Cytokines, apoptosis and complement in 22q11.2 deletion syndrome (DiGeorge syndrome)

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Cytokines, apoptosis and

complement in 22q11.2 deletion syndrome (DiGeorge syndrome)

Dina Mikhailovna Aresvik

Department of Pediatric Research Oslo University Hospital

Faculty of Medicine UNIVERSITY OF OSLO

2019

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© Dina Mikhailovna Aresvik, 2020

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-668-3

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.

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Acknowledgements

This work was carried out at the Department of Pediatric Research, Rikshospitalet, Oslo University Hospital during the years 2011-2019. The work was mainly financed through a generous grant from The South-Eastern Norway Regional Health Authority. In addition, financial support was received from Renée and Bredo Grimsgaard's Foundation, Henrik Homans Foundation and Eckbos Foundation.

I want to thank all the people who have supported me through all these years of research.

First, I want to thank all the patients, controls and their families for participating in the study and contributing to the research.

I want to express my sincere thanks to my supervisors, who gave me this opportunity. First, to my main supervisor Professor emeritus Tore Gunnar Abrahamsen (MD, PhD) for excellent guidance consisting of tireless enthusiasm combined with an impressive depth of knowledge, for stimulating ideas based on an extensive experience and perspective, and for allowing me generous liberty in my pursuit projects for this thesis. I am deeply grateful to my co-

supervisor and head of Norwegian National Unit for Newborn Screening Rolf Dagfinn Pettersen (MSc, PhD) for introducing me to the world of flow cytometry and apoptosis, for constructive feedback on protocols, manuscripts, numerous questions and unexpected

challenges. I would also like to thank Marianne Wright (MSc, PhD), co-supervisor on the first part of this work, for believing in me and for introducing me to the world of research while I still was a medical student. I have appreciated your supportive, encouraging and enthusiastic inputs.

I am grateful to the former head of the Department of Pediatric Research Professor emeritus Ola Didrik Saugstad (MD, PhD) for welcoming med and giving me the opportunity to work at

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the institute. I would further like to thank the functioning head of the institute Runar Almaas (MD, PhD) for your never stopping enthusiasm and support.

I would like to express my gratitude to Kari Lima (MD, PhD) who was the first one to conduct a study on 22q11.2 del patients in Norway. You experience has been indispensable and your steps were easy to follow. I am also grateful to Torstein Øverland (MD) who performed an impressive work on building up the national reference center for 22q11.2 deletion syndrome. Special thanks go to nurse Kathleen Halvorsen for always being positive and for making patients feel well at the hospital. Many thanks also to Hans Christian Erichsen (MD, PhD) for sharing his knowledge and for stimulating new ideas. It has also been a great pleasure collaborating with PhD student Kiran Gul (MD). I am truly grateful for getting the opportunity to work with all of you.

I wish to thank Monica Atneosen-Åsegg (MSc) who welcomed me when I arrived at the Department of Pediatric Research, helped me plan the first experiment and taught me all about laboratory work and cell culturing. I also owe great thanks to the laboratory engineer Grethe Dyrhaug for helping me with ELISA. I would further like to thank Grethe Dyrhaug and Cecilie Aas (Msc) for careful blood sampling of controls. Without help from all of you I could not have completed this work.

A special gratitude to the fruitful collaboration we had with Professor Tom Eirik Mollnes (MD, PhD). His help in guiding me through the world of cytokines and complement, giving us the opportunity to perform the multiplex and complement analysis and correcting my manuscripts, including tables that needed small, but time consuming adjustments, have been invaluable. I would also like to thank his research group and especial Judith K. Ludviksen, Julie K. Lindstad and Camilla Schjalm for excellent technical assistance.

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Finally, I want to thank my family and friends for their support. My mother Natalia Aresvik (MD, PhD) has supported me in everything I do and inspired me with her own research and her conscientious work as a physician. My grandparents have always been proud of me, no matter what. My beloved husband Martin Grinde has been extremely patient; he encouraged me and reminded me of what really matters in life. I could not have done this without you.

Dina Grinde, previous Aresvik

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Publications

This thesis is based on three publications named by roman numbers throughout the text:

I. Aresvik DM, Lima K, Overland T, Mollnes TE, Abrahamsen TG. Increased Levels of Interferon-Inducible Protein 10 (IP-10) in 22q11.2 Deletion Syndrome. Scand J Immunol.

2016;83(3):188-94. doi:10.1111/sji.12406.

II. Aresvik DM, Overland T, Lima K, Pettersen RD, Abrahamsen TG. Lymphocyte

Apoptosis and FAS Expression in Patients with 22q11.2 Deletion Syndrome. J Clin Immunol.

2019;39(1):65-74. doi:10.1007/s10875-018-0579-7.

III. Aresvik DM, Overland T, Lima K, Schjalm C, Mollnes TE, Abrahamsen TG.

Complement Activation in 22q11.2 Deletion Syndrome. Submitted manuscript.

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Abbreviations

22q11.2 del 22q11.2 deletion syndrome

7-AAD 7-Amino-Actinomycin

ACAD Activated cell autonomeous death

ADHD Attention deficit hyperactivity disorder

AICD Activation induced cell death

AnnV Annexin-V

ASD Autism spectrum disorders

bFGF Basic fibroblast growth factor

BMI Body mass index

C1-C9 Complement system components

CD Cluster of differentiation

CHD Congenital heart diseases

ELISA Enzyme-linked immunosorbent assay

FISH Fluorescence in situ hybridisation

FITC Fluorescein isothiocyanate

G-CSF Granulocyte-colony stimulating factor

GM-CSF Granulocyte macrophage colony stimulating factor

IFN Interferon

Ig Immunoglobulin

IL Interleukin

IP-10 Interferon-inducible protein 10

LCR Low copy number repeat

Mb Megabase

MBL Mannose-binding lectin

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MCP-1 Monocyte chemotactic protein

MIP Macrophage inflammatory protein

MoAb Monoclonal antibody

PBMC Peripheral blood mononuclear cells

PBS Phosphate-buffered saline

PDGF-BB Platelet derived growth factor-BB

PE Phycoerithryn

PHA Phytohemagglutinin

PS Phosphatidylserine

RANTES Regulated upon activation T cell expressed and secreted

TCC Terminal complement complex

TCR T cell receptor

TNF Tumor necrosis factor

TRAIL TNF-related apoptosis-inducing ligand

VCFS Velocardiofacial syndrome

VEGF Vascular endothelial growth factor

VPI Velopharynegeal insufficency

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Introduction

The 22q11.2 deletion syndrome

Historical background

The first description of a patient probably affected by a 22q11.2 deletion syndrome (22q11.2 del) was made in 1671 by a Danish scientist and bishop Niels Stensen, who described a patent with cleft palate and truncus arteriousus [1, 2]. He wrote:

“There was a cleft palate and hare-lip on the right side, and the mother attributed this anomaly to the fact that she was fond of rabbit stew… the unusual form of the arteries arising from the heart attracted the chief attention and called for admiration. In particular, the

pulmonary artery was much narrower than the aorta … when I opened the right ventricle, the probe that was passed forward and upward along the interventricular septum entered directly into the aorta just as readily as the probe passed from the left ventricle into the aorta”.

Later, well before the role of the thymus was appreciated, L. H. Harrington described in 1829 a child with a syndrome that resembles 22q11.2 del with an absent thymus and hypoplastic parathyroid glands [3].

