genetic and acquired abnormalities in c3 glomerulopathy and primary immune complex-mediated

211 Pages·2017·6.09 MB·English

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Genetic and Acquired Abnormalities in C3
Glomerulopathy and Primary Immune
Complex-Mediated MPGN
Thesis
How to cite:
Piras, Rossella Alberta (2018). Genetic and Acquired Abnormalities in C3 Glomerulopathy and Primary Immune
Complex-Mediated MPGN. PhD thesis The Open University.
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GENETIC AND ACQUIRED ABNORMALITIES
IN C3 GLOMERULOPATHY AND PRIMARY
IMMUNE COMPLEX-MEDIATED MPGN

Thesis submitted by
Rossella Alberta Piras, Chem.Pharm D.

for the degree of
Doctor of Philosophy

Discipline of Life and Biomolecular Sciences

The Open University, United Kingdom
IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Italy

Director of Studies
Dr. Marina Noris, PhD

Supervisor
Prof. Matthew Pickering, PhD

February, 2018

ABSTRACT

Membranoproliferative glomerulonephritis (MPGN) is an uncommon cause of
glomerular injury that mainly occurs in children and young adults.
MPGN is currently classified in immune complex-mediated MPGN (IC-MPGN),
characterized by activation of the complement classic pathway, and C3 Glomerulopathy
(C3G), with predominant complement alternative pathway (AP) activation. C3G is
further classified in Dense Deposit Disease (DDD) and C3 glomerulonephritis (C3GN).
The first part of the thesis describes a large cohort of patients with IC-MPGN (n=96),
DDD (n=26) and C3GN (n=77). Data obtained from genetic and biochemical analysis
were correlated with histological and clinical parameters. We found that the majority of
patients across the three histology groups (from 70 to 85 %) showed low C3 and normal
C4. About 18% of patients carried likely pathogenic variants (LPVs) in complement
genes, mainly in CFH regulatory gene and in C3 and CFB, encoding the two convertase
components. Interestingly, two LPVs in THBD gene, were identified in two patients
with C3G. C3NeF, an autoantibody stabilizing AP convertase, resulted abundant in
DDD patients (79%) but was also present in patients with IC-MPGN (40%) or C3GN
(39%).
To classify IC-MPGN and C3G patients based on the underlying pathogenesis, a three-
step algorithm based on histological, genetic and biochemical data was used to classify
patients in 4 clusters identifying 4 different pathogenetic patterns.
In the second part of the thesis, copy number variation (CNV) studies, disclosed
abnormal CNVs both in IC-MPGN and C3G. Interestingly, we describe, for the first
time, genomic rearrangements involving the CFHR4 gene. Finally, Western Blot studies
in DDD patients showed the presence of abnormal FHR molecular pattern in patients
with normal CNVs and without CFHR LPVs.
In conclusion, the present study increased our understanding in IC-MPGN and C3G and
provided new insights in the pathogenetic mechanisms underlying these complex
glomerular diseases.
To my family
ACKNOWLEDGEMENTS

I am grateful to Prof. Silvio Garattini and to Prof. Giuseppe Remuzzi, Director and
Research Coordinator of the IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”,
respectively, for giving me the opportunity to attend the PhD course.
My sincere thanks go to Dr. Marina Noris, my Director of Studies, for her continuous
suggestions that enriched my research and for her immense knowledge; to Dr. Matthew
Pickering, my Supervisor, for all the time he kindly dedicated to supervise my research.
Especially thanks to all my colleagues of the “Laboratory of Immunology and Genetic
of Rare Diseases”, and of the “Laboratory of Rare Diseases Documentation and
Research”, in particular Paraskevas Iatropoulos, Elisabetta Valoti, Caterina Mele,
Marta Alberti, Serena Bettoni, Roberta Donadelli, Matteo Breno , Paola Cuccarolo,
Manuela Curreri and Elena Bresin for their precious and helpful contribution to my
research.
A very special gratitude goes to Fabrizio Spoleti, president of the “Progetto DDD Onlus
- Associazione per la lotta alla DDD” for supporting my research and for his
enthusiasm for the Research. I express my gratitude to Prof. Richard Smith who gave
me the possibility to spend time in his laboratory, an experience that helped me to grow
professionally and personally.
This thesis would not have been possible without the incessant encouragement of my
parents, the understanding and love of Massy and the magical energies transmitted from
my little Filippo.
Contribution to the Thesis by other Researchers
Part of the thesis project was carried on with the collaboration of other researchers that
contributed to the research as follows:

Internal collaborations in the IRCCS Istituto di Ricerche Farmacologiche Mario
Negri
- All kidney reports were centrally reviewed by pathologists at Mario Negri Institute.
- Clinical data and biological samples were collected through the MPGN registry by
Dr. Paraskevas Iatropoulos, Dr. Manuela Curreri, Dr. Bresin, Dr. Daina, and Ms.
Sara Gamba.
- Regarding to the six gene NGS panel for IC-MPGN/C3G cohort, Dr Paraskevas
Iatropoulos and Dr. Caterina Mele participated in the "wet-lab" and the
bioinformatic set-up. The screening of the 6 complement genes was performed by
the candidate together with Dr. Elisabetta Valoti, Dr. Caterina Mele, Dr. Paraskevas
Iatropoulos and Ms Marta Alberti. Analysis of the NGS sequencing was performed
by Dr. Matteo Breno.
- SC5b-9 assays were performed by Dr. Serena Bettoni and Dr Paola Cuccarolo.
- FH quantification by ELISA in DDD patients was performed by Dr Paola
Cuccarolo.
- The presence of anti-FH autoantibodies was evaluated by Dr Elisabetta Valoti.
- C3 and C4 assays were performed by Dr. Flavio Gaspari and the Antonio Nicola
Cannata.
External collaborations
- Marina Vivarelli, Francesco Emma Division (both at the Nephrology and Dialysis,
Bambin Gesù Pediatric Hospital, Roma, Italy) and Luisa Murer (at the Unit of
Pediatric Nephrology, Dialysis and Transplantation, Azienda Ospedaliera of
Padova, Italy) participated in the recruitment of IC-MPGN/C3G patients.
- C3NeF assays were performed by Dr. Veronique Fremaux-Bacchi’s group at the
Department of Immunology, Assistance Publique-Hopitaux de Paris, Hopital
Europeen George-Pompidou and INSERM UMRS 1138, Cordelier Research
Center, Complement and Diseases Team, Paris, France.
- Library preparation using CasCADE panel was performed by the candidate at the
Molecular Otolaryngology and Renal Research Laboratories of University of Iowa
while NGS sequencing was performed by the University staff.

All other experiments and analyzes described in this thesis were performed by the PhD
candidate, Dr Rossella Alberta Piras.
Research Funding
This work was supported by:
o Progetto DDD Onlus-Associazione per la lotta alla DDD, Milan, Italy;
o Fondazione ART per la Ricerca sui Trapianti ART ONLUS (Milano, Italy);
o Fondazione Aiuti per la Ricerca sulle Malattie Rare ARMR ONLUS (Bergamo,
Italy);
o Telethon grant GGP09075;
o European Union Seventh Framework Programme FP7-EURenOmics project
number 305608;
o Kidneeds grant 2016.