- Email: [email protected]
Painful Bladder Syndrome on Chromosome 3
Author's Accepted Manuscript Significant Linkage Evidence for Interstitial Cystitis/Painful Bladder Syndrome on Chromosome 3 Kristina Allen-Brady , Kerry Rowe , Melissa Cessna , Sara Lenherr , Peggy Norton
PII: DOI: Reference:
S0022-5347(17)77178-9 10.1016/j.juro.2017.07.068 JURO 14877
To appear in: The Journal of Urology Accepted Date: 8 July 2017 Please cite this article as: Allen-Brady K, Rowe K, Cessna M, Lenherr S, Norton P, Significant Linkage Evidence for Interstitial Cystitis/Painful Bladder Syndrome on Chromosome 3, The Journal of Urology® (2017), doi: 10.1016/j.juro.2017.07.068. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.
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Significant Linkage Evidence for Interstitial Cystitis/Painful
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Bladder Syndrome on Chromosome 3
Kristina Allen-Bradya, Kerry Roweb, Melissa Cessnac, Sara Lenherrd, and Peggy Nortone
Division of Genetic Epidemiology, Department of Internal Medicine; University of Utah; Salt Lake City, Utah, USA, [email protected]
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Department of Pathology, Intermountain Healthcare Central Region and Intermountain Healthcare BioRepository, Salt Lake City, Utah, USA , [email protected]
Division of Urology, Department of Surgery; University of Utah, Salt Lake City, Utah USA, [email protected]
Division of Female Pelvic Medicine and Reconstructive Surgery, Department of Obstetrics and Gynecology; University of Utah, Salt Lake City, Utah, USA, [email protected]
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Homer Warner Center for Informatics Research, Intermountain Healthcare, Salt Lake City, Utah, USA, [email protected]
Financial Support: Supported by a grant from the Interstitial Cystitis Association
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Short title: Genetic Linkage Evidence for Interstitial Cystitis
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Presentation: Presented as an oral presentation at the American Urology Association (AUA) 112th Annual Scientific Meeting, Boston Massachusetts, May 12-16, 2017. Corresponding Author: Kristina Allen-Brady Genetic Epidemiology, University of Utah 391 Chipeta Way, Suite D Salt Lake City, UT 84108 Phone: (801) 585-5333 Fax: (801) 581-6052 [email protected] Key words: interstitial cystitis, genetic linkage, pedigree, chromosome 3 Word Count: 2498
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Abstract PURPOSE: Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic pelvic pain condition with unknown etiology. We hypothesized that related IC/PBS cases were more likely
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to have a genetic etiology. The purpose of this study was to perform a genetic linkage analysis. MATERIALS AND METHODS: IC/PBS cases were identified using diagnostic codes linked to a population-based genealogy resource (Utah Population Database) and electronic medical
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records. For this analysis, 13 high-risk pedigrees (defined as having a statistical excess number of IC/PBS cases among descendants compared to matched hospital population rates) were used.
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Case status was confirmed in medical records using natural language processing. DNA was obtained from stored, non-neoplastic, formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Each pedigree had at least two cases with DNA available. Parametric linkage analysis was performed.
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RESULTS: Pedigrees ranged in size from 2 to 12 genotyped cases (n=48 total cases). Significant genome-wide linkage evidence was found on chromosome 3p13-p12.3 (maximum hetTLOD = 3.56) under a dominant model. There were 2 pedigrees with at least nominal linkage
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evidence (LOD >0.59) in this region; the most informative pedigree included 12 IC/PBS cases (pedigree TLOD = 2.1). Other regions with suggestive linkage evidence included 1p21-q25,
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3p21.1-p14.3, 4q12-q13, 9p24-p22, and 14q24-q31, all under a dominant model. CONCLUSIONS: While the etiology of IC/PBS is unknown, this study provides evidence that a genetic variant(s) on chromosome 3, and possibly chromosomes 1, 4, 9, and 14, contribute to IC/PBS predisposition. Sequence analysis of affected cases in identified pedigrees may provide insight into genes contributing to IC/PBS.
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Introduction Interstitial cystitis/ painful bladder syndrome (IC/PBS) is a chronic pelvic pain condition that includes urinary frequency, urgency, and bladder discomfort.1 IC/PBS occurs primarily in
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women; prevalence rates are estimated at 197 cases per 100,000 women and 41 cases per
100,000 men.2 As IC/PBS is a diagnosis by exclusion of other similar diseases, these prevalence
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rates may be underestimated.3
While the etiology of IC/PBS is unclear, family history of IC/PBS is one of the strongest known
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risk factors suggesting a genetic component. First-degree relatives are at increased risk,4 and twin studies have shown a high concordance in monozygotic twins.5-7 Previous genetic studies have generally focused on candidate genes8 and gene expression studies using bladder biopsies.911
Genes involved in allergic or autoimmune disease such as the beta2-adrenoceptor (ADRB2)12
and genes involved in inflammation including FGF7,9 CCL21,9 IgE,11 and JNK pathway genes10
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have been implicated. A previous linkage analysis of panic disorder and bladder/renal problems, potentially interstitial cystitis,13 found significant evidence for linkage on chromosome
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13q.14
We hypothesized that related IC/PBS cases, sampled from pedigrees with a statistical excess
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number of cases, are likely to have a stronger genetic contribution to their disease. If there is a genetic contribution to IC/PBS, study of high-risk families increases power to detect a genetic effect. The purpose of this study was to perform a genetic linkage analysis to identify shared genetic regions in high-risk, related IC/PBS cases.
