A Major Effect QTL on Chromosome 18 for Noise Injury to the Mouse Cochlear Lateral Wall

Abstract

We recently demonstrated a striking difference among inbred mouse strains in the effects of a single noise exposure, whereby CBA/J and CBA/CaJ (CBA) mice show moderate reversible reduction in the endocochlear potential (EP) while C57BL/6J (B6) mice do not (Ohlemiller, K.K., Gagnon, P.M. 2007. Genetic dependence of cochlear cells and structures injured by noise. Hearing Res. 224, 34-50). Acute EP reduction in CBA was reliably associated with characteristic pathology of the spiral ligament and stria vascularis, both immediately after noise and 8 weeks later. Analysis of B6×CBA F1 hybrid mice indicated that EP reduction and its anatomic correlates are co-inherited in an autosomal dominant manner. Further analysis of N2 mice resulting from the backcross of F1 hybrids to B6 mice led us to suggest that the EP reduction phenotype principally reflects the influence of a small number of quantitative trait loci (QTLs). Here we report the results of QTL mapping of the EP reduction phenotype in CBA/J using 106 N2 mice from a (CBA×B6) × B6 backcross. Correlation of acute post-noise EP with 135 markers distributed throughout the genome revealed a single major effect QTL on chromosome 18 (12.5 cM, LOD 3.57) (Nirep, for Noise-induced reduction in EP QTL), and two marginally significant QTLs on chromosomes 5 and 16 (LOD 1.43 and 1.73, respectively). Our results underscore that fact that different cochlear structures may possess different susceptibilities to noise through the influence of non-overlapping genes. While Nirep and similar-acting QTLs do not appear to influence the extent of permanent hearing loss from a single noise exposure, they could reduce the homeostatic ‘reserve’ of the lateral wall in protracted or continual exposures, and thereby influence long term threshold stability.

Introduction

Vulnerability of the mammalian cochlea to permanent noise-induced hearing loss (NIHL) varies between species (Saunders and Tilney, 1980), and within species is associated with genetically-based variation in susceptibility (Borg, 1982; Davis et al., 2001; Erway et al., 1996; Henry, 1982; Li, 1992; Ohlemiller, 2006; Ohlemiller et al., 2000; White et al., 2009; Yoshida et al., 2000). In addition to the organ of Corti, cochlear targets of noise can include the spiral limbus, spiral ligament, and stria vascularis (Hirose and Liberman, 2003; Liberman and Mulroy, 1982; Wang et al., 2002), although it is not clear whether and how injury to these contributes to the extent of NIHL (Ohlemiller, 2008). We recently demonstrated that the cochlear lateral wall and spiral limbus of two inbred strains, CBA/J and C57BL/6J (B6), respond very differently to a single severe noise exposure (broadband, 110 dB SPL, 2 hr) (Ohlemiller and Gagnon, 2007). Although B6 mice showed greater permanent NIHL than CBA/J, presumably due to greater injury to the organ of Corti, CBA/J and CBA/CaJ mice in our sample exhibited a ∼30-40 mV reversible reduction in the endocochlear potential (EP), while B6 mice did not. EP reduction in CBA mice was reliably accompanied by acute changes in the appearance of the cochlear lateral wall, while B6 mice showed little change. Likewise, CBA cochleas exhibited significant permanent reductions in strial basal cell density, strial capillary density, ligament fibrocyte density, and limbus fibrocyte density, while B6 cochleas showed little change in these metrics. Repeat experiments in F1 hybrid mice formed from interbreeding B6 and CBA/J mice revealed a noise phenotype essentially identical to that of CBA/J mice, indicating that the underlying CBA alleles are dominant over B6 alleles for the major genes controlling this trait. Additional tests in N2 mice, resulting from backcrosses of F1 hybrid mice to B6 mice, suggested that a small number of quantitative trait loci (QTLs) are primarily responsible for the CBA-like noise-induced cochlear phenotype. In the N2 mice, acute EP reductions were well correlated with injury metrics for the spiral ligament and stria, supporting the interpretation that these features share a common process. Here we describe results from a genome-wide linkage scan to map QTLs that promote the CBA noise-related phenotype using EP data and tail DNA samples from 106 N2 backcross mice. Linkage analysis revealed a major effect QTL (Nirep) on Chromosome 18 (LOD 3.57) that could explain about 12% of the EP variation among the N2 backcross mice. The 95% confidence region for this QTL spans about 30 cM and includes several plausible candidate genes, including Aqp4 (Aquaporin 4) and Slc12a2 (Na⁺/K⁺/Cl^- exchanger).