In the modern era, the first description of the 22q11.2 del is made by Dr. Eva Sedláčková, an otolaryngologist from Czechoslovakia, who described a syndrome of velopharyngeal

insufficiency and developmental delay [4]. However, with medical and scientific publications dominated by the English language, the publication of Sedláčkovás work in her native

Czechoslovakian passed almost unnoticed [5]. Four years later, in 1959, the pathologic association of absent parathyroid and thymus glands was reported by David Lobdell [6]. In 1965, Dr. Angelo DiGeorge, a pediatric endocrinologist from Philadelphia, commented on a

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paper by Dr. Max Cooper and colleagues regarding the congenital absence of the thymus in chicken [7]. Dr. DiGeorge wrote that there was a group of infants with congenital absence of the thymus who might represent a human homologue of the thymectomised chickens.

DiGeorge and his co-worker, Dr. James Arey, had observed three infants with congenital absence of the parathyroid glands who also had no evidence of thymic tissue. Dr. DiGeorge wrote:

"The concurrent absence of both structures is not surprising if one recognizes that both are derived from common primordia. Furthermore, this association has been previously recorded although its physiologic significance has not been recognized."

In addition, Dr. DiGeorge and his colleagues, Drs Harold Lischner, Catherine Dacou- Voutetakis, and Hope Punnett, observed an infant with congenital hypoparathyroidism who was predicted to have absence of the thymus. In addition to the absence of the thymic shadow on the chest x-ray, the infant had abnormal cellular immunity, although the lymphocyte count, plasma cell numbers in lymph nodes, and serum immunoglobulins were normal. DiGeorge suggested that all infants with congenital hypoparathyroidism should be studied for defects in cellular immunity [8]. Combination of those features was then named “DiGeorge syndrome”

by Dr. Robert A. Good. Interestingly, DiGeorges first published paper on the subject actually did not appear until 1968 [9].

Subsequently, facial dysmorphia, conotruncal cardiac malformation and speech delay were included in the spectrum and various other names came to be applied to this constellation of phenotypic features. Thus, in 1969 Dr. Cayler described cardiofacial syndrome with

congenital heart disease and facial weakness [10]. In 1976 in Japan, Kinouchi et al, and later Takao and coworkers, described a conotruncal face syndrome, also named Takaos syndrome [11]. Recognition of congenital heart disease, especially involving the outflow tract by the

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Japanese as a key feature of DiGeorge contributed to the theory that a mechanism leading to perturbation of neural crest cell migration, particularly affecting the third and fourth

pharyngeal arches, may be involved [12]. In 1978, a speech and language pathologist Robert Shprintzen, described a new syndrome involving cleft palate, cardiac anomalies, typical faces and learning disabilities [13]. Dr. Shprintzen named it velo-cardio-facial syndrome (VCFS);

however, Shprintzen syndrome is also used.

In early 1980s, cytogenetically visible chromosome 22q11.2 deletions were reported, and authors suggested that, for at least some cases, DiGeorges syndrome could be caused by deletion of a gene located in chromosome 22, probably in band 22q11 [14, 15]. However, it was not until the development of fluorescence in situ hybridization (FISH) studies in the early 1990s that investigators were able to see the big picture and recognize that many previously described clinical conditions were actually caused by the 22q11.2 deletion. First, FISH studies using probes within the commonly deleted region identified submicroscopic 22q11.2 deletions as the most frequent cause of DiGeorge syndrome and velocardiofacial syndrome [16-18].

Then this preceded recognition that several seemingly unrelated conditions with overlapping phenotypic features were also caused by a 22q11.2 deletion, including conotruncal anomaly face syndrome, Cayler Cardiofacial syndrome, as well as subsets of patients with Opitz G/BBB syndrome [19-21].

Nomenclature

Right after the discovery of the common 22q11.2 deletion the acronym CATCH22 was introduced focusing on the main features; Cardiac defects, Abnormal faces, Thymic

hypoplasia, Cleft palate and Hypocalcemia resulting from 22q11 deletions [22]. The term was

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illustrative, but brought up negative associations to the satirical novel CATCH22, and is no longer used [23].

Reflecting the fact that the syndrome was described by clinicians concentrating on the specific areas of expertise, for example: DiGeorge syndrome by an endocrinologist and VCFS by a speech pathologist; there are still controversies and discussions about the naming of the syndrome. In an effort to be as systemic and precise as possible, it is recommended that patients with the classic chromosome 22q11.2 deletion are described according to the genetic nomenclature, that is, 22q11.2 deletion syndrome (22q11.2 del). While the term DiGeorge syndrome is now reserved for those rare patients who share clinical symptoms with 22q11.2 del but do not harbor a 22q11.2 deletion [3].

Demographics

22q11.2del is regarded as the most common chromosomal microdeletion syndrome in humans [24]. Based on the diagnosis of patients with major birth defects and population studies conducted between 1990s and early 2000s, the prevalence of the 22q11.2 del has been

estimated to range from 1: 3 000 to 1: 6 000 live births [25-29]. Those studies were performed using FISH technology, and later studies using single nucleotide polymorphism arrays have suggested that the true prevalence may be higher[30]. This is because atypical deletions are not detected by FISH probes, and, therefore, not included in the early population studies.

Further, prenatal studies conducted in 2012 and 2015 using invasive prenatal testing reported 22q11.2 deletions in 1:347 fetuses with abnormal ultrasonographic findings and in 1:992 of normal fetuses [31, 32]. The prevalence was higher (1:100) in fetuses with congenital heart diseases (CHD) or other major structural abnormalities, while in anatomically normal fetuses

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the observed prevalence was approximately 1:1000. Thus, considering the fact that many patients may remain undiagnosed due to the broad phenotype of the syndrome, it is possible that the true prevalence of 22q11.2 del is underestimated and the true live birth incidence remains to be identify by global newborn screening [24].

Moreover, the prevalence of the syndrome is expected to rise due to increasing numbers of familial cases [30]. Currently, existing research suggests that about 6-10% of new cases are familial, while 90-95% newly identified patients with 22q11.2 del are found to have de novo deletions [33] .However, before the mid-1980s patients who had severe cardiac anomalies did not survive. Now there is a large cohort of adults who have these anomalies, and they are raising their own families. Since 22q11.2 del is haplosufficiency disorder inherited in an autosomal dominant fashion, half of the children of individuals with 22q11.2 del will have the deletion [30].

There is no known predisposition based on ethnicity or gender, and both sexes and all races and ethnic groups are affected [34]. However, de novo deletion is slightly often maternal in origin [35]. Further, non-white patients may be diagnosed less often due to less recognizable dysmorphic facial features in these populations [36, 37]. On the other hand, one study suggest that Hispanics have a higher prevalence of 22q11.2 del than non-Hispanic Americans [25].

Pathophysiology Deletion

The 22q11.2 deletion occurs on band 11 on the long (q) arm of chromosome 22 [18, 38]. The 22q11.2 region is probably one of the most structurally complex areas of the genome, primary

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due to several large blocks of low copy number repeats (LCR) or segmental duplications [24, 39, 40]. Sequence analysis has identified four LCRs in this region and each block is

comprised of multiple repeats. Those blocks are named LCR22A-D, with A being most proximal (Fig. 1) [40]. Although the LCRs differ in content and organization of shared

modules, those modules that are common between them share 97-98% sequence identity with one another, thereby making the locus vulnerable to meiotic error [40-42]. Thus, the de novo deletions are caused by non-allelic recombination events between flanking LCRs during meiosis [39].

