1. Chitinases in Asthmatic Children Undergoing Tonsillectomy
“This study is a collaboration between the YCAAD and CPO investigating the effect of adenotonsillectomy on inflammation and asthma control in pediatric patients.”
Maria Koenigs Yale School of Medicine Class of 2014
Maria B. Koenigs, BA1, Jonathan C Levin, BS1, Lisa Gagnon, APRN2, Eric D Baum, MD2, David E Karas, MD2 and Geoffrey L Chupp, MD1
1Section of Pulmonary and Critical Care, Yale University School of Medicine, New Haven, CT, 2Section of Otolaryngology, Yale University School of Medicine, New Haven, CT

2. Chitin and Chitinases
Chitin is the 2nd most abundant polysaccharide in nature
Contributes to Th2 inflammation associated with asthma
(Ober and Chupp, Curr Opin Allergy Clin Immunol 2009)
Chitinases break down chitin and modulate host immune response
Chitotriosidase (true chitinase), YKL-40 (chitinase-like)
Seibold, J Allergy Clin Immunol 2008
Humans don’t have chitin but have conserved chitinases to deal with chitin in environment
Chitotriosidase- primary human chitinase in the lung, found to be elevated in serum of asthmatics and decreased in BAL fluid, found to be mutant copy in
Lung
Chitin
Chitinases
Chitin
Chitotriosidase
YKL-40
Th2 Inflammation

3. Human chitinase and chitinase-like proteins are associated with asthma in children
Chitotriosidase
YKL-40
Primary chitinase of the lung
Higher activity in the serum in asthmatics
(Bargagli, Respiration 2010)
Truncated form of protein found in asthmatic children
(Vicencio, Pediatrics 2010)
Elevated in serum of asthmatics (Chupp et al NEJM 2007)
Elevated levels associated with increased hospitalization in asthmatic children
(Cunningham, Annals of Aller, Asthma, and Immuno, 2010)
YKL-40
Chitotriosidase

4. Chitotriosidase is overexpressed in hypertrophied adenoid tissue in children
Chitotriosidase found to be over-expressed in adenoids 41 children with
Chronic rhinosinusitis
Otitis Media
Allergic rhinitis
The presence of Chitotriosidase in the upper airway suggests that there may be a relationship between inflammation in the upper and lower airways
Heo, Otolaryngol Head Neck Surg, 2011

5. Adenotonsillectomy and Asthma
% improved asthma control (Garabedian, Ann Otolaryngol Chir Cervicofac)
Since then- small mostly single-armed studies
renewed interest
Decreased ER visits and pediatrician visits
Decreased medication usage
Increased ACT score
(Ulis et al Unpublished data, (Piessens, Int Journal of Pediatric Otolaryngology, Busino, Laryngoscope)
Common surgical procedure in children, performed most commonly for symptoms of sleep disordered breathing or chronic/recurrent tonsillar infections
Observational study of 55 asthmatic patients, “a remarkable finding, 40-45% of cases it was markedly improved.”
1996 – Observational study
2011 – Retrospective chart review - 2.2% of asthmatics had tonsillectomy for chronic tonisllitis (versus 9.9%) of controls
2012 – Retrospective chart review
However, whether or not AT has a biological effect on airway inflammation and asthma has not yet been studied
Biological effect of adenotonsillectomy on asthmatic inflammation has not yet been studied

6. Hypothesis and Aim Hypothesis
Adenotonsillectomy will improve asthma control and modulate inflammatory pathways associated with asthma
Aim
A prospective study to investigate the clinical and biological effect of adenotonsillectomy on asthma, chitotriosidase activity, and YKL-40 levels before and after surgery

7. (Physician Diagnosis)
Study Design
Baseline - Asthma Questionnaires - Genotypes - Chitinase Levels - PFTs (age ≥6)
6 month follow-up - Asthma Questionnaires - Chitinase Levels - PFTs (age ≥6)
184 pediatric patients (ages 2-18) undergoing adenotonsillectomy enrolled
107 Asthmatics
(Physician Diagnosis)
77 Controls
137 enrolled, Physician diagnosis of asthma, 71 asthmatics vs 66 controls
Baseline – Clinical (Qs, PFTs), Lab (Genotypes, Chitinase, IgE)
6-mo – Repeat Qs, PFTs, Labs
Total 51 follow-ups (91 eligible for 6 month)
Mean 6.8 months (range 5-12) until follow-up
Asthmatics – 27 (19 labs)
Controls – 24 (9 labs)

