Patent Application
20220178944
The present disclosure relates to methods of analyzing epigenetic alterations in the germline DNA of animals. More particularly, the present disclosure relates to methods of analyzing histone retention involved in epigenetic alterations as well as the identification and treatment of diseases associated with such epigenetic alterations.
Generally, epigenetics refers to molecular factors and processes around DNA that regulate genome activity but are independent of DNA sequences. Various epigenetic processes include DNA methylation, histone modifications, non-coding RNA, chromatin structure, and RNA methylation. Epigenetics is becoming increasingly important for evaluation of diseases that may be induced by exposure to environmental toxicants. For instance, a variety of environmental factors can promote epigenetic transgenerational inheritance of disease or phenotypic alterations through epigenetic changes in the germline (sperm or eggs). Although direct exposure of an animal to a particular toxicant may not result in a disease pathology, it is important to recognize that subsequent offspring of the animal may have an increased propensity to develop a disease as a result of the toxicant exposure to a prior generation of relation.
For example, glyphosate is one of the most commonly used herbicides in agriculture. A low level of glyphosate direct exposure to animals (i.e., below the No Observational Adverse Effect Level (NOAEL) dose of 50 mg/kg per day) has no effect on the pathologies of the directly exposed individuals using a mammalian rat model. When the exposed gestating female F0 generation and F1 generation offspring were examined later in life, both generations were found to have negligible detectable alteration in pathology compared to controls. However, evaluation of the subsequent F2 generation grand offspring and F3 generation great-grand offspring demonstrated a significant increase in the frequency of pathology and disease. In particular, the F2 generation demonstrated an increase in testis and kidney disease, altered pubertal onset, increased obesity, increased ovarian disease, mammary tumors, altered pubertal onset, obesity, premature birth abnormalities, and the presence of multiple disease. The glyphosate induced transgenerational disease in the F3 generation pathology included prostate disease, obesity, weaning weight alterations, ovarian disease, kidney disease, parturition abnormalities, obesity, and increased presence of multiple disease.
However, there exists a need for further mechanisms to evaluate epigenetic alterations in animals, especially following exposure to toxicants. Accordingly, the present disclosure provides evaluation of differential histone retention sites (DHRs) as disease biomarkers. The present disclosure provides methods for evaluating DHRs in sperm as disease biomarkers as well as the use of overlapping differential DNA methylation region (DMR) DMR and DHR biomarkers between different diseases or pathologies.
In illustrative embodiments, a method of inducing an epigenetic alteration in germline DNA of a male progeny animal is provided. For these embodiments, the method comprises administering one or more toxicants to a female parental animal wherein the female parental animal subsequently gives birth to the male progeny animal, and wherein the male progeny animal comprises the epigenetic alteration in germline DNA.
In illustrative embodiments, a method for preparing a DNA fraction from an animal useful for analyzing histone retention involved in an epigenetic alteration is provided. For these embodiments, the method comprises (a) extracting DNA from a germline sample of an animal, (b) producing a fraction of the DNA extracted in (a) by selecting DNA comprising histone retention, and (c) analyzing the histone retention in the fraction of DNA produced in (b).
In illustrative embodiments, a method of identifying a disease or a disease propensity in a male progeny animal is provided. For these embodiments, the method comprises identifying a profile of histone retention DNA in the male progeny animal, wherein the profile is associated with the disease or the disease propensity in the male progeny animal, and wherein the disease or the disease propensity is associated with an epigenetic alteration in germline DNA resulting from contact of the germline DNA of the male progeny animal with one or more toxicants during gestation of the male progeny animal.
Additional features of the present disclosure will become apparent to those skilled in the art upon consideration of illustrative embodiments exemplifying the best mode of carrying out the disclosure as presently perceived.
The detailed description particularly refers to the accompanying figures in which:
In an illustrative aspect, a method of inducing an epigenetic alteration in germline DNA of a male progeny animal is provided. For these embodiments, the method comprises administering one or more toxicants to a female parental animal wherein the female parental animal subsequently gives birth to the male progeny animal, and wherein the male progeny animal comprises the epigenetic alteration in germline DNA.
Any toxicant capable of inducing an epigenetic alteration is contemplated by the present disclosure. For instance, the examples described herein utilize glyphosate and atrazine as exemplary environmental toxicants. Without being bound by any hypothesis, it is contemplated that other toxicants are capable of inducing an epigenetic alteration according to the methodology of the present disclosure.