Figure 1. Low copy repeats (LCR22A-D) and genes within the 22q11.2deletion [24].

Common commercial FISH probes are indicated. The protein coding and selected non-coding genes are indicated with respect to their relative position along chromosome 22.

Reprinted by permission from Springer Nature.

The most common deletion, which is seen in approximately 85% of patients, is 3 megabase (Mb) and occurs between LCR22-A and LCR22-D [40, 39]. Nested proximal deletions are

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less frequent; 1.5 Mb deletion located between LCR22-A and LCR22-B account for about 8%

of 22q11.2 deletions, while 2Mb LCR22-A-LCR22-C deletion is seen in about 2% of patients [40, 43]. Nested central deletions, which are either 1.5 Mb deletion of the region located between LCR22ǦB and LCR22ǦD or 0.7 Mb deletion between LCR22-C and LCR22-D seem to be relatively rare [44].

Genes

There are 90 known or predicted genes present in the typical 3 Mb 22q11.2 locus that are hemizygously deleted, while the smaller proximal 1.5 Mb deletion encompassed 55 of these genes [45].

The most studied gene of interest in the 22q11.2 del is TBX1, encoding a T-box transcription factor [24, 30]. Using multiple mouse model approaches, Tbx1 was found to be a critical determinant of cardiac, thymus and parathyroid phenotypes. Thus, heterozygous loss-of- function mutations of Tbx1 in the mouse result in partially penetrant cardiovascular, thymic and parathyroid defects that are reminiscent of congenital defects in 22q11.2 del [46, 47].

Whereas homozygous Tbx1 mutants die at birth with severe defects, including cardiac outflow tract malformations, cleft palate and absence of thymus and parathyroid glands. In addition, Tbx1 has also been implicated in brain microvascular development, which may count for the neuropsychiatric symptoms [48, 49].

Other genes within the deleted region have also been proposed to contribute to the phenotype of the patients (Fig.1). For example, haplosufficiency for GpIbβ, coding for a platelet

membrane glycoprotein, may contribute to the mild thrombocytopenia seen in patients [50].

DGCR8, encoding the DGSR8 microprocessor complex subunit, may modify the expression

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of genes outside of the 22q11.2 deletion region that contribute to the neuropsychiatric

phenotype [51]. Haplosufficiency for COMT, which encodes cathechol-O-methyltransferase, one of the several enzymes that degrade catecholamines, has also been implicated as

contributing to behavioral and psychiatric problems [52]. The CRKL gene, encoding a cytoplasmic adaptor protein to growth factor signaling, may be responsible the aetiology of cardiac anomalies in patients with atypical (LCR22B-LCR22D) [53]. CRKL seems also to modulate natural killer cell function [54].

Developmental aspects

The structures primarily affected in the 22q11.2 del - the thymus, the parathyroid gland, the aortic arch and the cardiac outflow tract, as well as craniofacial structures - are all derivatives of the pharyngeal arch system and receive contributions from all three classic germ layers of the embryo – the endoderm, mesoderm and ectoderm – together with neural crest cells derived from the closing neural tube [24, 55]. The TBX1 gene, described in pervious section, is important for the development of pharyngeal apparatus [56, 57].

During the embryogenesis, the parathyroid glands and the thymus derive from tissue interactions between the pharyngeal endoderm and neural crest cells, while the craniofacial skeleton and musculature and bony palate are derived from neural crest cells or the anterior mesoderm (Fig. 2) [58]. The pharyngeal mesoderm progenitor cells give rise to craniofacial muscles and second heart field derivatives, including the cardiac outflow tract [24]. Thus, the compromised arch development leads to aberrant development of thymus, parathyroid gland and the facial structures [30]. Congenital cardiac abnormalities seen in 22q11.2del are related to defects in the arteries formed within the pharyngeal apparatus and in the cardiac outflow tract [58]. The most specific cardiovascular defects associated with 22q11.2 del are

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interrupted aortic arch type B, which is due to aplasia of the left fourth pharyngeal artery and tetralogy of Fallot, which can be ascribed is related to defects in the development of the pulmonary infundibulum [59, 24].

Other recognized malformations, such as behavioral and psychiatric disturbances, as well as renal and distal skeletal abnormalities, are not directly due to defects in the pharyngeal arches [30].

Figure 2. Migration of neural crest cells from the hindbrain to the brachial arch/pharyngeal pouch system and cardiac outflow tract in a human embryo at 4–6 weeks of gestation.

Malformations associated with 22q11.2 del due to disturbance of this process are listed [55].

Reprinted by permission from Oxford University Press.

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Notably, 22q11.2 hemizygosity alone cannot explain the genetic mechanism of the highly variable phenotypic expression of 22q11.2 del. Patients with smaller nested deletions have major phenotypic features in common with patients with the typical LCR22A-LCR22D deletion [39, 44, 60]. Thus, a smaller deletion does not indicate milder symptoms, making genotype–phenotype correlations difficult. Moreover, several studies have described phenotypic discordance between the monozygotic twins with 22q11.2 del [61-63].

Following mechanisms have been suggested to explain this phenomenon:

1) Combined effect of multiple deleted genes, where different genes in the deleted region cause each of the major anomalies [50, 52].

2) Stochastic events during fetal development [64] and environmental factors [65]

3) Sensitivity of individual genes within the 22q11.2 region to gene dosage [66, 67]

4) Variants in genes on the non-deleted 22q11.2 [68]

5) Additional modifying variants outside the 22q11.2 region, involving both protein- coding genes and regulatory mechanisms [69, 70].

Clinical features

22q11.2 del syndrome leads to significant morbidity with frequent multi-organ system

involvement [24]. Patients with the 22q11.2 del display a wide phenotypic variation and more than 180 signs and symptoms have been describe[71]. None of the findings occur with 100%

frequency, and none are obligatory [72]. However, in spite of variable clinical expression, patients with 22q11 deletion share a number of major features and have a characteristic

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phenotype [73]. Most individuals have a combination of 4-6 of the most common features and the median number of lifetime features per adult patent is 9 [74].

Many clinical features are characteristic at certain ages. First, the organ malformations dominate, while neuropsychological problems appear later in life [75].

Cardiovascular abnormalities

Congenital heart diseases are seen in about 77 % of patients with 22q11.2 del [30]. They became evident in the prenatal or neonatal period and are often the initial manifestation that leads to diagnosis [30, 76]. The most common CHD seen in 22q11.2 del are conotruncal anomalies, that are defects confined to the outflow segment of the heart and to the aortic arch, such as tetralogy of Fallot, truncus arteriosus, interrupted aortic arch type B and ventricular septal defect [77]. Anomalies of the aortic arch and/or the pulmonary arteries may occur as isolated entities or in association with conotruncal defects [78]. Other types of CHD are rare in patients with 22q11.2 del, however, virtually every type of CHD has been described in the context of 22q11.2 del [34, 77].