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Materials and Methods Subjects
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We used a novel research approach to take advantage of a population-based genealogy database that has been linked to medical records and existing sources of DNA. Relationships between IC/PBS cases were identified using the Utah Population Database (UPDB), a computerized
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genealogy for Utah that has been linked to electronic medical records and other public data sources.15 Genealogy information is available for over 8 million unique individuals. The
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majority of families living in Utah are represented in the UPDB, most with at least five generations of information available. All involved subjects were required to have genealogy information available for at least three generations. The Utah population has previously been shown to be mostly of Northern European descent,16 and has experienced high migration rates
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and low levels of inbreeding among the founding population.17
To identify potential IC/PBS cases, we used the UPDB genealogy links to electronic medical records (ICD-9) diagnostic codes for both inpatient and outpatient visits at Intermountain
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Healthcare (IH), the largest healthcare provider in Utah. IH electronic medical records were available since 1995 for approximately 4 million records. Potential IC/PBS cases were identified
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as having a diagnostic code of chronic interstitial cystitis, ICD-9 code: 595.1 (n=3,864 patients). High-risk pedigrees were defined as pedigrees where the number of observed IC/PBS cases was significantly greater (p<0.05) than expected. The expected number of cases was calculated as the population rate of disease for a specific cohort, defined by sex and birth year in 5-year intervals. Family members in a pedigree were assigned the population rate of IC/PBS based on their birth year and sex cohort. Rates of disease were summed across all family members to equal the
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expected number of cases in a pedigree. We identified 1,533 IC/PBS cases who were members of pedigrees with a statistical excess of IC/PBS (p<0.05) and which included at least two cases per pedigree. To confirm IC/PBS case status, co-author and IH bioinformaticist (KR) used
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natural language processing (NLP) to verify the existence of at least one of the following terms in the medical record: “interstitial cystitis” (not “rule out interstitial cystitis”), “chronic pelvic pain”, “painful bladder (syndrome)”, “bladder pain syndrome”, “hypersensitive bladder
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syndrome” and/or “Hunner’s ulcers”. Approximately 50 records were manually confirmed to check the query. Of 1,533 potentially high-risk IC/PBS cases, 718 cases were confirmed. For
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unconfirmed IC/PBS cases, diagnostic information may be contained in paper charts; however, these records are not electronically searchable and were not included in this study. Of 718 confirmed cases, 580 fit into a pedigree with at least two confirmed, affected cases. This study was approved by the University of Utah Institutional Review Board (IRB), Intermountain
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Healthcare IRB, and the Resource for Genetic Epidemiology, which oversees use of UPDB data. All subjects and their DNA samples were de-identified to the primary investigators (KAB, PN,
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and SL) on this project.
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DNA isolation and quantification
We used existing formalin-fixed, paraffin embedded (FFPE) tissue blocks as the source of DNA for this project. The Intermountain Healthcare BioRepository (IBR) has archived FFPE blocks for almost all pathology samples collected since the 1970s, which are linked to the clinical record. We required tissue from FFPE blocks from at least two NLP confirmed IC/PBS cases in a pedigree. These patients were linked to their pathology records and screened for non-
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neoplastic FFPE tissue samples suitable for preparation and DNA extraction. As we were interested in germline DNA, any non-neoplastic tissue obtained for any indication (e.g., appendectomy) was acceptable for obtaining DNA.
Archived hematoxylin and eosin (H&E)
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stained slides from candidate FFPE blocks were reviewed histologically (MC) to ensure that the tissue was non-cancerous and suitable for DNA extraction. Either tissue scrolls (8 x 10 υm) or directed punches (6 x 1 mm) were prepared from the FFPE blocks. The prepared FFPE tissue
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was de-paraffinized, dehydrated, and DNA was isolated from dried tissue using Qiagen QIAamp DNA FFPE Tissue kit (Qiagen, Valencia, CA). DNA yield, purity, and integrity were assessed
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using Qubit (Life Technologies, Grand Island, NY). Genotyping Assay
Extracted DNA samples were genotyped on the OmniExpress BeadChips platform (Illumina)
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which includes approximately 710,000 single nucleotide polymorphisms (SNPs). The BeadChips were run according to the manufacturer’s instructions, using 200 ng of DNA per sample unless insufficient material was available (i.e., three samples).
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Genotype Data and Quality Control
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The OmniExpress BeadChip contains SNPs that are in linkage disequilibrium with each other (i.e., correlated), which can result in false positive linkage findings. To avoid spurious findings, SNPs in high linkage disequilibrium were removed (r2 >0.16 over a sliding 500,000 basepair window in the publically available HapMap CEPH/Utah data).18 All included samples were required to have at least 95% genotyping success rate and sex of each subject was confirmed based on X and Y genotype data. All SNPs met the following quality control criteria to ensure
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good coverage across the genome: minimum spacing 0.1 cM, minimum heterozygosity 0.3, and
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individual call rate of 95% for all genotyped subjects.