Materials and Methods

Animals

All procedures were approved by the Washington University Institutional Animal Care and Use Committee. Mice of both genders were noise exposed and examined at 3-4 mos of age. Mice were derived from CBA/J (CBA), C57BL/6J (B6), and B6.CAST-Cdh23^Ahl+/Kjn (B6.CAST) mice purchased from The Jackson Laboratory (JAX). Our original study (Ohlemiller and Gagnon, 2007) used 42 N2 mice solely to ascertain the distribution of EPs after noise exposure. In that study we also wished to ensure that the ahl allele of Cdh23 carried by B6 mice was not responsible for the observed strain differences in EP. Thus we examined both B6 and B6.CAST mice, plus N2s formed by backcrossing B6.CAST × CBA F1s to B6.CAST mice. As we reported, no difference was found between the B6 and B6.CAST noise-related EP phenotype, arguing against any role for the Cdh23^ahl allele. For the purpose of mapping, it was advantageous to revert to B6 × CBA crosses due to poor breeding of the B6.CAST mice. Initial mapping analyses included EP data and DNA samples from 80 [B6 × CBA] × B6 N2 mice. Based on those results, the sample was expanded to include an additional 26 of the original B6.CAST-derived N2s using a higher marker density on Chr. 18 (see below). The 106 total cases analyzed were taken from a larger sample of 120 animals we then restricted to include only mice with post-noise EPs ≤80 mV or ≥90 mV (see Fig. 4). Control comparison data are presented for 7 non-exposed N2 mice aged 4-5 mos.

Figure 4.

Distribution of EPs in all 120 N2 mice from which acute EP recordings were obtained. Roughly half the mice show an essentially normal EP (≥90 mV). Two other modes are suggested, each containing roughly one quarter of the sample. Unfilled bars indicate 14 mice with ‘borderline’ EPs not included in mapping analyses.

Noise exposure

Noise exposures were performed in a foam-lined, single-walled soundproof room (IAC). The noise exposure apparatus consisted of a 21 × 21 × 11 cm wire cage mounted on a pedestal inserted into a B&K 3921 turntable. To ensure a uniform sound field, the cage was rotated at 1 revolution/80 s within a 42 × 42 cm metal frame. A Motorola KSN1020A piezo ceramic speaker (four total) was attached to each side of the frame. Opposing speakers were oriented non-concentrically, parallel to the cage, and driven by separate channels of a Crown D150A power amplifier. Broadband noise was generated by General Radio 1310 generators and band-passed at 4-45 kHz by Krohn-Hite 3550 filters. Noise levels at various points in the exposure cage, measured using a B&K 4135 ¼ inch microphone in combination with a B&K 2231 sound level meter, ranged from 110-113 dB SPL. Mice were exposed in pairs for 2.0 hrs.

CAP recording

Compound action potential (CAP) recordings were conducted as terminal procedures within 1-3 hrs after noise exposure. Animals were anesthetized (60 mg/kg sodium pentobarbital, IP) and positioned ventrally in a custom headholder. Core temperature was maintained at 37.5±1.0 °C using a thermostatically-controlled heating pad. A tracheostomy was performed and the musculature over the bulla was cut posteriorly to expose the bone overlying the round window. A small hole was made over the round window using a hand drill, and an insulated silver wire electrode was inserted into round window antrum using a micromanipulator. Additional electrodes inserted into the neck musculature and hind leg served as reference and ground, respectively. Electrodes were led to a Grass P15 differential amplifier (100-3,000 Hz, ×100), then to a custom amplifier providing another ×1,000 gain, then digitized at 30 kHz using a Cambridge Electronic Design Micro1401 in conjunction with SIGNAL™ and custom signal averaging software. Sinewave stimuli generated by a Hewlett Packard 3325A oscillator were shaped by a custom electronic switch to 5 ms total duration, including 1 ms rise/fall times. The stimulus was amplified by a Crown D150A power amplifier and output to a KSN1020A piezo ceramic speaker located 7 cm directly lateral to the left ear. Stimuli were presented freefield and calibrated using a B&K 4135 ¼ inch microphone placed where the external auditory meatus would normally be. Toneburst stimuli at each frequency and level were presented 100 times at 3/sec. The minimum sound pressure level required for visual detection of a response (N₁) was determined at 5, 10, 20, 28.3, and 40 kHz, using a 5 dB minimum step size. For purposes of averaging and plotting (Fig. 1A), in cases where no response could be elicited in the acute phase after noise exposure, a threshold value of 105 dB was assigned.