Immunodeficiency

Immunodeficiency affects up to 77% of pediatric patients with 22q11.2 del and is thought to arise due to thymic hypoplasia and impaired T cell production [30, 79]. The size of the thymus does not predict circulating T-cell counts, partly due to microscopic rests of thymic epithelial cells at aberrant locations [80]. The condition is heterogeneous, ranging from patients with normal T-cell counts to complete DiGeorge syndrome with very low T-cell

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counts [81, 82]. Less than 1% of the patients have true thymic aplasia requiring a transplant, while about 20% have no evidence of diminished T cells [3, 50]. Most patients have mild to moderate reduction in circulating T cells [83]. Those patients are slightly

immunocompromised and have increased frequency of infections such as recurrent respiratory infections, including sinusitis, otitis media, and pneumonia [84, 85]. They do not develop opportunistic infections [75]. Abnormal palatal anatomy may lead to compromised drainage and an increased susceptibility to upper airway bacterial infections [30]. The increased infection susceptibility decreases during early childhood [75]. Many infants with low T cell counts demonstrate improve in the first year of life [85, 86]. The age dependent decline in T cell numbers is slower in patients and many adult patients have T cells counts comparable to unaffected individuals [87]. Homeostatic proliferation of existing T cells appears to be responsible for the initial rise in infancy as well as the slower age-determined decline of T cells [88, 89].

The humoral immune system is largely intact in 22q11.2 del patients [90]. However, immunoglobulin (Ig) A deficiencies, impaired response to vaccines and transient hyppogammaglobulinemia have been described [91-93].

Palatal abnormalities

The palate is affected in approximately 80% [94]. Majority of patients present with

velopharyngeal insufficiency, submucosal cleft palate or bifid uvula, while overt cleft palate is seen in 11% of patients, of whom only 1-2% have cleft lip or cleft palate [30, 34, 95].

Magnetic resonance imaging studies have shown that patients with 22q11.2 del have a

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different velopharyngeal anatomy with predisposition to velopharyngeal insufficiency (VPI) [96].

Palatal abnormalities contribute to poor feeding, speech quality and acquisition [95, 97].

Nasopharyngeal reflux can also lead to persistent otorrhea or to recurrent and chronic sinus infection due to repeated contamination of nasal cavity [24, 95].

Endocrine abnormalities

Hypocalcemia which is caused by hypoparathyroidism due to congenital parathyroid aplasia or hypoplasia is seen in about 50% of 22q11.2 del patients at some point in their live [98, 99].

Studies have shown that neonatal hypocalcemia is not associated with development of permanent hypoparathyroidisme later in life [99]. The reserve capacity of the parathyroid glands is limited in patients with 22q11.2 and transit neonatal hypocalcemia or new-oncet hypocalcemia often occurs during times of stress, such as during illness, preoperatively or during adolescence and pregnancy [99, 100]. As hypoparathyroidism may present at any age, and since asymptomatic hypoparathyroidism may occur in patients with 22q11 del, screening of this population have been advocated [99, 101].

As both the follicular cells and C cells of the thyroid gland are at least in part derived from neural crest cells of the fourth and fifth pharyngeal pouches, congenital abnormalities of the thyroid would not be unexpected in patients with 22q11.2 del [102]. Thyroid abnormalities in 22q11.2 del patients was confirmed by autopsy [103]. Thus, hypothyroidism is seen in about 20% of patients, while hyperthyroidism due to Graves disease has been reported in about 5%

of subjects with a 22q11.2 del [104].

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Obesity has been reported in approximately 40% of 22q11.2 del patients, often with onset during childhood or adolescence [74, 105, 106]. Consequently, incidence of type 2 diabetes is higher in obese 22q11.2 patients [105].

Autoimmunity

The frequency of autoimmune disease is significantly increased in 22q11.2 and is seen in about 10% of patients. There is a general susceptibility to autoimmunity rather than an association with a specific disease [30, 99, 107]. Juvenile rheumatoid arthritis, idiopathic thrombocytopenia, hemolytic anemia and thyroid disease are common [50, 108-110]. Celiac disease may be more common than in general population [111]. Homeostatic expansion of self-reactive T cells and decrease in regulatory T cells are thought to contribute to the predisposition to autoimmunity [30].

Psychiatric aspects

The syndrome is associated with variable cognitive delays, learning disabilities, behavioral phenotypes and psychiatric illnesses such as attention deficit hyperactivity disorder (ADHD), anxiety and schizophrenia [112-114]. In a large-scale international study on psychiatric disorders from childhood to adulthood in 22q11.2 del Schneider et al demonstrated that

ADHD is the most frequent disorder in childhood, affecting as many as 37% of patients [114].

Mood and anxiety disorders were common in 22q11.2 del and affected almost 31% of participants at all ages, but especially in children and adolescents. The frequency of major depressive disorder increased with age. Schizophrenia spectrum disorders were present in as many as 41% of adults over age 25, and early-onset psychosis was relatively common in

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individuals with 22q11.2 del, making 22q11.2 del the strongest known molecular genetic risk factor of schizophrenia [114]. Anxiety, obsessive-compulsive disorder and depression in childhood have been identified as strong predictive factors for subsequent development of psychotic disorders in adulthood [115]. In addition, early cognitive decline predicts development of psychotic illness [116]. The high frequency of behavioral problems and psychiatric disorders in patients with 22q11.2 del suggests a very specific effect of the deletion [24].

Other

Both gastrointestinal and genitourinary abnormalities are frequent and are seen in about one-third of 22q11.2 del patients [74]. Skeletal anomalies such as clubfoot and overlying toes have been described [117]. The risk of developing scoliosis is increased [75]. Anomalies of the eyes have been reported [118]. Dental issues due to enamel disturbances are frequently seen [119].

Cytokines

Cytokines are soluble hormone-like proteins that allow for communication between cells [120]. It is an umbrella term which includes interleukins, chemokines, lymphokines,

monokines, colony stimulating factors, tumor necrosis factor and interferons [121, 122]. They are secreted by leukocytes as well as a variety of other cells and are key components of the innate and adaptive immune systems. Cytokines have multiple biological properties and are also involved in processes such as hematopoiesis, angiogenesis and embryonic development [120-123]. Thus, it is widely accepted that a disturbed cytokine balance contributes in the

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pathogenesis of various disorders, including inflammation, cancer, autoimmunity and psychiatric disorders [124-126].

Cytokines and 22q11.2 del

Several studies have examined cytokines in 22q11.2 del. Plasma levels of IL-7 are higher in 22q11.2 del patients with “low T-lymphocytes” compared to patients with “normal T- lymphocytes” and are inversely correlated with T cell receptor excision circles values, indicating a positive feedback on the production of the naïve T-lymphocytes [127]. Further, serum levels of IL-1β, INF-γ, IL-12(p70) and ratio between IL-6 and IL-10 correlate with autism-related behavior [128]. Moreover, pediatric 22q11.2 del patients have elevated

percentage of CD3+CD4+IL-4-IFN-γ+ lymphocytes compared to controls [129]. However, an increased percentage of CD3+CD4+IL-4+INF-γ- are seen in adults. Furthermore, in another study no difference in gene expression of IFN-γ, as well as IL-10 and TNF-β in highly purified T lymphocytes between patients and controls was found [130]. Taking into account the diversity of the 22q11.2 del, all those findings highlight the importance of the cytokines in the pathogenesis of the syndrome.

Apoptosis

Apoptosis, or programmed cell death, is essential for tissue development and homeostasis while deregulation of apoptosis may contribute to diseases such as cancer, autoimmunity and degenerative diseases [131-133]. Caspases, which play a central role in the regulation and execution of apoptotic cell death, can be activated by two distinct, but interconnected

signaling cascades: either the extrinsic receptor mediated pathway which is triggered by death

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receptor ligands or the intrinsic, mitochondrial pathway which is initiated by cellular stress such as growth factor withdrawal, ultraviolet- or γ-irradiation and chemotherapeutic drugs and regulated by pro- and anti-apoptotic BCL-2-like as proteins [134-136]. Once activated,

caspases are responsible for the proteolytic cleavage of a broad spectrum of cellular targets, which leads to cell death [137].

