Determining the Association of Chromosomal Variants With Non-PV Triggers and Ablation-outcome in AF (DECAF)

Specific Aim: This prospective pilot study aims to examine the association of specific
genetic variants (single nucleotide polymorphisms) namely rs2200733, rs6843082, rs10033464,
rs17042171, rs2106261 and rs13376333 on chromosome 1, 4 and 16, with presence of
non-pulmonary vein triggers (NPVT) as well as ablation-outcome in AF patients.

Hypothesis: Genetic variants predict prevalence of non-PV triggers as well as long-term
procedure-outcome.

Background: Atrial fibrillation (AF) is the most common clinical arrhythmia affecting nearly
3.0 million people in the United States. Its significant contribution to population
morbidity and mortality is amplified by the fact that AF is associated with 3-5 fold
increase in the prevalence of cerebro-vascular stroke and 2-fold increase in the risk of
death. Limited efficacy of the available therapeutic strategies makes the matter worse;
failures being attributed to lack of clear-understanding of the pathophysiology of this
complex arrhythmia. In addition to the traditional risk factors including advancing age,
obesity, metabolic syndrome, ischemic/valvular heart disease and hyperthyroidism etc that
predict the occurrence of AF, genetic predisposition to AF has been reported in recent
years.

Common AF often occurs with structural heart diseases but not all individuals with the same
cardiac pathology develop AF, indicating that there must be genetic factors predisposing
some individuals to AF. In 2007, the first Genome wide association study (GWAS) for AF in
subjects of European descent was reported by investigators from Iceland. Two common variants
on chromosome 4q25 were found that were strongly associated with AF. Following this initial
report, several research groups provided independent replication analyses. So far, there are
at least three distinct genomic loci, 4q25, 16q22, and 1q21 that have a strong association
with AF.

Although an SNP is generally not sufficient to cause AF, it may act in combination with
other SNPs or pathological conditions (e.g., ischemia and stretch) to increase
susceptibility to AF or it may have some regulatory role in the expression of nearby genes
that are potential candidate genes for AF.

Recent GWAS findings have left us with promising novel molecular pathways for AF and provide
a starting point for the dissection of these novel pathways related to AF. However, the
majority of these studies have not been replicated in independent populations or in
different ethnic groups and none of the molecular pathways through which these SNPs lead to
AF have been definitively determined. A better understanding and characterization of the
genetic variants associated with AF in general population would facilitate new approaches to
the diagnosis and efficient treatment of this arrhythmia.

On the other hand, catheter ablation has become an established invasive procedure treating
patients with AF and has offered the promise to free patients of symptoms as well as
eliminate the need for use of chronic drug therapy with agents that sometimes have
significant risks, cost, and inconvenience for the patient. However, despite of the rapid
progress of this technique in the past decade, the success rate and outcome of AF ablation
remain suboptimal and largely variable depending on the case volume and ablation strategy
used at individual centers. Many risk factors have been associated with the procedural
failure and arrhythmia recurrence after AF ablation, including the type of AF, size of left
atrium, cycle length of AF, etc. Recently, we have demonstrated that the presence of NPVT
strongly predicts failure of AF ablation while elimination of NPVTs during the procedure
significantly improves the long term freedom from arrhythmia after ablation of AF of any
type. Non-PV triggers are defined as ectopic triggers originating from sites other than
pulmonary veins such as left atrial posterior wall, superior vena cava, left atrial
appendage, ligament of Marshall and coronary sinus.

Lately, a few published researches have shown that certain common genetic variants on
different chromosomes are associated not only with an increased risk of AF itself, but also
with the response to AF treatment or prognosis of AF patients. As we have observed a high
incidence of NPVTs in AF patients and their role in determining the AF ablation outcome, we
hypothesize that some common genetic variants at different chromosomal loci are associated
with the occurrence of NPVTs, by which they subsequently predispose the development of AF
and influence its response to management.

Study Design:

This prospective pilot study will enroll 400 consecutive AF patients undergoing catheter
ablation. Baseline blood sample will be collected from all patients for genetic analysis.
Quality of Life (QoL) surveys will be conducted at baseline and 1-year follow-up. All
patients will be followed up for recurrence for one year.

The study will be conducted in collaboration with Dr. V. Iyer, professor at University of
Texas at Austin. All genetic analyses will be performed at the UT core facility.