Linkage Analysis
To compute linkage statistics, the multipoint Markov-chain Monte Carlo (MCMC) linkage
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analysis package, MCLINK, was used.19 MCLINK computes logarithm of the odds (LOD) scores, which provide statistical evidence for whether a presumed disease gene and genetic
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marker are linked. That is, they are nearby on a chromosome and more likely to co-segregate than random, which would be 50%. MCLINK uses blocked Gibbs sampling to provide haplotype reconstructions for extended pedigrees and performs multi-point analysis; the resultant theta LOD (TLOD) scores are robust to model misspecification as model misspecification is
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"absorbed" by the estimate of theta. We report heterogeneity TLODs (hetTLOD) to account for possibly multiple heterogeneous loci contributing to IC/PBS. A parametric linkage analysis was performed using general dominant and recessive models. The disease allele frequency was
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assumed to be 0.001 for the dominant model and 0.01 for the recessive model. Penetrance estimates were assumed to be 0.5 and 0.0005 for carriers and non-carriers, respectively. As the
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true underlying genetic model for IC/PBS is not known, the use of general parametric models is appropriate, because while a general model is less powerful than the true underlying genetic model, it does not result in an increase in Type I error rates.20 The Lander and Kruglyak genome-wide criteria were used to determine suggestive (LOD > 1.86) and significant linkage evidence (LOD >3.30).21 For regions of the genome with significant evidence of linkage, the
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number of pedigrees that obtained at least a nominal point-wise significance (i.e., pedigree specific TLOD>0.59) at that specific locus was reported.
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Results A total of 67 punch or scroll FFPE tissue specimens from high-risk IC/PBS pedigrees (3 male, 64 female) were obtained from the following locations: appendix (n=6), breast (n=2), cervix
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(n=5), colon (n=1), fallopian tube (n=5), gallbladder (n=28), lung (n=1), lymph nodes (n=1), ovary (n=4), prostate (n=1), salivary gland/parotid (n=1), seminal vesicle (n=1), skin (n=2),
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subcutis/glandular epithelium (n=1), uterus (n=7), and vulva (n=1). These 67 samples were a subset of 233 FFPE tissue blocks screened for potential use out of 580 total confirmed cases in pedigrees; of 166 FFPE tissue blocks excluded, 120 FFPE tissue blocks had insufficient tissue, 14 had no sample available, 23 had no normal tissue, and 9 were excluded for other reasons.
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After receiving the 67 FFPE tissue, five samples were excluded because they were the only confirmed IC/PBS case in their pedigree with DNA available. After DNA extraction and genotyping, 14 additional samples were excluded because their SNP genotype call rate was less
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than 95%. The resultant number of DNA samples that met all inclusion criteria was 48 (Figure 1). These 48 samples were part of 13 high-risk pedigrees that included between 2 and 12
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sampled and confirmed IC/PBS cases (Table 1); all 48 samples were obtained from females, although male IC/PBS cases were present in some families. Significant genome-wide linkage evidence was found on chromosome 3p13-p12.3 under a dominant model with a maximum hetTLOD score of 3.56 at rs7614618 (Figure 2). The 1-LOD drop range surrounding the maximum hetTLOD score (i.e., LOD scores >2.5) was from rs12330322 to rs7431017. There were two pedigrees (#12 and #13) with individual TLOD
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scores >0.59 in the 1-LOD drop range. The most informative pedigree included 12 IC/PBS cases and had an individual TLOD score of 2.1 and the other pedigree had a TLOD score of 1.39 and
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included 5 genotyped IC/PBS cases. There was suggestive evidence for linkage (LOD >1.86) in the following locations: 1p21-q25 (maximum hetTLOD=2.34), 3p21.1-p14.3 (maximum hetTLOD=2.12), 4q12-q13 (maximum
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hetTLOD=1.97), 9p24-p22 (maximum hetTLOD=2.31), and 14q24-q31 (maximum
hetTLOD=2.06), all under a dominant model. The suggestive chromosome boundary ranges
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were defined by hetTLOD scores >1.5.
Discussion
We have identified significant evidence for genetic linkage on 3p13-p12.3 for IC/PBS. Although
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this study originated as a pilot study with a relatively small number of cases, two of the thirteen pedigrees involved were linked to this chromosome 3 region providing evidence from different kindreds that a predisposition gene for IC/PBS likely resides here. We also identified five
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chromosomal regions with suggestive evidence for linkage. To date, no definitive genes predisposing to IC/PBS have been identified, although genes involved in inflammatory pathways
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have been implicated.8-12
IC/PBS is regarded as a heterogeneous disease with likely multiple etiologies. While the pathophysiology of IC/PBS is unclear, chronic inflammation of the bladder is likely a strong contributor to IC/PBS, and interactions of inflammation with urothelial barrier dysfunction, neural hyperactivity, exogenous substances, and extra bladder disorders are considered contributors to IC/PBS.22 We hypothesized that familial IC/PBS cases sampled from pedigrees
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with an excess number of cases are more likely to have a genetic etiology for their disease. As variants can be shared by many affected relatives, this results in increased frequency of variant
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observation and increased power to detect the causal variant.23 There are good candidate genes for IC/PBS in the chromosomal regions identified showing
significant and suggestive evidence for linkage. In the chromosome 3p13-p12.3 region which
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achieved genome-wide significance, the gene CNTN3 (Contactin 3) is a strong candidate gene for IC/PBS. As one of the contactin genes, it mediates cell surface interactions during nervous
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system development and is thought to function in sensory processing.24 Significant increased expression of CNTN3 has been observed in a study of 20 diseases with the highest disease-pain ratios, which included vulvar vestibulitis and vulvodynia.25
In the chromosomal regions that achieved suggestive evidence for linkage, the chromosome
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1p21-q25 region contains the candidate gene NGF (Nerve Growth Factor) gene, which has previously been shown to be a urinary biomarker for IC/PBS,26 and increased expression of this gene has been observed in bladder biopsies of IC/PBS patients.27 The chromosome 1p21-q25
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region also contains other genes involved in inflammation including IL6 (interleukin 6) and CRP (c-reactive protein), which have been observed to be increased in the serum and urine of IC/PBS
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subjects.28 The chromosome 9p24-p22 contains the gene IL33 (interleukin 33) which is an important mediator of innate immunity and is involved in stimulating mast cell activation and proliferation; urinary levels of IL33 are significantly increased in patients with IC/PBS.29 The chromosome 14q24-q31 region includes the gene FOS (FBJ murine osteosarcoma viral oncogene homolog), which has been shown to be upregulated early in animal models of induced genitourinary inflammation9 and has been shown to be expressed in the spinal horn neurons as part of an immediate response to bladder inflammation.30
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A previous linkage analysis involving panic disorder and bladder/renal disorders identified significant linkage evidence on chromosome 13q.14 While we did not observe significant or suggestive evidence for linkage on chromosome 13, there was one pedigree with an individual
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TLOD score of 1.831 in the chromosome 13q33 region. Although the existence of panic
disorder cases in this pedigree is not known, we plan to include this pedigree in future genetic
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studies.