Apoptosis and the immune system

T-cell apoptosis plays an essential role in the immune system: It eliminates developing lymphocytes that fail to express an antigen receptor, removes lymphocytes with antigen receptors that recognize autoantigens and regulates the size and duration of immune responses [138].

During an immune response, naïve T cells undergo expansion and differentiate into effector cells [139]. Naïve T cells and T cells in the early expansion phase are resistant towards apoptosis [140]. After reaching the peak of the immune response T cells become sensitive towards cell death and T cell numbers decline during the contraction phase (Fig. 3) [139, 141]. Induction of activation induced cell death (AICD) depends on re-stimulation via the T cell receptor (TCR) and involves engagement of death receptors like FAS, tumor necrosis factor (TNF) or TNF-related apoptosis-inducing ligand (TRAIL) [142]. T cell activation itself influences the susceptibility to FAS-induced apoptosis because initial T cell activation leads to up-regulation of FAS [141]. Moreover, TCR re-stimulation results in the expression of FASL, which induces FAS-mediated apoptosis either of the same cell that expresses FAS or of neighboring cells [142]. Death receptor independent, intrinsic pathways, are also described for AICD [141]. Activated T cells which are not re-stimulated, die by activated cell

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autonomeous death (ACAD). In contrast to AICD, ACAD does not require re-stimulation of the TCR upon activation and is not mediated by death receptors [141]. Some T cells surviving AICD enter the memory T cell pool [139].

Figure 3. Scheme of a T cell immune response [141]. Reprinted by permission from Elsevier.

Dysregulation of apoptosis in the immune system may lead to immunodeficiency,

autoimmunity or cancer [143-145]. For example, increased AICD of T cells was described in patients with cartilage-hair hypoplasia [146] and several studies have reported increased spontaneous apoptosis and AICD in lymphocytes from patients with common variable immunodeficiency and selective Ig(A) deficiency [147]. On the other hand, mutations affecting FAS apoptotic pathway are associated with loss of apoptotic signaling and leads to an autoimmune disorder called autoimmune lymphoproliferative syndrome [148]. Further, several studies have reported that T cells undergo increased spontaneous apoptosis and AICD in aged individuals as compared with young controls [149]. This can explain a decline in T

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cell functions during aging, along with decreased thymic output and reduced proliferative potential which leads to increased frequency of infections, autoimmunity and cancer.

Apoptosis and 22q11.2 del

Increased spontaneous apoptosis of both CD4+ and CD8+ T cells from one patient with 22q11.2del have been described [150]. Peripheral blood mononuclear cells (PBMCs) of that patient expressed increased levels of FAS and FASL and decreased levels of BCL-2. At the same time, Pierdominici et al. found a reduction of both spontaneous, as well as CD3- and FAS-mediated apoptosis of lymphoid cells from thymic tissue obtained from two patients with 22q11.2del compared to controls [151]. In addition, Zhou et al found no difference in FAS, BCL-2 and tumor supressor P53 expression in the hypoplastic thymuses in 22q11.2del compared to non-22q11.2del thymic tissue [152]. Thus, the role of apoptosis in the

pathogenesis of 22q11.2 del is not clear.

Complement System

The complement system is an integral component of the innate immunity and can be activated through the classical, lectin, and alternative pathways [153]. All three pathways are activated by different stimuli, and they converge on Complement 3 (C3), generating convertases that catalyze the conversion of C3 into its active fragments C3a and C3b. This activation can be detected by different activation products of C3, including the C3b, iC3b and C3c products (C3bc) exposing the same neoepitope in all fragments [154, 155]. C3b in the alternative pathway is the amplification step that leads to all downstream complement events in the cascade, which ends with the formation of the terminal C5bǦ9 complement complex (TCC)

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[156]. C5b-9 can be inserted into a membrane as the membrane attack complex and lyse bacteria and cells. It can be made in the fluid phase as soluble TCC and used as a marker indicating that the terminal pathway has been activated to its very end [154].

Since its discovery in the 1890s when complement was found to “complement” the killing of bacteria by heat-stable antibodies present in normal serum, complement has been primarily viewed as supportive first line defense against microbial intruders [157]. However, in recent years, it become clear that complement system exerts much broader functions in homeostasis then earlier anticipated [156, 158, 159]. Through a close communication of the complement system with other regulatory systems, complement is involved in the number of processes, such as clearance of immune complexes, cellular debris and apoptotic cells, and it has also been associated with early development of tissue repair [160-163].

Complement activation is normally highly regulated, however, a disturbed complement activation can lead to the interaction and activation of immune cells with release of pro- inflammatory mediators such as cytokines [164]. Thus, disturbed complement activation can contribute to the pathology of several autoimmune and inflammatory diseases, such us systemic lupus erytomatosus, antiphospholipid antibody syndrome or ANCA-associated vasculities [165-167]. Activation of the complement system has also been linked to obesity, and dysregulation of complement has been demonstrated in both schizophrenia and bipolar disorders [168-171]. On the other hand, deficiency of complement components might increase the overall susceptibility to infectious disease [172].

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31 Complement and 22q11.2 del

To your knowledge, the role of complement system in the pathology of 22q11.2 del have not been investigated systemically. Elevated levels of C3 and high-normal levels of C4 were described in one 22q11.2 del patient with uveitis [173]. While complement studies were normal in two 22q11.2 del patients with polyarthritis [108]. However, it is not specified what kind of complement studies were performed. Thus, involvement of the complement in the pathogenesis of 22q11.2 del can not be excluded.

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Aims of the Study

The general aim of this study was to increase the general knowledge about the

pathophysiology of the 22q11.2 del in order to get a better understanding of the disease and to facilitate an improved treatment and follow up. The study was planned to describe cytokine levels, apoptosis and complement in patients with 22q11.2 del compared to controls. The main goal of this study was to access whatever 22q11.2 del patients in general have different inflammatory profile than healthy individuals.

Specific aims of the present study were:

Paper I:

x Examine whether 22q11.2 del patients have a different cytokine profile compared to healthy individuals.

Paper II:

x Identify if patients with 22q11.2 del have increased spontaneous apoptosis of lymphocytes, as earlier suggested.

x Study activated cell death by both AICD and ACAD in lymphocytes.

Paper III:

x Investigate the role of the complement system in the pathology of 22q11.2 del by studying the degree of complement activation in vivo.

x Explore functional capacity of complement and measure Mannose-binding lectin (MBL) levels in patients in order to access a possible complement deficiency.

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Materials and Methods

Patients

Patients from all over Norway with a proven heterozygous 22q11.2 deletion by fluorescent in situ hybridization or multiplex ligation-dependent probe amplification were included in the study. They routinely attended the Pediatric Outpatient Clinic at Rikshospitalet, Oslo University Hospital during the time period 2011-14 (Paper I) or 2015 (Paper II and III).

Patients with atypical and known additional deletions were not included. Two patients, twins, with an additional 1q21.1 microdeletion were excluded. A total of 55 patients with 22q11.2 del were included in the analysis in Paper I, 67 patients were included in paper II and 69 patients were included in Paper III.

Patients underwent routine medical examination performed by either Torstein Øverland, Kari Lima or Dina Aresvik. The medical records were scrutinized by Dina Aresvik to confirm patients medical history and clinical features.