Figure 1.

A. Mean(-SD) CAP thresholds for 90 noise-exposed N2 mice and 7 non-exposed comparison mice. B. The distribution of CAP thresholds at 20 kHz in the noise-exposed N2s. NR: No response.

Endocochlear potential recording

The EP was measured using a ventral approach immediately after CAP recording. Using a fine drill, a hole was made in the left cochlear capsule directly over scala media of the lower basal turn. Glass capillary pipettes (40-80 MΩ) filled with 0.15 M KCl were mounted on a hydraulic microdrive (Frederick Haer) and advanced until a stable positive potential was observed that did not change with increased electrode depth. The signal from the recording electrode was led to an AM Systems Model 1600 intracellular amplifier.

Tissue processing for histology

To confirm previous anatomic observations (Ohlemiller and Gagnon, 2007), qualitative light microscopic evaluation was carried out for 13 mice. At the end of recording, animals were overdosed and perfused transcardially with cold 2.0% paraformaldehyde/2.0% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Each cochlea was rapidly isolated, immersed in the same fixative, and the stapes was immediately removed. Complete infiltration of the cochlea by fixative was ensured by making a small hole at the apex of the cochlear capsule, and gently circulating the fixative over the cochlea using a transfer pipet. After decalcification in sodium EDTA for 72 hours, cochleas were post-fixed in buffered 1% osmium tetroxide, dehydrated in an ascending acetone series, and embedded in Epon. Cochleas were sectioned in the mid-modiolar plane at 4.0 μm, then stained with toluidine blue for bright field viewing with a Nikon Optiphot™ light microscope. Typically 50 sections were obtained from the mid-modiolar ‘core’.

Morphometry

In examining the present material, we noted a tendency for the nuclei of mesothelial cells lining the scala vestibuli side of Reissner's membrane to become enlarged when the EP was reduced. To confirm this as an inherited tendency, as well as one that distinguishes CBA/J and B6 mice following noise exposure, the 13 sectioned N2 cochleas plus archival cochleas from 5 noise exposed B6 or B6.CAST mice and 5 exposed CBA/J or CBA/CaJ mice were examined by light microscopy. In each cochlea, 10 sections evenly spaced over 200 μm distance through the mid-modiolar ‘core’ were scored with regard to whether any Reissner's mesothelial cells in the cochlear upper basal turn appeared abnormal. For each animal, the proportion of sections scored as normal was paired with the EP recorded, and the result for 23 animals was plotted (Fig. 3). Scoring for any mouse was blind with respect to EP.

Figure 3.

Proportion of sections showing normal mesothelial cells on the scala vestibuli side of Reissner's membrane versus EP in N2 and comparison mice from the parental strains. There was no overlap in the prevalence of abnormal cells for EPs <80 mV and >90 mV, and no overlap in range for the parental strains. N2 mice were either ‘CBA-like’ or ‘B6-like’.

Mapping

QTL mapping analyses were carried out on a contractual basis by the JAX QTL Mapping Analysis Service (http://jaxservices.jax.org/qtl/index.html). DNA samples from tail tips of the experimental N2 backross mice were sent to The Jackson Laboratory (Bar Harbor, ME) for genotyping of markers distributed throughout the genome, and linkage was analyzed using the R/QTL v. 1.08-56 computer program (Broman et al., 2003). Mapping was carried out in two phases. To enhance the odds of identifying QTLs with the largest effects, and to avoid confounds possibly posed by marginal recordings, only samples associated with EPs ≤80 mV or ≥90 mV were included. For the initial analysis 80 samples and 100 chromosome markers were selected, and a whole genome scan was performed. This analysis revealed a single moderately significant QTL (LOD 2.91, p<05) on Chr. 18. Based on these results, 35 additional markers on Chr. 18 were selected and a whole genomic scan was repeated using an additional 26 samples. Both scans used a 2 cM pseudomarker grid with 128 imputations and an assumption of normality for EP (Sen and Churchill, 2001). One thousand permutations were performed to determine the significance thresholds for QTL detection (Doerge and Churchill, 1996), and four thresholds (1%, 5%, 10% and 63%) were calculated. Given the skewness of the EP distribution (Fig. 4), a non-parametric model was also evaluated.