There are limitations of this study. This study was designed as a pilot study to investigate the
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feasibility of using FFPE tissue samples for genetic analyses of IC/PBS and hence, our sample size was limited. Despite the small sample size, significant and suggestive results were observed in regions of the genome with strong IC/PBS candidate genes. There were also limitations because we used FFPE tissue samples to obtain DNA; only IC/PBS cases with prior biopsies and confirmed IC/PBS status were included and a large percentage of FFPE samples failed
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genotyping quality control and could not be used for analyses. As samples were all deidentified, a thorough investigation of the clinical phenotypes of subjects was not possible including investigation of biomarkers and other comorbid conditions. In the future, we plan to
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sequence candidate regions using the FFPE tissue, recruit additional subjects who are from high-
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risk pedigrees, and continue genetic studies to confirm findings and identify IC/PBS candidate predisposition genes.
Conclusions
In conclusion, this study provides evidence that a genetic variant(s) on chromosome 3 and possibly chromosomes 1, 4, 9, and 14 contribute to IC/PBS predisposition. Further study of the
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pedigrees underlying these significant and suggestive linkage peaks, and sequence analysis of the
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affected cases in these pedigrees may provide insight into genes contributing to IC/PBS.
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Acknowledgements This study was supported by a grant from the Interstitial Cystitis Association. Partial support for
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all data sets within the Utah Population Database (UPDB) was provided through a grant (P30 CA2014) from the Huntsman Cancer Foundation, the University of Utah, and the University of Utah Center for Clinical and Translational Science (CCTS). UPDB research is also supported in
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part by an NCRR grant (R01 RR021746) with additional support from the Utah State
Department of Health and the University of Utah. The authors thank Eric Johnson and Thom
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Jensen from Intermountain Healthcare BioRepository for assistance with case selection and
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technical expertise. We further thank four anonymous reviewers for their helpful suggestions.
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Figure Legends
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Figure 1. Flow chart showing IC/PBS screening and sample selection.
Figure 2. Genome-wide linkage results for dominant (blue) and recessive (red) models for 13 high-risk IC/PBS pedigrees. Chromosome number is shown on the horizontal axis; hetTLOD
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scores are shown on the vertical axis.
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Table 1. Distribution of 48 confirmed IC/PBS cases in 13 pedigrees Number of descendants
Number of male IC/BPS cases / Total number of IC/PBS cases
Number of confirmed IC/PBS cases included in study
1 2 3 4 5 6 7 8 9 10 11 12 13
884 1640 3698 1856 18440 1861 11360 17093 58607 36080 18664 30873 90724
2/5 1/6 0/7 0/4 4 / 22 0/4 0 / 16 0 / 19 6 / 61 1 / 36 1 / 23 5 / 33 11 / 88
2 2 2 2 2 2 3 3 4 4 5 5 12
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Pedigree
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Patients with an ICD-9 code indicating IC/PBS within Intermountain Healthcare n=3,864
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IC/PBS cases who are members of a high-risk (p<0.05) IC/PBS pedigree n=1,533
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IC/PBS cases with case status confirmed using Natural Language Processing (NLP) n=718
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IC/PBS cases who are part of a pedigree that includes at least two NLP confirmed IC/PBS cases n=580
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IC/PBS cases screened for availability of FFPE tissue n=233
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IC/PBS FFPE tissue samples received from the Intermountain Biorepository n=67
IC/PBS samples genotyped and meeting all quality control criteria n=48
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Abbreviation Key Formalin-fixed, paraffin embedded (FFPE)
Heterogeneity theta logarithm of the odds (hetTLOD)
Intermountain Healthcare (IH) Intermountain Healthcare BioRepository (IBR)
Institutional Review Board (IRB) Logarithm of the odds (LOD) Natural language processing (NLP)
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Interstitial cystitis/painful bladder syndrome (IC/PBS)
Single nucleotide polymorphisms (SNPs)
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Theta logarithm of the odds (TLOD)
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Utah Population Database (UPDB)
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International Classification of Diseases, Ninth Revision (ICD-9)
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Hematoxylin and eosin (H&E)
PII: DOI: Reference:
S0022-5347(17)77178-9 10.1016/j.juro.2017.07.068 JURO 14877
To appear in: The Journal of Urology Accepted Date: 8 July 2017 Please cite this article as: Allen-Brady K, Rowe K, Cessna M, Lenherr S, Norton P, Significant Linkage Evidence for Interstitial Cystitis/Painful Bladder Syndrome on Chromosome 3, The Journal of Urology® (2017), doi: 10.1016/j.juro.2017.07.068. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to The Journal pertain.