The patients did not have any clinically apparent infection when sampled.

Controls

Controls were healthy volunteers recruited among health care workers, their families and friends. In paper I, the control group comprised 54 healthy individuals, while 57 controls were included in Paper II and 56 were included in Paper III. They had no known infection,

inflammation, allergic disease or other acute or chronic illness at the time of blood sampling.

Controls were age and sex matched.

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Ethics

The study was conducted according to the guidelines at Oslo University Hospital and was approved by the Regional Committee for Research Ethics, reference number 2011/1741.

Before inclusion, written informed consent was obtained from either the participants (age >

16 years), the parents (patient age < 12 years) or both patients and parents (patient age 12-16 years).

Weight

Trained nurses measured weight and height of the patients. Body mass index (BMI) was derived from height and weight based on the standard formula kg/m². Adults were then classified as underweight (BMI < 18.5 kg/m2), normal weight (18.5 ≤ BMI < 25 kg/m²), overweight (25.0 ≤ BMI < 30.0 kg/m2) or obese (BMI ≥ 30.0 kg/m2) according to the World Health Organization criteria. For children and teens ages 2 through 19 years, normal weight was defined from 5^th percentile to less than the 85^th BMI-for-age percentile.

Clinical chemistry and immunology

Clinical chemistry analysis, including leukocyte differential count, high sensitive C-Reaktive protein (hsCRP), levels of total and albumin corrected calcium (Ca²⁺), parathyroidea hormone (PTH), thyroidea stimulating hormone (TSH), free thyroxine (free T4) and hemoglobin (Hb) were performed in both patients and controls at the Department of Medical Biochemistry, Oslo University Hospital. Quantitative immunoglobulin testing (IgG, IgA, IgM, and IgE) and thrombocyte counts were routinely performed at the Department of Medical Biochemistry,

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Oslo University Hospital in patients only. Lymphocyte subpopulation phenotyping in patients was routinely assayed at the Department of Immunology, Oslo University Hospital.

Blood sampling

Peripheral venous blood was drawn into three different tubes containing either Serum Sep Clot Activator, Ethylenediaminetetraacetic acid (EDTA) or Sodium Heparin. For serum preparation, peripheral venous blood was drawn into tubes containing Serum Sep Clot Activator. Blood was allowed to clot before centrifugation at 2000 x g for 15 min (Paper I, II and III). For plasma preparation, peripheral venous blood was drawn into tubes containing EDTA. Samples were stored on ice immediately after blood sampling and centrifuged at 2000 x g for 15 min at 4° C (Paper III). Both serum and plasma samples were aliquoted, stored at – 80° C and thawed < 3 times. For PBMC isolation, peripheral venous blood was drawn into tubes containing Sodium Heparin. Samples were placed on ice immediately after blood sampling and PBMCs were isolated (Paper II).

Cytokine assay

In paper I, serum samples were analyzed using a multiplex cytokine assay (Bio-Plex Human Cytokine 27-Plex Panel; Bio-Rad Laboratories Inc., Hercules, CA) containing the following interleukins (IL), chemokines and growth factors: IL-1β, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, eotaxin, basic fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interferon (IFN)-γ, interferon-inducible

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protein (IP-10), monocyte chemotactic protein (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, platelet derived growth factor-BB (PDGF-BB), regulated upon activation T cell expressed and secreted (RANTES), tumor necrosis factor (TNF), and vascular

endothelial growth factor (VEGF). The samples were analyzed on a Multiplex Analyzer (Bio- Rad Laboratories) according to instructions of the manufacturer. Two of the analytes were excluded (RANTES and PDGF) since serum was used and these analytes are released from platelets in vitro during preparation. Seven of the analytes were generally low or below the lower detection limit and therefore excluded from further analysis (IL-1β, Il-2, IL-4, IL-5, IL- 13, IL-15, and MIP-1α). Thus, a total of 18 analytes were included in the study.

Serum concentrations of IP-10 and G-CSF were also measured by Enzyme-Linked Immuno- Sorbant Assay (ELISA) obtained from R&D Systems (Minneapolis, MN) and by Bio-Plex Human Cytokine 2-Plex Panel (Bio-Rad Laboratories Inc., Hercules, CA). This was done since these two markers were found to be higher in the patient group in the multiplex assay.

Thus confirmation assay were required to avoid statistical Type I errors.

Cell culture

In paper II, PBMCs were isolated from heparinized whole blood with Lymphoprep (Axis- Shield PoC, Norway), counted and resuspended in RPMI 1640 medium (Lonza, Belgium), containing penicillin/streptomycin (Pen-Strep, Lonza, Belgium) and 10% heat inactivated fetal bovine serum (FBS) (Lonza, Belgium).

For activation, freshly isolated PBMCs (1 x 10⁶ cells/ml) were cultured in 24-well cell culture plates (Corning Inc., NY) in the presence or absence of monoclonal anti-CD3 antibody (OKT3) (Sigma-Aldrich, MO) at concentration 1 μg/ml and recombinant human interleukin 2

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(IL-2) (BD Biosciences, CA) at concentration 30 U/ml for 72 hours at 37°C in a humidified 5% CO2 atmosphere. Cells were then washed and re-stimulated with a secondary monoclonal antibody (2.MoAb) anti-CD3 Ab (OKT3, 1μg/ml) or with human activating anti-FAS

antibody clone CH11 at concentration 500ng/ml (Millipore, CA) or left untreated for 24 hours at 37°C in a humidified 5% CO2 atmosphere (Fig. 4).

For analysis of FASL in supernatants, cell supernatants obtained after incubation were aliquoted and stored at -80° C.

Fig. 4 Schematic presentation of in vitro culture in order to mimic spontaneous apoptosis, activation induced cell death (AICD), activated cell autonomous death (ACAD) and FAS mediated apoptosis

Determination of cell death

During apoptosis, phosphatidylserine (PS) translocate from the inner part of the plasma membrane to the out layer and becomes exposed on the external surface of the cell. Annexin- V (AnnV) has a high affinity for PS and is therefore suited to detect apoptotic cells. In conjunction with a permeability probe 7-Amino-Actinomycin (7-AAD), a distinction can be made between viable AnnV ^– 7-AAD ^– cells, apoptotic cells with intact plasma-membrane integrity (early apoptotic AnnV⁺7-AAD^–cells) and cells with leaky plasma membrane (late

Spontaneous apoptosis

+anti-CD3 + IL-2 72 h

- 2. MoAb

+ anti-CD3

+ anti-FAS

ACAD

AICD

Anti-FAS induced apoptosis

Restimulation

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apoptotic/necrotic AnnV⁺ 7AAD⁺ cells) [174]. For analysis of cell death, cells were stained with Phycoerithryn (PE) Annexin V Apoptosis Detection Kit I (BD Biosciences, CA) according to manufacturer’s instructions. Briefly, cells were harvested, washed twice in ice cold phosphate-buffered saline (PBS) and then re-suspended in 1X binding buffer. Then the solution was transferred to BD Trucount Tubes (BD Biosciences, CA) and PE AnnV and 7- AAD were added. Cells were gently vortexed and incubated for 15 min at room temperature in the dark. After incubation more binding buffer was added. Samples were then kept on ice and analyzed within one hour using FACSCalibur (BD Biosciences, CA). Data were collected on 50 000 Truecount bead events and analyzed using CXP Analysis software (Beckman Coulter, CA) gated on lymphocytes.