In an embodiment, the epigenetic alteration is differential histone retention of DNA in the germline DNA of the male progeny animal. In an embodiment, the epigenetic alteration is differential histone retention of DNA in the germline DNA of the male progeny animal. In an embodiment, the epigenetic alteration in the germline DNA of the male progeny animal is associated with a disease in the male progeny animal. In an embodiment, the disease is selected from the group consisting of kidney disease, prostate disease, male infertility, immune cell activation, cancer, and any combination thereof.
In an embodiment, the epigenetic alteration in germline DNA is associated with in reduced fertility of the male progeny animal or sterility of the male progeny animal. In an embodiment, the reduced fertility or sterility of the male progeny animal is associated with abnormal testicular development, abnormal spermatogenesis, decreased sperm motility, decreased forward sperm movement, or any combination thereof.
In an embodiment, the animal is a human. In an embodiment, the animal is a companion animal. In an embodiment, the companion animal is a dog or a cat.
In an illustrative aspect, a male progeny animal formed according to the method of claim 1. In an embodiment, the male progeny animal is of a generation selected from the group consisting of a F1 generation, a F2 generation, a F3 generation, and a F4 generation.
In an illustrative aspect, a method for preparing a DNA fraction from an animal useful for analyzing histone retention involved in an epigenetic alteration. The method comprises (a) extracting DNA from a germline sample of an animal, (b) producing a fraction of the DNA extracted in (a) by selecting DNA comprising histone retention, and (c) analyzing the histone retention in the fraction of DNA produced in (b).
In an embodiment, the germline sample is sperm. In an embodiment, the sperm comprises purified sperm. In an embodiment, the sperm comprises sonicated sperm.
In an embodiment, analyzing the histone retention comprises histone antibody chromatin immunoprecipitation (ChIP) analysis. Briefly, ChIP analysis is well known to the skilled artisan as a means to analyze histone retention. In an embodiment, analyzing the histone retention comprises PCR. In an embodiment, analyzing the histone retention comprises ChIP and PCR
In an embodiment, the animal is a male progeny animal born from a female parental animal, wherein the female parental animal is administered one or more toxicants. In an embodiment, the epigenetic alteration is associated with in reduced fertility of the male progeny animal or sterility of the male progeny animal. In an embodiment, the reduced fertility or sterility of the male progeny animal is associated with abnormal testicular development, abnormal spermatogenesis, decreased sperm motility, decreased forward sperm movement, or any combination thereof.
In an embodiment, the histone retention comprises an altered histone retention DNA profile associated with a disease. In an embodiment, the disease is selected from the group consisting of kidney disease, prostate disease, male infertility, immune cell activation, cancer, and any combination thereof. In an embodiment, the method further comprises identification of one or more genes associated with the altered histone retention DNA profile. In an embodiment, the histone retention analysis is combined with a differential DNA methylation region analysis.
In an embodiment, the animal is a human. In an embodiment, the animal is a companion animal. In an embodiment, the companion animal is a dog or a cat.
In an illustrative aspect, a method of identifying a disease or a disease propensity in a male progeny animal is provided. The method comprises identifying a profile of histone retention DNA in the male progeny animal. In an embodiment, the profile is associated with the disease or the disease propensity in the male progeny animal, and In an embodiment, the disease or the disease propensity is associated with an epigenetic alteration in germline DNA resulting from contact of the germline DNA of the male progeny animal with one or more toxicants during gestation of the male progeny animal.
In an embodiment, the male progeny animal is administered a pharmaceutical composition to treat the identified disease or the disease propensity. In an embodiment, the disease is selected from the group consisting of kidney disease, prostate disease, male infertility, immune cell activation, cancer, and any combination thereof.
In an embodiment, the profile of histone retention DNA is identified according to a method for preparing a DNA fraction from the male progeny animal comprising (a) extracting DNA from a germline sample of the male progeny animal, (b) producing a fraction of the DNA extracted in (a) by selecting DNA comprising histone retention, and (c) analyzing the histone retention in the fraction of DNA produced in (b).
In an embodiment, the histone retention analysis is combined with a differential DNA methylation region analysis. In an embodiment, the animal is a human. In an embodiment, the animal is a companion animal. In an embodiment, the companion animal is a dog or a cat.
The following numbered embodiments are contemplated and are non-limiting:
Evaluation of Epigenetic Alterations Induced by Glyphosate
In the instant example, glyphosate is utilized as an exemplary toxicant. Rats were used as the exemplary animal.