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Significant Linkage Evidence for Interstitial Cystitis/Painful
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Bladder Syndrome on Chromosome 3
Kristina Allen-Bradya, Kerry Roweb, Melissa Cessnac, Sara Lenherrd, and Peggy Nortone
Division of Genetic Epidemiology, Department of Internal Medicine; University of Utah; Salt Lake City, Utah, USA, [email protected]
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Department of Pathology, Intermountain Healthcare Central Region and Intermountain Healthcare BioRepository, Salt Lake City, Utah, USA , [email protected]
Division of Urology, Department of Surgery; University of Utah, Salt Lake City, Utah USA, [email protected]
Division of Female Pelvic Medicine and Reconstructive Surgery, Department of Obstetrics and Gynecology; University of Utah, Salt Lake City, Utah, USA, [email protected]
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Homer Warner Center for Informatics Research, Intermountain Healthcare, Salt Lake City, Utah, USA, [email protected]
Financial Support: Supported by a grant from the Interstitial Cystitis Association
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Short title: Genetic Linkage Evidence for Interstitial Cystitis
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Presentation: Presented as an oral presentation at the American Urology Association (AUA) 112th Annual Scientific Meeting, Boston Massachusetts, May 12-16, 2017. Corresponding Author: Kristina Allen-Brady Genetic Epidemiology, University of Utah 391 Chipeta Way, Suite D Salt Lake City, UT 84108 Phone: (801) 585-5333 Fax: (801) 581-6052 [email protected] Key words: interstitial cystitis, genetic linkage, pedigree, chromosome 3 Word Count: 2498
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Abstract PURPOSE: Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic pelvic pain condition with unknown etiology. We hypothesized that related IC/PBS cases were more likely
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to have a genetic etiology. The purpose of this study was to perform a genetic linkage analysis. MATERIALS AND METHODS: IC/PBS cases were identified using diagnostic codes linked to a population-based genealogy resource (Utah Population Database) and electronic medical
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records. For this analysis, 13 high-risk pedigrees (defined as having a statistical excess number of IC/PBS cases among descendants compared to matched hospital population rates) were used.
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Case status was confirmed in medical records using natural language processing. DNA was obtained from stored, non-neoplastic, formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Each pedigree had at least two cases with DNA available. Parametric linkage analysis was performed.
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RESULTS: Pedigrees ranged in size from 2 to 12 genotyped cases (n=48 total cases). Significant genome-wide linkage evidence was found on chromosome 3p13-p12.3 (maximum hetTLOD = 3.56) under a dominant model. There were 2 pedigrees with at least nominal linkage
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evidence (LOD >0.59) in this region; the most informative pedigree included 12 IC/PBS cases (pedigree TLOD = 2.1). Other regions with suggestive linkage evidence included 1p21-q25,
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3p21.1-p14.3, 4q12-q13, 9p24-p22, and 14q24-q31, all under a dominant model. CONCLUSIONS: While the etiology of IC/PBS is unknown, this study provides evidence that a genetic variant(s) on chromosome 3, and possibly chromosomes 1, 4, 9, and 14, contribute to IC/PBS predisposition. Sequence analysis of affected cases in identified pedigrees may provide insight into genes contributing to IC/PBS.
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Introduction Interstitial cystitis/ painful bladder syndrome (IC/PBS) is a chronic pelvic pain condition that includes urinary frequency, urgency, and bladder discomfort.1 IC/PBS occurs primarily in
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women; prevalence rates are estimated at 197 cases per 100,000 women and 41 cases per
100,000 men.2 As IC/PBS is a diagnosis by exclusion of other similar diseases, these prevalence
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rates may be underestimated.3
While the etiology of IC/PBS is unclear, family history of IC/PBS is one of the strongest known
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risk factors suggesting a genetic component. First-degree relatives are at increased risk,4 and twin studies have shown a high concordance in monozygotic twins.5-7 Previous genetic studies have generally focused on candidate genes8 and gene expression studies using bladder biopsies.911
Genes involved in allergic or autoimmune disease such as the beta2-adrenoceptor (ADRB2)12
and genes involved in inflammation including FGF7,9 CCL21,9 IgE,11 and JNK pathway genes10
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have been implicated. A previous linkage analysis of panic disorder and bladder/renal problems, potentially interstitial cystitis,13 found significant evidence for linkage on chromosome
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13q.14
We hypothesized that related IC/PBS cases, sampled from pedigrees with a statistical excess
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number of cases, are likely to have a stronger genetic contribution to their disease. If there is a genetic contribution to IC/PBS, study of high-risk families increases power to detect a genetic effect. The purpose of this study was to perform a genetic linkage analysis to identify shared genetic regions in high-risk, related IC/PBS cases.