Measurement of FAS

For analysis of FAS expression on cell surface, PBMC were stained with Fluorescein isothiocyanate (FITC) Mouse Anti-Human FAS antibody or FITC Mouse IgG1, N Isotype Control (BD Biosciences, CA) according to manufacturer`s instructions. Briefly, cells were washed with ice cold PBS, re-suspended in FBS Stain Buffer (BD Bioscinces, CA) and pre- incubated for 15 min with purified human IgG (Sigma-Aldrich, MO) before staining in order to avoid unspecific MoAb binding. FAS antibody or isotype control was then added and cells were incubated for 30 min on ice in the dark. After incubation cells were washed in staining buffer and analyzed immediately using FACSCalibur (BD Biosciences, CA). Data were collected on 45 000 events and analyzed using CXP Analysis software (Beckman Coulter, CA) gated on lymphocytes.

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Measurement of FAS Ligand

Human FAS Ligand was measured both in serum and cell culture supernatants by Human FAS Ligand ELISA kit obtained from R&D Systems (Minneapolis, MN). Manufacturer’s instructions were followed during the procedure. The optical density was determined using a microplate reader (Multiscan Ascent) set to 450 nm and the wavelength correction set to 540 nm. Results were analyzed using Ascent Software (Thermo Electron, Finland).

Functional complement capacity

In paper III, assay of functional capacity of the classical, lectin and alternative pathways in the complement system was routinely assayed at the Department of Immunology, Oslo University Hospital using Wielisa COMPL300 Total Complement Functional Screen kit from Wieslab AB, Lund, Sweden [175]. Classical pathway deficiency was defined as < 40% capacity and lectin and alternative pathway deficiency was defined as < 10% capacity.

Serum concentrations of MBL were quantified if lectin pathway deficiency was found using an MBL ELISA kit (BIOPORTO Diagnostics A/S, Hellerup, Denmark) according to the manufactures instructions. Low MBL value was defined as <500 ng/mL, and complete deficiency below 100 ng/mL.

Complement activation products

Complement activation in paper III was measured using activation products from C3 (C3bc) and TCC using ELISA assays described in detail previously [176]. The C3bc assay is based on a capture monoclonal antibody (bH6) reacting with an epitope exposed in C3b, iC3b and

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C3c. The TCC assay is based on a capture monoclonal antibody (aE11) exposed in C9 when incorporated into TCC. Thus, both assays are highly specific for the activation products and not influenced by the amount of the native component.

Statistics

SPSS for Windows release 18 (Paper I) and 25 (Paper II and III) (Chicago, IL,) was employed for the statistical analysis. For comparison of two groups, the non-parametric Mann-Whitney U test was used. When more than two groups of individuals were compared, the non-

parametric Kruskall-Wallis test was used. If a significant difference was found, Mann-

Whitney U test was used to calculate the difference between each pair of groups. Coefficients of correlation (r) were calculated by the non-parametric Spearman’s rank test. The strength of correlation was interpreted as previously described [177].Categorical data were compared using a Chi-squared test. Repeated measurements were compared using Wilcoxon signed rank test. Data are given as median and interquartile range unless otherwise stated. Results were considered significant when p<0.05. For cytokines measured by multiplex, correction for multiple testing using the Bonferroni adjustments for 18 parameters was applied, and results with p<0.003 were considered significant. Figures were generated using GraphPad Prism version 6.04 (Paper I) and 7.04 (Paper II and III) for Windows (GraphPad Software, La Jolla, CA).

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Summary of Results

Paper I:

In order to investigate cytokine profile in patients with 22q11.2 del, we measured serum levels of 27 cytokines, including chemokines and growth factors, using multiplex technology.

InterferonǦinducible protein 10 (IPǦ10) was also measured by ELISA to confirm the multiplex results. The 22q11.2 del patients (n=55) had distinctly and significantly raised levels of proǦ inflammatory and angiostatic chemokine IPǦ10 (P < 0.001) compared to controls (n=54). The patients with congenital heart defects (n = 31) had significantly (P = 0.018) raised serum levels of IPǦ10 compared to patients born without heart defects (n = 24). The other cytokines investigated were either not detectable (IL-1β, Il-2, IL-4, IL-5, IL-13, IL-15, and MIP-1α) or did not differ (IL-1ra, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-17, eotaxin, bFGF, GM-CSF, INF-γ, MCP-1, MIP-1β, TNF, and VEGF) between patients and controls.

Paper II:

In order to clarify if spontaneous apoptosis of lymphocytes is truly increased in patents with 22q11.2 del, we performed flow cytometry analysis of spontaneous apoptosis in 26 patients and 19 controls. We did not find increased spontaneous apoptosis (p=0.132) in patients with 22q11.2 del compared to healthy individuals. However, upon activation, anti-FAS-induced apoptosis was significantly increased (p=0.035) in patients with 22q11.2del (n=13) compared to those in controls (n=9), while there were no difference in activation induced cell death or activated cell autonomous death. We also found a significant increase in expression of FAS on freshly isolated lymphocytes from patients (n=23, p=0.004). There was no difference (p=0.269) in serum levels of FASL in patients (n=63) versus controls (n=56), but patients

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with CHD (n=35) had significantly higher (p=0.022) levels of FASL compared to those in patients without CHD (n=28).

Paper III:

In order to access a possible complement deficiency, we first studied classical, lectin and alternative complement pathways by functional assays in 62 patients. All patients had normal complement activation in both the classical and the alternative pathways. Eight percent had pathologically low activation of lectin pathway due to low levels of MBL.

In order to address if these patients had pathologic complement activation, we studied the complement activation products C3bc and TCC. The patients (n=64) had significantly

(p=0.007) raised plasma levels of C3bc compared with controls (n=45). TCC was slightly, but not significantly (p=0.062), increased in patients compared with controls. Patients with

psychiatric disorders (n=21) had significantly raised (p=0.044) plasma levels of C3bc compared to patients with cognitive abnormalities (n=19) and healthy individuals.

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Discussion

Methodological considerations

Cohort of patients

All patients in this study was recruited from the reference center for 22q11.2 del at the Department of Pediatrics, Rikshospitalet, Oslo University Hospital. This national center was established in 2007 in order to ensure better and more systemic follow-up of 22q11.2 del patients in Norway. Despite that the reference center is located at the Department of Pediatrics, it offers a follow up for all patient groups with Torstein Øverland taking care of pediatric population and Kari Lima taking care of our adult patients. The reference center is widely known, among others due to existence of national guidelines, and patients from all over the Norway is referred to this center. Thru the collaboration with genetic institutions, all patients newly diagnosed with 22q11.2 are referred to the center. Previous study done by Kari Lima on 22q11.2 del largely contributed to recruitment of patients to the reference center, as, using all the genetic institute in the country, all diagnosed patients between 1996 and 2003 were invited to participate. As of today, 229 patients with 22q11.2 del are registered at the reference center. Thus, we believe that the majority of patients diagnosed with 22q11.2 del receive a follow-up at the center. Recruitment of patients from this national reference center has the advantage that the results may be more representative of the condition than reports from specialized clinics.

Patients with duplications and atypical deletions, as well as patients diagnosed with additional syndrome have not been included in this study as they may have a different phenotype than those with a typical deletion [30, 178]. All patients, both newly diagnosed and follow-up, who attended the Pediatric Outpatient Clinic at Rikshospitalet, Oslo University Hospital during the

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time period 2011-15 and fullfield the inclusion criteria were invited and agreed to participate.