Primary endpoints:

1. Pulmonary vein antrum isolation (PVAI) and isolation of all non-PV triggers

2. Recurrence of arrhythmia

Study Procedure:

1 ml of whole blood will be collected from each patient in a 4 ml Sodium-heparin tube before
the ablation procedure. The blood sample will be labeled with an anonymous patient
identifier that can only be identified by research staff. Following collection, blood sample
will be stored at -200 C at the clinical lab of St. David's Medical Center until it is
transferred (twice weekly in nitrogen bucket) to the lab of Dr. V. Iyer (Molecular Genetics
and Microbiology, UT at Austin) where it will be stored at -70 degree C freezer until the
end of the study, when all samples will be simultaneously analyzed. Genomic DNA purification
and SNP analysis by Taqman assay will be performed for all samples at the end of the study,
at the UT facility.

DNA purification protocol:

Qiagen QiaAMP 96 well blood kit will be used to lyse the white blood cells and purify
genomic DNA.

Overview of the procedure for using TaqMan SNP Genotyping Assays:

Purified DNA samples will be used in a TaqMan qPCR assay. Briefly, a small quantity of each
sample of DNA will be added to six wells in a 384 well qPCR plate. Next, Taqman fluorescent
labeled probe will be addeded to the wells. Lastly, TaqMan master mix will be put in each
well. This qPCR plate will be run on a Life Technologies ViiA7 real-time qPCR machine. To
detect the presence or absence of each SNP in each patient, a plate reader will be used to
detect fluorescence in each well.

Ablation procedures:

Standard mapping and ablation procedures will be performed at the discretion of the
physician.

Follow-up:

Following ablation, patients will be discharged on their previously ineffective AADs which
will be continued for 90 days (blanking period). The blanking period allows time for the
inflammatory process to subside. AADs will be discontinued after the blanking period for all
patients. If a patient suffers a recurrence of an atrial arrhythmia, AAD therapy may be
administered at the discretion of the physician.

All patients will be followed-up for minimum of 1 year following the PVAI. Recurrence will
be assessed by event recording for 5 months and Holter monitoring at 1, 3, 6 and 12 months.
Recurrence will be defined as freedom from atrial flutter (AFL), AF or atrial tachycardia
(AT) of > 30 seconds duration, in the absence of anti-arrhythmic drugs (AADs) at follow-up.

Risk Analysis:

This study does not pose any additional risk to the patient. The risks are the same as those
for a standard AF ablation.

Minor risks associated with a venous blood draw may include fainting or bruising, pain or
discomfort and a 1/1000 risk of infection at the site where blood is drawn.

Benefits:

The subject may not incur any benefit by participating in this study.

Potential benefit for future patients: This information will enrich the knowledge about the
molecular mechanism of AF and help in developing personalized ablation strategies for future
patients that would be more effective in eliminating this arrhythmia.

Statistical Methodology:

Sampling Plan:

Consecutive eligible patients will be approached for enrollment.

Analysis Plan:

Continuous variables will be reported as mean ± standard deviation (SD). The categorical
variables will be reported as frequencies and percentage. Data analysis will be performed
using the unpaired Student's t-test for continuous variables and chi-square test for
categorical variables.

For genotype-rhythm outcome correlations, 3 different models will be applied. The variant
alleles will be assumed to be either dominant or recessive or having additive effects in
these models. In the dominant model, an identical effect is expected in heterozygous and
homozygous variant carriers. In the recessive model, an effect is only seen in homozygous
variant carriers. Lastly, in the additive model, heterozygous variant carriers are assumed
to have an intermediate effect as compared to the homozygotes.

Multivariate regression analysis will be performed using Cox proportional hazards model to
test the association of the SNPs with the prevalence of non-PV triggers and outcome
variable. SAS 9.2 (SAS Institute Inc., Cary, NC) will be used for statistical analysis

Inclusion Criteria:

1. Age ≥ 18 years

2. AF patients undergoing catheter ablation

3. Able and willing to provide written informed consent

Exclusion Criteria:

1. Previous left atrial catheter ablation or MAZE procedure

2. Reversible causes of atrial arrhythmia such as hyperthyroidism, sarcoidosis,
pulmonary embolism etc