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Materials and Methods Subjects
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We used a novel research approach to take advantage of a population-based genealogy database that has been linked to medical records and existing sources of DNA. Relationships between IC/PBS cases were identified using the Utah Population Database (UPDB), a computerized
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genealogy for Utah that has been linked to electronic medical records and other public data sources.15 Genealogy information is available for over 8 million unique individuals. The
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majority of families living in Utah are represented in the UPDB, most with at least five generations of information available. All involved subjects were required to have genealogy information available for at least three generations. The Utah population has previously been shown to be mostly of Northern European descent,16 and has experienced high migration rates
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and low levels of inbreeding among the founding population.17
To identify potential IC/PBS cases, we used the UPDB genealogy links to electronic medical records (ICD-9) diagnostic codes for both inpatient and outpatient visits at Intermountain
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Healthcare (IH), the largest healthcare provider in Utah. IH electronic medical records were available since 1995 for approximately 4 million records. Potential IC/PBS cases were identified
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as having a diagnostic code of chronic interstitial cystitis, ICD-9 code: 595.1 (n=3,864 patients). High-risk pedigrees were defined as pedigrees where the number of observed IC/PBS cases was significantly greater (p<0.05) than expected. The expected number of cases was calculated as the population rate of disease for a specific cohort, defined by sex and birth year in 5-year intervals. Family members in a pedigree were assigned the population rate of IC/PBS based on their birth year and sex cohort. Rates of disease were summed across all family members to equal the
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expected number of cases in a pedigree. We identified 1,533 IC/PBS cases who were members of pedigrees with a statistical excess of IC/PBS (p<0.05) and which included at least two cases per pedigree. To confirm IC/PBS case status, co-author and IH bioinformaticist (KR) used
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natural language processing (NLP) to verify the existence of at least one of the following terms in the medical record: “interstitial cystitis” (not “rule out interstitial cystitis”), “chronic pelvic pain”, “painful bladder (syndrome)”, “bladder pain syndrome”, “hypersensitive bladder
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syndrome” and/or “Hunner’s ulcers”. Approximately 50 records were manually confirmed to check the query. Of 1,533 potentially high-risk IC/PBS cases, 718 cases were confirmed. For
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unconfirmed IC/PBS cases, diagnostic information may be contained in paper charts; however, these records are not electronically searchable and were not included in this study. Of 718 confirmed cases, 580 fit into a pedigree with at least two confirmed, affected cases. This study was approved by the University of Utah Institutional Review Board (IRB), Intermountain
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Healthcare IRB, and the Resource for Genetic Epidemiology, which oversees use of UPDB data. All subjects and their DNA samples were de-identified to the primary investigators (KAB, PN,
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and SL) on this project.
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DNA isolation and quantification
We used existing formalin-fixed, paraffin embedded (FFPE) tissue blocks as the source of DNA for this project. The Intermountain Healthcare BioRepository (IBR) has archived FFPE blocks for almost all pathology samples collected since the 1970s, which are linked to the clinical record. We required tissue from FFPE blocks from at least two NLP confirmed IC/PBS cases in a pedigree. These patients were linked to their pathology records and screened for non-
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neoplastic FFPE tissue samples suitable for preparation and DNA extraction. As we were interested in germline DNA, any non-neoplastic tissue obtained for any indication (e.g., appendectomy) was acceptable for obtaining DNA.
Archived hematoxylin and eosin (H&E)
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stained slides from candidate FFPE blocks were reviewed histologically (MC) to ensure that the tissue was non-cancerous and suitable for DNA extraction. Either tissue scrolls (8 x 10 υm) or directed punches (6 x 1 mm) were prepared from the FFPE blocks. The prepared FFPE tissue
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was de-paraffinized, dehydrated, and DNA was isolated from dried tissue using Qiagen QIAamp DNA FFPE Tissue kit (Qiagen, Valencia, CA). DNA yield, purity, and integrity were assessed
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using Qubit (Life Technologies, Grand Island, NY). Genotyping Assay
Extracted DNA samples were genotyped on the OmniExpress BeadChips platform (Illumina)
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which includes approximately 710,000 single nucleotide polymorphisms (SNPs). The BeadChips were run according to the manufacturer’s instructions, using 200 ng of DNA per sample unless insufficient material was available (i.e., three samples).
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Genotype Data and Quality Control
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The OmniExpress BeadChip contains SNPs that are in linkage disequilibrium with each other (i.e., correlated), which can result in false positive linkage findings. To avoid spurious findings, SNPs in high linkage disequilibrium were removed (r2 >0.16 over a sliding 500,000 basepair window in the publically available HapMap CEPH/Utah data).18 All included samples were required to have at least 95% genotyping success rate and sex of each subject was confirmed based on X and Y genotype data. All SNPs met the following quality control criteria to ensure
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good coverage across the genome: minimum spacing 0.1 cM, minimum heterozygosity 0.3, and
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individual call rate of 95% for all genotyped subjects.