Outbred Sprague Dawley SD male and female rats were fed a standard diet with water ad lib and mated. Gestating female rats were exposed to glyphosate and offspring bred for three generations in the absence of exposure. Female and male rats of an outbred strain Hsd:Sprague Dawley SD (Harlan) at 70 to 100 days of age were fed ad lib with a standard rat diet and ad lib tap water. Timed-pregnant females on days 8 through 14 of gestation were administered daily intraperitoneal injections of glyphosate (25 mg/kg BW/day dissolved in PBS) (Chem Service, Westchester Pa.) or dimethyl sulfoxide (DMSO) or Phosphate Buffered Saline (PBS).
The instant example is designed to examine glyphosate induced transgenerational disease. To control dose and not cause stress from gavage oral administration, an IP exposure was utilized. Twenty-five mg/kg for glyphosate is 0.4% of rat oral LD50 and 50% of the NOAEL and considering glyphosate rapid metabolism approximately twice the occupational exposure 3-5 mg/kg per daily exposure A portion of original control colony was excluded from the study due to the obesity founder effects as previously identified. These animals were replaced with offspring from DMSO-treated controls from a concurrent study. Disease phenotypes were compared from both DMSO lineage and PBS lineage controls, with no significant differences observed with histopathology evaluations between the two populations.
The gestating female rats treated were designated as the F0 generation. Breeding for each generation occurred at 70-90 days and 5 different litters were generated with no cousin or sibling breeding to avoid any inbreeding artifacts. Litters were culled to 10 pups (5 males and 5 females) between days 3-5 of birth to avoid litter effects. F1-F3 generation control and glyphosate lineages were housed and aged in the same room and racks with lighting, food and water. The F3 generation males were aged to 1 year of age for pathology analysis.
The F3 generation male rats were euthanized at 12 months of age by CO2 inhalation and cervical dislocation for tissue harvest. Testis, prostate, ovary, kidney, and gonadal fat pads were fixed in Bouin's solution (Sigma) for less than 6 hours, followed by 70% ethanol, then processed for paraffin embedding and hematoxylin, and eosin (H & E) staining by standard procedures for histopathological examination. Paraffin five micron sections were processed and stained (Nationwide Histology, Spokane Wash., USA).
Full necropsies as required on animals that died prior to the time of scheduled sacrifice at one year were undertaken and tumor classifications were also performed. Upon dissection, a brief examination of abdominal and thoracic organs was performed to look for obvious abnormalities. The current study found no significant gross pathology of heart, lung, liver, gastro-intestinal track, or spleen. The tissues evaluated histologically were selected from previous literature showing them to have pathology in transgenerational models, with an emphasis on reproductive organs. Histopathology readers were trained to recognize the specific abnormalities evaluated for this study in rat testis, ventral prostate, ovary and kidney. Three different pathology readers were used for each tissue that were blinded to the treatment groups. A set of quality control (QC) slides was generated for each tissue and was read by each reader prior to evaluating any set of experimental slides. These QC slide results are monitored for reader accuracy and concordance.
Testis histopathology criteria included the presence of vacuoles in the seminiferous tubules, azoospermic atretic seminiferous tubules, and ‘other’ abnormalities including sloughed spermatogenic cells in center of the tubule and a lack of a tubule lumen. Prostate histopathology criteria included the presence of vacuoles in the glandular epithelium, atrophic glandular epithelium and hyperplasia of prostatic gland epithelium. Kidney histopathology criteria included reduced size of glomerulus, thickened Bowman's capsule, and the presence of proteinaceous fluid-filled cysts >50 μm in diameter. A cut-off was established to declare a tissue ‘diseased’ based on the mean number of histopathological abnormalities plus two standard deviations from the mean of control group tissues, as assessed by each of the three individual observers blinded to the treatment groups. This number (i.e. greater than two standard deviations) was used to classify rats into those with and without testis, prostate, or kidney disease in each lineage. A rat tissue section was finally declared ‘diseased’ only when at least two of the three observers marked the same tissue section ‘diseased’.
Obesity was assessed with an increase in adipocyte size (area), body mass index (BMI) and abdominal adiposity. BMI was calculated with weight (g)/length (cm)2 with the length of the animal measured from the nose to the base of the tail. Gonadal fat pad slides were imaged using a Nikon Eclipse E800 microscope (10×) with an AVT Prosilica GE1050C Color GigE camera. Five field of view image captures were taken per slide in varying parts of the fat pad. Adipocyte size was measured converting pixels into microns using Adiposoft. Measurements of the 20 largest cells from each image for a total of 100 were averaged as hypertrophic cells are the most metabolically relevant and susceptible to cell death. Obesity and lean phenotypes were determined utilizing the mean of the control population males and females, and a cut off of 1.5 standard deviations above and below the mean.