All possible pairs of QTL locations on each chromosome were tested for association with EP. The likelihood from the full model (pseudomarker pair and the interaction between them) and the null model (no genetic effect) was compared and LOD scores were calculated. LOD scores obtained by comparing the likelihood from the full model and an additive model (no interaction) were also calculated. Possible QTL×QTL interactions identified from a single QTL scan and pair-wise scan were fit to multiple regression models, and associated variations in EP was estimated. P values for terms in the multiple regression model were calculated. Terms were dropped sequentially until all of the terms in the model were significant at 1% level for main QTL effects and 0.1% for the interaction effects.

Results

CAP thresholds

Based on comparison of exposed and non-exposed mice (Fig. 1A), acute threshold shifts in N2s often exceeded 90 dB in the mid-frequency region. Nearly half of 90 mice tested showed no response at the highest test frequencies. At 20 kHz, for example, 38 of 90 mice tested (42%) showed no acute CAP response (Fig. 1B). Both acute and permanent shifts are anticipated to vary in N2 mice according to the distribution of alleles at many unknown loci that influence acute and permanent NIHL. Although there need be no overlap between loci that impact NIHL and those that promote EP changes in noise, some degree correlation of thresholds with EP is expected, since the EP forms part of the driving force for hair cell receptor currents (Wangemann, 2002, 2006). We previously observed significant correlations between EP and CAP thresholds in acute N2s with regression slopes of approximately -0.4 dB/mV (Ohlemiller, 2009; Ohlemiller and Gagnon, 2007). In the present sample, CAP thresholds at all seven test frequencies were significantly correlated with EP (p<.04), with regression slopes ranging from -0.11 dB/mV (10 kHz) to -0.39 dB/mV (2.5 kHz) (plots not shown).

Histologic correlates of EP reduction

Qualitative light microscopy supported previous observations regarding the histologic correlates of acute EP reduction. Figure 2 compares the lateral wall of the upper basal cochlear turn in two N2 mice showing different EPs after noise. The cochlea shown in Figure 2A had a normal EP (111 mV) and displayed no visible anomalies of the spiral ligament or stria vascularis. Although the animal featured in Fig. 2B showed only a mildly depressed EP (78 mV), this was accompanied by the features we previously described, including vacuolization of strial basal cells and Type II fibrocytes (arrows in C and E) and shrinkage of Type I fibrocytes in ligament (arrows in D). We also report here for the first time that a depressed EP after noise also seems reliably associated with a change in appearance of the nuclei of mesothelial cells lining the scala vestibuli side of Reissner's membrane (compare Fig. 2 F,G). Affected nuclei become more rounded and lighter in color. Even though similar cells line much of the perilymphatic space, this change appears limited to cells that line Reissner's. To confirm that changes in the appearance of these nuclei reliably track EP after noise, and that such changes distinguish B6 and CBA mice, cochleas from 13 noise-exposed N2 mice, 5 B6 or B6.CAST mice, and 5 CBA/J or CBA/CaJ mice were scored for the presence or absence of abnormal mesothelial cells. Figure 3 shows that EPs over 90 mV were solely associated with preponderantly normal mesothelial cell profiles, while EPs below 80 mV were exclusively associated with mostly abnormal profiles. Moreover, trends in N2 mice follow those for the parent strains. This analysis suggests that the effects of noise on both EP and mesothelial cells are co-inherited, presumably through their relation to a common trait. Examination of archival material obtained 24 hrs and 8 wks after noise (not shown) showed typical normal appearance of these cells.

Figure 2.