Linkage Analysis
To compute linkage statistics, the multipoint Markov-chain Monte Carlo (MCMC) linkage
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analysis package, MCLINK, was used.19 MCLINK computes logarithm of the odds (LOD) scores, which provide statistical evidence for whether a presumed disease gene and genetic
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marker are linked. That is, they are nearby on a chromosome and more likely to co-segregate than random, which would be 50%. MCLINK uses blocked Gibbs sampling to provide haplotype reconstructions for extended pedigrees and performs multi-point analysis; the resultant theta LOD (TLOD) scores are robust to model misspecification as model misspecification is
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"absorbed" by the estimate of theta. We report heterogeneity TLODs (hetTLOD) to account for possibly multiple heterogeneous loci contributing to IC/PBS. A parametric linkage analysis was performed using general dominant and recessive models. The disease allele frequency was
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assumed to be 0.001 for the dominant model and 0.01 for the recessive model. Penetrance estimates were assumed to be 0.5 and 0.0005 for carriers and non-carriers, respectively. As the
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true underlying genetic model for IC/PBS is not known, the use of general parametric models is appropriate, because while a general model is less powerful than the true underlying genetic model, it does not result in an increase in Type I error rates.20 The Lander and Kruglyak genome-wide criteria were used to determine suggestive (LOD > 1.86) and significant linkage evidence (LOD >3.30).21 For regions of the genome with significant evidence of linkage, the
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number of pedigrees that obtained at least a nominal point-wise significance (i.e., pedigree specific TLOD>0.59) at that specific locus was reported.
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Results A total of 67 punch or scroll FFPE tissue specimens from high-risk IC/PBS pedigrees (3 male, 64 female) were obtained from the following locations: appendix (n=6), breast (n=2), cervix
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(n=5), colon (n=1), fallopian tube (n=5), gallbladder (n=28), lung (n=1), lymph nodes (n=1), ovary (n=4), prostate (n=1), salivary gland/parotid (n=1), seminal vesicle (n=1), skin (n=2),
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subcutis/glandular epithelium (n=1), uterus (n=7), and vulva (n=1). These 67 samples were a subset of 233 FFPE tissue blocks screened for potential use out of 580 total confirmed cases in pedigrees; of 166 FFPE tissue blocks excluded, 120 FFPE tissue blocks had insufficient tissue, 14 had no sample available, 23 had no normal tissue, and 9 were excluded for other reasons.
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After receiving the 67 FFPE tissue, five samples were excluded because they were the only confirmed IC/PBS case in their pedigree with DNA available. After DNA extraction and genotyping, 14 additional samples were excluded because their SNP genotype call rate was less
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than 95%. The resultant number of DNA samples that met all inclusion criteria was 48 (Figure 1). These 48 samples were part of 13 high-risk pedigrees that included between 2 and 12
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sampled and confirmed IC/PBS cases (Table 1); all 48 samples were obtained from females, although male IC/PBS cases were present in some families. Significant genome-wide linkage evidence was found on chromosome 3p13-p12.3 under a dominant model with a maximum hetTLOD score of 3.56 at rs7614618 (Figure 2). The 1-LOD drop range surrounding the maximum hetTLOD score (i.e., LOD scores >2.5) was from rs12330322 to rs7431017. There were two pedigrees (#12 and #13) with individual TLOD
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scores >0.59 in the 1-LOD drop range. The most informative pedigree included 12 IC/PBS cases and had an individual TLOD score of 2.1 and the other pedigree had a TLOD score of 1.39 and
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included 5 genotyped IC/PBS cases. There was suggestive evidence for linkage (LOD >1.86) in the following locations: 1p21-q25 (maximum hetTLOD=2.34), 3p21.1-p14.3 (maximum hetTLOD=2.12), 4q12-q13 (maximum
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hetTLOD=1.97), 9p24-p22 (maximum hetTLOD=2.31), and 14q24-q31 (maximum
hetTLOD=2.06), all under a dominant model. The suggestive chromosome boundary ranges
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were defined by hetTLOD scores >1.5.
Discussion
We have identified significant evidence for genetic linkage on 3p13-p12.3 for IC/PBS. Although
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this study originated as a pilot study with a relatively small number of cases, two of the thirteen pedigrees involved were linked to this chromosome 3 region providing evidence from different kindreds that a predisposition gene for IC/PBS likely resides here. We also identified five
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chromosomal regions with suggestive evidence for linkage. To date, no definitive genes predisposing to IC/PBS have been identified, although genes involved in inflammatory pathways
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have been implicated.8-12
IC/PBS is regarded as a heterogeneous disease with likely multiple etiologies. While the pathophysiology of IC/PBS is unclear, chronic inflammation of the bladder is likely a strong contributor to IC/PBS, and interactions of inflammation with urothelial barrier dysfunction, neural hyperactivity, exogenous substances, and extra bladder disorders are considered contributors to IC/PBS.22 We hypothesized that familial IC/PBS cases sampled from pedigrees
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with an excess number of cases are more likely to have a genetic etiology for their disease. As variants can be shared by many affected relatives, this results in increased frequency of variant
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observation and increased power to detect the causal variant.23 There are good candidate genes for IC/PBS in the chromosomal regions identified showing
significant and suggestive evidence for linkage. In the chromosome 3p13-p12.3 region which
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achieved genome-wide significance, the gene CNTN3 (Contactin 3) is a strong candidate gene for IC/PBS. As one of the contactin genes, it mediates cell surface interactions during nervous
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system development and is thought to function in sensory processing.24 Significant increased expression of CNTN3 has been observed in a study of 20 diseases with the highest disease-pain ratios, which included vulvar vestibulitis and vulvodynia.25
In the chromosomal regions that achieved suggestive evidence for linkage, the chromosome
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1p21-q25 region contains the candidate gene NGF (Nerve Growth Factor) gene, which has previously been shown to be a urinary biomarker for IC/PBS,26 and increased expression of this gene has been observed in bladder biopsies of IC/PBS patients.27 The chromosome 1p21-q25
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region also contains other genes involved in inflammation including IL6 (interleukin 6) and CRP (c-reactive protein), which have been observed to be increased in the serum and urine of IC/PBS
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subjects.28 The chromosome 9p24-p22 contains the gene IL33 (interleukin 33) which is an important mediator of innate immunity and is involved in stimulating mast cell activation and proliferation; urinary levels of IL33 are significantly increased in patients with IC/PBS.29 The chromosome 14q24-q31 region includes the gene FOS (FBJ murine osteosarcoma viral oncogene homolog), which has been shown to be upregulated early in animal models of induced genitourinary inflammation9 and has been shown to be expressed in the spinal horn neurons as part of an immediate response to bladder inflammation.30
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A previous linkage analysis involving panic disorder and bladder/renal disorders identified significant linkage evidence on chromosome 13q.14 While we did not observe significant or suggestive evidence for linkage on chromosome 13, there was one pedigree with an individual
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TLOD score of 1.831 in the chromosome 13q33 region. Although the existence of panic
disorder cases in this pedigree is not known, we plan to include this pedigree in future genetic
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studies.