8. YCAAD Asthma Questionnaire (Pediatric Version)
Study Methods
YCAAD Asthma Questionnaire (Pediatric Version)
Asthma Symptoms: Symptoms history, Triggers, Medication usage, ED visits
Comorbidities: sinusitis, atopy (allergies and eczema), GERD
Environmental, home, and product exposures
Personal and family med history
Socioeconomic data, school and work days missed
Asthma Control Test (ACT)
Objective Data
Chitinase Genotype
Chitinase Activity
YKL-40 Level
IgE Levels
Pulmonary Function Testing
(age ≥ 6, and otherwise able)
Asthma Questionnaire surveys symptoms, comorbidities, exposures, personal and family med hx, SES data, school & work days missed, and ACT
Baseline ACT collected retrospectively on initial group of patients, however, data was consistent with ACTs collected prospectively
Objective data
Asthma Symptoms: Symptoms history, Triggers, Medication usage, ED visits
Comorbidities: sinusitis, atopy (allergies and eczema), GERD
Environmental, home, and product exposures
Personal and family med history
Socioeconomic data, school and work days missed
Asthma Control Test
Genotype
CHIT1 exon 10 duplication (rs )
CHI3L1 Promoter SNP (rs )
Chitinase Activity – measure of Chitotriosidase (Enzyme Assay)
YKL-40 Level (ELISA)
IgE Levels
Pulmonary Function Testing
(age ≥ 6, and otherwise able)

9. Study Population at 130 patients
Mean Age: 6.2 (2-18 range)
Follow up rate: 80.7%
Primary Indication for tonsillectomy
75% for tonsillar hypertrophy and OSA
23% recurrent infection
Differences in asthmatics versus controls
Hispanic ethnicity
Comorbities: Sinusitis, hx of GERD, Atopy
Decreased FEV1
No sig diff in age, gender, race, second hand smoke, T&A primary indication
Sig: Hispanics/Latinos in asthmatics, Atopy, Hx GERD, Sinusitis, FEV1% (consistent)
IgE higher in asthmatics, not sig

10. Clinical Response to T&A in Asthmatics
In asthmatics – sig decreased chitinase activity
Follow-up data for asthmatics showed…
Sig increases ACT score; fewer ED visits, steroid courses, ear infections, other antibiotic use, missed school and parental work days
Levin et al Unpublished Data

11. Chitinase Activity is modified by T&A
Correspondingly, Chitinase activity significantly lower in Asthmatics, YKL-40 levels NS
Levin et al Unpublished Data

12. Subpopulation Analysis Poorly controlled asthmatics
Poor asthma control is defined as
ACT ≤ 19
More than 2 ED/Urgent care visits per year
More than 2 oral steroid courses per year
More than 2x/week rescue SABA use
Poorly controlled asthma- ED visits and ACT(N Engl J Med April 9; 360(15): 1487–1499.)
Steroid use in > 2 Ann Allergy Asthma Immunol 2004; 92:312-9
Frequent exacerbations: Aidan Long Mass general, hospital

13. Composite Variable Asthma improvement in uncontrolled asthmatics at baseline
Asthma Improved in 36/48 (75%)
SABA use decreased
ACT Score Increase > 3
Number Oral Steroid Courses Decreased
ED/Urgent Care Visits Decreased
18/28 (64%)
16/20 (80%)
We defined improvement in asthmatic symptoms by any of the following:ED visits decreased, steroid courses decreased, ACT +3, Albuterol use in last month decreased, controller stopped
24/35 (68%)
Levin et al Unpublished Data
19/22 (86%)

14. Baseline chitinase activity is elevated in poorly controlled asthmatics who improve with T&A
Levin et al Unpublished Data

15. Hypertrophy/ Inflammation
Concept: Tonsillectomy induces changes in systemic inflammation associated with asthma
Tonsil
Hypertrophy/ Inflammation
Before Tonsillectomy
  Serum Chitinases
Chitin
 Systemic Th2 Response
Asthma
Severity
After Tonsillectomy
 Serum Chitinases
Our primary study results suggest that patients who have a higher chitinase level (animate) at baseline with tonsilar hypertrophy/inflammation & asthma are more likely to see improvement after tonsillectomy
Animate
Given high serum levels, indicates a systemic component to inflammation at baseline
After tonsillectomy, serum chitinase levels fall
Suggesting a decreased systemic inflammatory response along with decreased asthma severity
Chitin
 Systemic Th2 Response
Asthma
Severity