Cochlear upper basal turn lateral wall in two example N2 mice showing different EPs 1-3 hrs after noise exposure. A. Mouse having a normal EP shows normal spiral ligament and stria vascularis. B. Mouse with moderate depression of the EP shows anomalies of both stria and ligament. These include shrinkage of strial basal cells (Inset and panel C), shrinkage of Type I fibrocytes (Inset and panel D), and vacuolization of Type II fibrocytes (Inset and panel E). F and G compare Reissner's membrane in the two mice. Mesothelial cells lining the top of Reissner's show enlarged nuclei (arrows) when the EP is reduced. T_I and T_II: Type I and II fibrocytes; SpL: Spiral ligament; StV: Stria vascularis; RM: Reissner's membrane.

Distribution of EPs

Figure 4 shows the distribution of EPs in the acute phase after noise exposure. Data from 14 animals with EPs ranging 81-89 mV that were not included in mapping analyses are indicated by unfilled bars. Key similarities between the distribution shown and that previously published (Ohlemiller and Gagnon, 2007) include 1) multiple modes and 2) essentially normal EPs in half (53%) of the animals tested. Additional modes near 50 mV and 75 mV support a visual impression of three modes. While it is not possible to determine with certainty whether there are two or three modes, it is interesting that each of the two lower modes contain roughly one quarter of the animals tested. Among cases with an EP ≥90 mV, the mean EP was 103.5±7.5 mV, compared to 110.0±4.1 mV for unexposed N2s. Coat color (black or agouti) and EP appeared unrelated. Similarity of EP distributions in the present and previous study further suggested that the difference in noise phenotype between CBA and B6 mice is established by a small number of QTLs.

Mapping of QTLs

Figure 5 presents the results from a genome-wide one-dimensional QTL scan for EP under an assumption of normality. Three models were tested. The first model (shown) included QTL as the only factor. Additional models that tested sex as a covariate and sex*QTL interactions yielded similar results, indicating that gender was not a significant factor. Three QTLs were identified (Table I), a single major effect QTL on chromosome 18 (12.5 cM, LOD 3.57) (Nirep, for Noise-induced reduction in EP QTL), and two marginally significant QTLs on chromosomes 5 and 16 (LOD 1.43 and 1.73, respectively). Figure 6 shows the 95% confidence intervals for these QTLs, along with the location of markers used for mapping. Two dimensional genome-wide analysis detected no epistasis between QTLs (not shown). Finally, multiple regression was used to estimate the percent EP variance explained and effect size of each QTL (Table I). The QTL identified on Chr. 18 was estimated to account for approximately 12% of EP variance, with an effect size of about 19 mV (difference in average EP between homozygous and heterozygous N2 mice). Analyses using non-parametric statistical methods gave essentially the same results as the model based on a normal distribution.

Figure 5.

LOD scores from whole genomic analysis of N2 mice showing the linkage associations of EP values with segregating genotypes of 135 chromosome markers. Small hash marks on X-axis show marker locations along each of the 19 autosomes (see Fig. 6 for greater detail.). Genome-wide significance levels are indicated by horizontal dashed lines.

Table I. Statistics for identified QTLs.

	One-dimensional Scan				Regression Analysis
Chr. #	Marker	*Position (cM)	LOD	P value	% variance	Effect size
5	05-030544560-G	27.9	1.43	>.10	7%	12 mV
16	c16.loc2	2.1	1.73	>.10	5%	13 mV
18	c18.loc8	12.5	3.57	<.001	12%	19 mV

Figure 6.

LOD scores for two suggestive QTLs on Chr. 5 and 16, and a strong QTL on Chr. 18. Black bars indicate 95% confidence intervals for each QTL. Hash marks on X-axis show marker locations.

Candidate genes

The 95% confidence intervals identified on Chr. 5 and 16 are too large to suggest candidate loci. The 95% confidence interval for Chr. 18 is also large, containing over 80 known genes (Mouse Genome Informatics, URL: http://www.informatics.jax.org). However, loci in this interval known to be related to hearing include intriguing candidates. Among these is Slc12a2 (32.0 cM), the Na⁺/K⁺/Cl^- exchanger located in strial marginal cells. Complete dysfunction of the exchanger results in the collapse of scala media and loss of the EP (Dixon et al., 1999; Pace et al., 2001). It also results in pathology within the organ of Corti, spiral ligament, and Reissner's membrane. The interval also includes Nr3c1 (20.0 cM), a glucocorticoid receptor (Bray and Cotton, 2003) whose expression pattern in the adult cochlea is not well defined. Glucocorticoids mediate protective processes against noise injury (Canlon et al., 2007; Le Prell et al., 2007). Aquaporin4 water channels (Aqp4, 6.0 cM) participate in fluid volume control in some epithelial cells. Within the cochlea, they are found in the lateral organ of Corti and in inner sulcus cells, and are required for normal hearing (Li and Verkman, 2001). Finally, lysyl oxidase (Lox, 29.0 cM) helps stabilize the extracellular matrix and mediates matrix cell-cell signaling (Lucero and Kagan, 2006), and could influence the response of the spiral ligament and limbus to noise.