There are limitations of this study. This study was designed as a pilot study to investigate the
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feasibility of using FFPE tissue samples for genetic analyses of IC/PBS and hence, our sample size was limited. Despite the small sample size, significant and suggestive results were observed in regions of the genome with strong IC/PBS candidate genes. There were also limitations because we used FFPE tissue samples to obtain DNA; only IC/PBS cases with prior biopsies and confirmed IC/PBS status were included and a large percentage of FFPE samples failed
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genotyping quality control and could not be used for analyses. As samples were all deidentified, a thorough investigation of the clinical phenotypes of subjects was not possible including investigation of biomarkers and other comorbid conditions. In the future, we plan to
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sequence candidate regions using the FFPE tissue, recruit additional subjects who are from high-
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risk pedigrees, and continue genetic studies to confirm findings and identify IC/PBS candidate predisposition genes.
Conclusions
In conclusion, this study provides evidence that a genetic variant(s) on chromosome 3 and possibly chromosomes 1, 4, 9, and 14 contribute to IC/PBS predisposition. Further study of the
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pedigrees underlying these significant and suggestive linkage peaks, and sequence analysis of the
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affected cases in these pedigrees may provide insight into genes contributing to IC/PBS.
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Acknowledgements This study was supported by a grant from the Interstitial Cystitis Association. Partial support for
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all data sets within the Utah Population Database (UPDB) was provided through a grant (P30 CA2014) from the Huntsman Cancer Foundation, the University of Utah, and the University of Utah Center for Clinical and Translational Science (CCTS). UPDB research is also supported in
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part by an NCRR grant (R01 RR021746) with additional support from the Utah State
Department of Health and the University of Utah. The authors thank Eric Johnson and Thom
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Jensen from Intermountain Healthcare BioRepository for assistance with case selection and
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technical expertise. We further thank four anonymous reviewers for their helpful suggestions.
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Figure Legends
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Figure 1. Flow chart showing IC/PBS screening and sample selection.
Figure 2. Genome-wide linkage results for dominant (blue) and recessive (red) models for 13 high-risk IC/PBS pedigrees. Chromosome number is shown on the horizontal axis; hetTLOD
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scores are shown on the vertical axis.
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Table 1. Distribution of 48 confirmed IC/PBS cases in 13 pedigrees Number of descendants
Number of male IC/BPS cases / Total number of IC/PBS cases
Number of confirmed IC/PBS cases included in study
1 2 3 4 5 6 7 8 9 10 11 12 13
884 1640 3698 1856 18440 1861 11360 17093 58607 36080 18664 30873 90724
2/5 1/6 0/7 0/4 4 / 22 0/4 0 / 16 0 / 19 6 / 61 1 / 36 1 / 23 5 / 33 11 / 88
2 2 2 2 2 2 3 3 4 4 5 5 12
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Pedigree
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Patients with an ICD-9 code indicating IC/PBS within Intermountain Healthcare n=3,864
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IC/PBS cases who are members of a high-risk (p<0.05) IC/PBS pedigree n=1,533
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IC/PBS cases with case status confirmed using Natural Language Processing (NLP) n=718
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IC/PBS cases who are part of a pedigree that includes at least two NLP confirmed IC/PBS cases n=580
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IC/PBS cases screened for availability of FFPE tissue n=233
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IC/PBS FFPE tissue samples received from the Intermountain Biorepository n=67
IC/PBS samples genotyped and meeting all quality control criteria n=48
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Abbreviation Key Formalin-fixed, paraffin embedded (FFPE)
Heterogeneity theta logarithm of the odds (hetTLOD)
Intermountain Healthcare (IH) Intermountain Healthcare BioRepository (IBR)
Institutional Review Board (IRB) Logarithm of the odds (LOD) Natural language processing (NLP)
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Interstitial cystitis/painful bladder syndrome (IC/PBS)
Single nucleotide polymorphisms (SNPs)
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Theta logarithm of the odds (TLOD)
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Utah Population Database (UPDB)
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International Classification of Diseases, Ninth Revision (ICD-9)
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Hematoxylin and eosin (H&E)