16. Summary and Conclusions
Tonsillectomy improves asthmatic control in a majority of pediatric patients
Chitinase activity is decreased in asthmatics following adenotonsillectomy
Higher baseline chitinase activity may predict which poorly controlled asthmatics will have improved asthma control after adenotonsillectomy
In summary, it was found that…

17. Limitations Seasonal variability
Batch effect – sample collection and processing
Limited PFT data in young population
Small loss to follow-up

18. Future Directions Currently: Genome-Wide Expression Analyses
on Tonsillar Tissue, Nasal Epithelial Tissue, and Blood
Prospective study to explore the use baseline chitinase activity to predict response to adenotonsillectomy
Comparison with matched non-surgical control group
Personalized Medicine
T&A on systemic inflammation

19. Acknowledgements Jon Levin, Yale School of Medicine ‘13
Lisa Gagnon, APRN
Dr. Geoffrey Chupp and Dr. David Karas
Dr. Susan He
Connecticut Pediatric Otolaryngology
Yale Center for Asthma and Airway Diseases
Yale-New Haven Hospital Pediatric Surgery Center
North Haven Surgical Center

20. Questions?

21. Adenotonsillectomy and Asthma
Mostly small, single-arm studies
1986: 40-45% showed improvement
(Garabedian, Ann Otolaryngol Chir Cervicofac)
2010: Prospective study of asthmatics and controls
Fewer ER/pediatrician visits, steroid courses, decreased asthma severity medication use post-op
(Ulis et al Unpublished data)
2011: Retrospective study of asthmatics and controls
Fewer hospital visits, steroid courses, medication use
Improved ACT scores
(Busino, Laryngoscope)
2012: Decreased respiratory meds after surgery
(Piessens, Int Journal of Pediatric Otolaryngology)
Common surgical procedure in children, performed most commonly for symptoms of sleep disordered breathing or chronic/recurrent tonsillar infections
Observational study of 55 asthmatic patients, “a remarkable finding, 40-45% of cases it was markedly improved.”
1996 – Observational study
2011 – Retrospective chart review - 2.2% of asthmatics had tonsillectomy for chronic tonisllitis (versus 9.9%) of controls
2012 – Retrospective chart review
However, whether or not AT has a biological effect on airway inflammation and asthma has not yet been studied
Biological effect of adenotonsillectomy on asthmatic inflammation has not yet been studied

22. Chitotriosidase (CHIT1) in Asthmatics
Primary active chitinase in the human lung
(Seibold, J Allergy Clin Immunol 2008)
CHIT1 24-bp exon 10 duplication (rs ): nonfunctional protein
 rate of mutant allele in 6 asthmatic children with fungal sensitization
(Vicencio, Pediatrics 2010)
Higher activity in serum of asthmatics (Bargagli, Respiration 2010)
Lower activity in BAL of asthmatics (Siebold, J All Clin Immuno 2008)
Hypertrophied Adenoids: chitotriosidase overexpression in 41 children with chronic rhinosinusitis, otitis media, and allergic rhinitis
(Heo, Otolaryngol Head Neck Surg, 2011) genotype for CHIT1 and asthma, hospitalizations, or ED visits
EXON 9
EXON 10
EXON 11
Wild Type
Mutant
24 bp duplication
CHIT1 encodes, duplication exists that makes nonfunctional protein
422 patients showed no association between genotype for CHIT1 and asthma, hospitalizations, or ED visits (Wu A, J All Clin Immunol 2010)
Additionally, Hypertrophied adenoids…in chronic upper airway disease

23. YKL-40 (CHI3L1) in Asthmatics
Elevated in the serum of asthmatics (Chupp et al., NEJM 2007)
CHI3L1 Promoter SNP (rs , -131CG)
Associated with elevated YKL-40 levels, severity of asthma, bronchial hyperresponsiveness, and reduced pulmonary function; minor allele is protective (Ober et al., NEJM 2008)
Associated with asthma hospitalizations in children
(Cunningham, Annals of Aller, Asthma, and Immuno, 2010)
Chitinase-like protein
In adults, Serum levels Correlated with severity, bronchial hyperresponsiveness, subepitithelial basement membrane thickening, and poor pulmonary function
Genotype – SNP (C allele)  elevated; G allele protective