Thus, this study should reflect the Norwegian 22q11.2 del population very well.

From 1998 to 2004 four or five individuals have been diagnosed with 22q11.2 del in Norway every year. With an estimated prevalence of 1:3000 – 1:6000 and approximately 60 000 births per year in Norway, it can be calculated that in the Norwegian population of approximately five million people, a higher number of patients remains to be identified. Researchers worldwide agree that due to a wide phenotypic variation the syndrome steel remains

underdiagnosed [30]. Studies in families have revealed mild cases not diagnosed until he or she got an affected child [33]. Thus, we can hypothesize that the total picture of the syndrome will probably change in the future as several patients with less recognized clinical features will be diagnosed. At the same time, Lima et al demonstrated that some patients with serious or several typical features got the diagnosis late in life [75].

Study design

The study was planned to describe cytokine levels, apoptosis and complement in patients with 22q11.2 del compared to controls. The main goal of this study was to access whatever

22q11.2 del patients in general have different inflammatory profile than healthy individuals.

In addition, we wanted to study apoptosis in 22q11.2 del as we believed that it was not

increased, despite previous findings [150]. This study was not design to conclude about causal relationship between our measurements and clinical features.

The background information regarding patient features was obtained from medical records.

All medical records studied, especially those from the reference center, were structured and of high quality. All patients were investigated by co-authors at the same day as blood samples

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were taken. However, recording and categorizing signs and symptoms in groups were

challenging. It was especially difficult to categorize psychiatric disorders, VPI and infections.

For example, number of patients presented with less well defined cognitive symptoms requiring some kind of a follow up. Another example is infants who clearly presented with VPI symptoms such feeding difficulties/regurgitation of milk, but who did not receive the diagnosis as they have not established speech yet. Infection severity was also difficult to define, especially in cases of mild or moderate affection, while it was easier to register the more serious infections such as pneumonias. It is also worth noting that it was no correlation at all between T-cell counts and infection susceptibility. Besides, one could speculate if some features attributed to our patients are biased due to the 22q11.2 del diagnosis. The threshold for admitting the patient to the hospital or prescribing antibiotics will probably be lower for patients having an immunodeficiency diagnosis. The same will most likely be true when it comes to involving of pedagogical or psychological follow-up in patients with known predisposition for developing psychiatric diseases. Another thing to consider is that both immunodeficiency and psychiatric disorder represents a continuum. While infection susceptibility tend to reverse with increasing age, patients who presented with behavioral abnormalities in childhood are likely to develop psychiatric diseases in the adulthood [75, 115]. Moreover, autoimmune disorders may appear during childhood, adulthood or late in life, depending on the type of disease and in 22q11.2 del a general susceptibility to

autoimmunity rather than an association with a specific disease is seen [30, 99, 107, 179]. In our cohort, six out of seven patients above age of 30 years had symptoms of autoimmune or inflammatory disease. Thus, in order to provide more details about onset and disappearance of conditions, changes with age and relationships between features and cytokines,

complement system and the apoptotic proteins, the longitudinal study should be conducted.

Such study should involve a multidisciplinary team.

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46 Analysis

Multiplex

In paper I, multiplex technology was used to detect serum cytokine levels. Traditionally, ELISA has been the gold standard for cytokine analysis [180]. Yet this approach of measuring a single protein in each sample limits the amount of information which can be obtained from a small human sample [181]. In this study it was a particular issue, as blood volume obtained was restricted due to the patient age and concurrent blood sampling for diagnostic purpose.

Multiplex assay become widely used during the last years and measures multiple cytokines in the same sample at the same time [180, 181]. In comparison to ELISA, multiplex assays in general are more sensitive, show a broader analytical range, are more rapid and require smaller sample volumes [182]. However, good correlations, but often poor concurrence of quantitative values between multiplex assays and corresponding ELISA measurements have been shown [183]. This is because the techniques, including the antibodies used, are different.

Further, by analyzing a large number of analytes there will be an increased risk of statistical Type 1 errors. We found two of the analytes, IP-10 and G-CSF, to be significantly higher in the patient population. However, only IP-10 was confirmed to be increased when using an ELISA assay. Thus we assume that the G-CSF might have been statistically false positive, whereas the IP-10 data seem solid and reliable since they were significantly higher in the patient group using both assays. Results obtained by Elisa and Multiplex also showed a strong correlation.

Flow cytometric analysis

In order to investigate lymphocyte apoptosis, flow cytometric analysis was employed in Paper II. The major bias of the work is the variability in the number of the blood samples tested in

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each experiment. Despite that we tried to include as many patients as possible, in some patients a restricted subset of tests was performed due to insufficient blood volume obtained.

This was both because of the age of the patients and because blood tests for clinical use were prioritized. In order to make a best possible comparison, age and sex matched controls were recruited. However, even if the patient number is lower in flow cytometry analysis then in ELISA, we still have a fair number of patients, which is rare for studies of 22q11.2del syndrome.

In order to activate T cells, stimulation of freshly isolated PBMCs was performed with OKT3 in combination with IL-2 (Paper II). This method is widely used [184]. However, several antigens can be used for T blasts generation. Along with OKT3, phytohemagglutinin (PHA) is frequently employed for that purpose. When PHA is used, T cells have to be cultured for 6 days instead of 3 days [185]. The longer incubation time with PHA can be explained by the difference in the activation pathway. OKT3 has also been shown to be more potent than PHA as a mitogen [186]. Prior to the study, pilots were performed with both OKT3 for 3 days and PHA for 6 days. In our hands, we did not observed any difference between those two agents in the activation of the T cells and the apoptosis rate after re-stimulation with 2.MoAb.

Complement analysis

In paper III, functional capacity of the classical, lectin and alternative pathways in the complement system was assayed. The functional capacity of the classical and alternative pathway was completely normal, while lectin pathway was low in five patients due to MBL deficiency. The functional capacity of the complement pathways was measured in patients only, and MBL was only measured when functional capacity of lectin was low. Thus, the measurements can not be compared to the controls and statistical analysis can not be

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performed in order to access a possible difference. However, MBL deficiency have been widely studied, revealing that variation in MBL concentration in apparently healthy individuals is large, and about oneǦthird of the Caucasian population possess genotypes conferring low levels of MBL [187]. Based on that, we could compare our findings with available background information.

Statistical analysis

For comparison of two groups, the non-parametric tests were used. Parametric tests are, in general, more powerful, that is require a smaller sample size, than nonparametric tests [188, 189]. However, they assume a normal distribution of values. Nonparametric tests are less powerful than parametric tests, but they can be necessary when the distribution is not normal, the distribution is not known, or the sample size is too small to assume a normal distribution.

Based on that, the use of the non-parametric tests in our study was more appropriate.

Despite the above mentioned shortcomings, we hope that our findings may contribute and inspire to further research on 22q11.2 del.

Discussion of major findings

Cytokine levels in 22q11.2 del

In paper I, a wide range of interleukins, chemokines and growth factors were studied in 55 22q11.2 del patients and 54 age and sex matched controls. Among 25 cytokines examined only IP-10 was significantly different between patients and controls. No difference in the serum levels of IL-6, IL-7, IL-8, IL-9, IL-10, GM-CSF, INF-γ, MCP-1, MIP-1β, TNF, and

Cytokines, apoptosis and complement in 22q11.2 deletion syndrome (DiGeorge syndrome)