Briefly, the epididymis was dissected free of fat and connective tissue, then, after cutting open the cauda, placed into 6 ml of phosphate buffer saline (PBS) for 20 minutes at room temperature. Further incubation at 4° C. will immobilize the sperm. The tissue was then minced, the released sperm pelleted at 4° C. 3,000×g for 10 min, then resuspended in NIM buffer and stored at −80° C. for further processing.
An appropriate amount of rat sperm suspension was used for DNA extraction. Previous studies have shown mammalian sperm heads are resistant to sonication unlike somatic cells. Somatic cells and debris were therefore removed by brief sonication (Fisher Sonic Dismembrator, model 300, power 25), then centrifugation and washing 1-2 times in 1×PBS. The resulting pellet was resuspended in 820 μL DNA extraction buffer and 80 μl 0.1M DTT added, then incubated at 65° C. for 15 minutes. Then, 80 μl proteinase K (20 mg/ml) was added and the sample was incubated at 55° C. for 2-4 hours under constant rotation. Protein was removed by addition of protein precipitation solution (300 μl, Promega A795A), incubation for 15 min on ice, then centrifugation at 13,500 rpm for 30 minutes at 4° C. One ml of the supernatant was precipitated with 2 μl of glycoblue (Invitrogen, AM9516) and 1 ml of cold 100% isopropanol. After incubation, the sample was spun at 13,500×g for 30 min at 4° C., then washed with 70% cold ethanol. The pellet was air-dried for about 5 minutes then resuspended in 100 μl of nuclease free water. For all generations, equal amounts of DNA from each individual's sample was used for methylated DNA immunoprecipitation (MeDIP) and chromatin immunoprecipitation (ChIP).
Genomic DNA was sonicated and run on 1.5% agarose gel for fragment size verification. The sonicated DNA was then diluted with TE buffer to 400 μl, then heat-denatured for 10 minutes at 95° C., and immediately cooled on ice for 10 min to create single-stranded DNA fragments. Then 100 μl of 5×IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added, and the mixture was incubated overnight on a rotator at 4° C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; Life Technologies 11201D) were pre-washed per manufacturer's instructions, and 50 μl of beads were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 h on a rotator at 4° C. After this incubation, the samples were washed three times with 1×IP buffer using a magnetic rack. The washed samples were then resuspended in 250 μl digestion buffer (5 mM Tris PH 8, 10. mM EDTA, 0.5% SDS) with 3.5 μl Proteinase K (20 mg/ml), and incubated for 2-3 hours on a rotator at 55° C. DNA clean-up was performed using a Phenol-Chloroform-Isoamyalcohol extraction, and the supernatant precipitated with 2 μl of glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol in −20° C. freezer for one to several hours. The DNA precipitate was pelleted, washed with 70% ethanol, then dried and resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with the ssDNA kit (Molecular Probes Q10212).
Histone chromatin immunoprecipitation with genomic DNA was performed. Individual rat sperm were collected, and the sperm counts were determined for each individual. A sonication of 10 seconds was performed for each sperm sample to remove any somatic cell contamination using a Sonic Dismembrator Model 300 (Thermo Scientific Fisher, USA) then centrifuged 1800×g for 5 minutes at 4° C. then resuspended and counted individually on a Neubauer counting chamber (Propper manufacturing Co., Inc., New York, USA). The sperm samples were reconstituted up to 1 ml with PBS (phosphate buffered saline). To reduce disulfide bonds, 50 μl of 1 M DTT was added to each sample and an incubation of 2 hours at room temperature under constant rotation followed. To quench any residual DTT (dithiothreitol, Fisher Scientific, NY USA) in the reaction, 120 μl of fresh 1 M NEM (N-Ethylmaleimide, Thermo Scientific, Rockford, USA) was then added and the samples were incubated for 30 min at room temperature under constant rotation. After 5 minutes of centrifugation at 450×g at room temperature, the sperm samples were pelleted and the supernatant discarded. Pellets were resuspended in PBS and then spun again at 450×g for 5 min at room temperature. The supernatant was discarded, and the pellets were resuspended in 130 μl of complete buffer supplemented with tergitol 0.5% and DOC 1%. The samples were then sonicated using the Covaris M220. Each tube in the experiment went through the “Chromatin shearing” program for 10 minutes.