Discussion

The present results confirm and extend our previous work (Ohlemiller and Gagnon, 2007) demonstrating nearly Mendelian quantitative inheritance of a specific response to noise by the cochlear lateral wall of CBA/J mice. After a single severe noise exposure CBA/J mice show EP reduction that is attended by both acute and permanent changes in the spiral ligament and stria vascularis, and by permanent changes in the spiral limbus. To this list we now add acute changes in the appearance of Reissner's membrane. Although CBA/CaJs were not examined in the same detail, our observations indicate that CBA/J and CBA/CaJ mice possess the same noise phenotype. For the same exposure, neither B6 nor the B6.CAST congenic mice undergo EP reduction, nor exhibit any of the anatomic changes exhibited by CBA mice, despite the fact that the extent of permanent NIHL is greater in both B6 and B6.CAST than in CBA/J or CBA/CaJ (Davis et al., 1999; Gagnon et al., 2007; Harding et al., 2005). Therefore genes that promote permanent NIHL and those that promote acute EP reduction need not overlap, and noise injury to the organ of Corti and lateral wall can occur independently, at least in mice. Bohne and colleagues (Ahmad et al., 2003) have characterized in chinchillas noise-related reversible EP reduction that has its origins in the organ of Corti, where focal outer hair cell lesions leave transient breaches in the reticular lamina. These are sealed over and the EP is restored, yet the process is clearly tied to the extent of permanent NIHL. Such striking differences between and within species in the basic features of cochlear noise injury make it clear that there is no single noise injury archetype for the mammalian cochlea.

Candidate genes and processes

The present mapping data confirm that the CBA lateral wall noise phenotype, (EP reduction combined with acute and permanent pathology of the spiral ligament and stria vascularis) is genetically linked, and that a major locus underlying this phenotype, Nirep, is located on Chr. 18. Explicit correlations demonstrated previously between EP and lateral wall injury metrics (Ohlemiller and Gagnon, 2007), and between EP and Reissner's mesothelial cell pathology (Fig. 3), argue that all cell types showing injury either express the same critical gene(s), or all participate in a common injury process. The latter interpretation is consistent with the ‘communal’ nature of cochlear ion homeostasis; Many supporting and auxiliary cell types contribute to this function, including fibrocytes of the spiral ligament and limbus, constituent cells of Reissner's membrane, and cells of the stria vascularis (Wangemann, 2002, 2006). Thus, interruption at multiple points along normal routes of ion transfer could give rise to the changes seen in CBA mice. We also recently reported both EP reduction and similar anatomic changes after noise in BALB/cJ mice (Ohlemiller and Gagnon, 2009). Notably, the trait is recessively inherited versus B6, and based on our ongoing studies in N2 mice, is equally dependent on multiple loci. Thus a similar trait to CBAs may arise in BALB/cJ via different genes or alleles, and through mechanisms that only partially overlap. In addition, application of both furosemide (which interferes with the Na⁺/K⁺/Cl^- exchanger) (Schmiedt et al., 2002) and metabolic inhibitors such as 3-nitropropionic acid (Hoya et al., 2004; Okamoto et al., 2005) can produce lateral wall pathology that somewhat resembles the CBA noise phenotype. From such genetic and pharmacologic evidence, we infer that Nirep and the other suggested QTLs need not be expressed in all of the affected cells and structures, and may be genes that govern metabolism or blood flow, or genes that encode ion channels, pumps or key extracellular matrix proteins. While these QTLs may not impact the extent of NIHL over short periods, they may reduce the homeostatic ‘reserve’ of the lateral wall through cumulative influence. Over time, continual noise or ototoxic exposures may thereby become more likely to decrease the EP permanently, or to critically disrupt cellular networks that regulate K⁺. Studies that probe longer term patterns and efforts to identify Nirep are underway in our laboratory.