M, Ali Z, Tariq M, Saba N, Hussain W, Budde B, Irshad S, Noegel AA, Honing S, Baig SM, Nurnberg P, and Hussain MS (2020). An update of pathogenic variants in ASPM, WDR62, CDK5RAP2, STIL, CENPJ, and CEP135 underlying autosomal recessive primary microcephaly in 32 consanguineous families from Pakistan. Molecular genetics & genomic medicine 8, e1408.

Scicluna BP, Uhel F, van Vught LA, Wiewel MA, Hoogendijk AJ, Baessman I, Franitza M, Nurnberg P, Horn J, Cremer OL, Bonten MJ, Schultz MJ, van der Poll T, Molecular D, and Risk Stratification in Sepsis c (2020). The leukocyte non-coding RNA landscape in critically ill patients with sepsis. eLife 9.

Ufartes R, Berger H, Till K, Salinas G, Sturm M, Altmuller J, Nurnberg P, Thiele H, Funke R, Apeshiotis N, Langen H, Wollnik B, Borchers A, and Pauli S (2020). De novo mutations in FBRSL1 cause a novel recognizable malformation and intellectual disability syndrome. Hum Genet 10.1007/s00439-020-02175-x.

Yigit G, Saida K, DeMarzo D, Miyake N, Fujita A, Yang Tan T, White SM, Wadley A, Toliat MR, Motameny S, Franitza M, Stutterd CA, Chong PF, Kira R, Sengoku T, Ogata K, Guillen Sacoto MJ, Fresen C, Beck BB, Nurnberg P, Dieterich C, Wollnik B, Matsumoto N, and Altmuller J (2020). The recurrent postzygotic pathogenic variant p.Glu47Lys in RHOA causes a novel recognizable neuroectodermal phenotype. Hum Mutat 41, 591-9.

Sur-Erdem I, Hussain MS, Asif M, Pinarbasi N, Aksu AC, and Noegel AA (2020). Nesprin-1 impact on tumorigenic cell phenotypes. Molecular biology reports 47, 921-34.