After the Covaris sonication, the fragmentation was checked by running 10 μl of each sample on a 1.5% agarose gel. Samples were then centrifuged at 12,500×g for 10 min at room temperature. The supernatant was transferred to a fresh microfuge tube and 65 μl of protease inhibitor cocktail (1 tablet dissolved in 500 μl, 20× concentrated) (Roche, cat. no. 11 873 580 001) were added in each sample as well as 3 μl of antibody (anti-histone H3 pan-monoclonal antibody, cat no. 05-928, with broad spectrum species specifically form Millipore Corp, Temecula Calif. USA). The DNA-antibody mixture was incubated overnight on a rotator at 4° C. The following day, magnetic beads (ChIP-Grade protein G magnetic beads, Cell Signaling 9006) were pre-washed as follows: the beads were resuspended in the vial, then 30 μl per sample was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of 1×IP buffer as the initial volume of beads. 30 μl of beads were added to each DNA-antibody mixture from the overnight incubation, then incubated for 2 h on a rotator at 4° C. After the incubation, the bead-antibody-DNA complex was washed three times with 1×IP buffer as follows: the tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then washed with 1×IP buffer 3 times. The washed bead-antibody-DNA solution was then resuspended in 300 μl of digestion buffer (1 M Tris HCl, pH 8.0, 0.5 M EDTA, 10% SDS) and 3 μl proteinase K (20 mg/ml). The sample was incubated for 3 h on a rotator at 56° C. After incubation the samples were extracted with Phenol-Chloroform-Isoamylalcohol and precipitated with 2 μl of Glycoblue (20 mg/ml), a one-tenth volume of 3 M sodium acetate and two volumes of ethanol overnight at −20° C. After a centrifugation at 18,000×g for 30 min at 4° C., the supernatant was removed without disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol, then centrifuged again at 18,000×g for 10 min at 4° C. and the supernatant was discarded. The tube was spun briefly to collect residual ethanol to bottom of tube and as much liquid as possible was removed with a gel loading tip. Pellet was air-dried at room temperature until it looked dry (about 5 min), then resuspended in 20 μl H2O. DNA concentration was measured in the Qubit (Life Technologies) with the BR dsDNA kit (Molecular Probes Q32853).
MeDIP DNA and H3 ChIP DNA was used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, Calif.) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA from the single-stranded DNA resulting from MeDIP. After this step, the manufacturer's protocol was followed indexing each sample individually with NEBNext Multiplex Oligos for Illumina. The library quality control used an Agilent 2100 Bio analyzer analysis for size, amount and quality. The samples were sequenced on the Illumina HiSeq 2500 at PESO, with a read size of approximately 50 bp and approximately 20 million reads per pool. Twelve libraries were run in one lane.
Sequencing data quality was assessed using the FastQC program. Sequencing reads were cleaned and filtered to remove adapters and low quality bases using Trimmomatic. The basic read quality was verified using summaries produced by the FastQC program. The reads for each MeDIP and H3 histone ChIP samples were mapped to the Rnor 6.0 rat genome using Bowtie2 with default parameter options. The mapped read files were then converted to sorted BAM files using SAMtools. The MEDIPS R package was used to calculate differential coverage between control and exposure sample groups. The edgeR p-value was used to determine the relative difference between the two groups for each genomic window. Windows with an edgeR p-value less than the selected stringent threshold were considered DMR or DHR sites. The site edges were extended until no genomic window with an edgeR p-value less than 0.1 remained within 1000 bp of the DMR or DHR. The edgeR p-value is optimal for individual epimutation identification and used to assess the significance of the DMR or DHR identified. A false discovery rate (FDR) analysis for the exposure versus control was performed and provided value less than 0.05. Due to the lower number of individuals with one specific disease type, an FDR analysis was generally not useful, nor permutation analysis for the specific disease biomarkers.
Differential epimutation sites were annotated using the biomaRt R package to access the Ensembl database. The DMR associated genes were then automatically sorted into functional groups using information provided by the publicly available DAVID and Panther databases incorporated with an internal curated database (www.skinner.wsu.edu under genomic data). The DAVID and Panther databases have a greater accuracy for experimental gene functional links than literature based searches such as GO categories. A Pathway Studio, Elsevier, database and network tool was used to assess physiological and disease process gene correlations (Pathway Studio software (Elsevier, Inc. 2020), Pathway Studio 12.3.0.16). All molecular data has been deposited into the public database at NCBI (GEO #GSE118557 and GSE152678) and R code computational tools.
Sprague Dawley gestating female rats (F0 generation) at 90 days of age were exposed in order to study the transgenerational effects of glyphosate. The pregnant rats were transiently exposed (25 mg/kg body weight glyphosate daily) between days 8-14 of gestation during fetal gonadal sex determination when germ cell epigenetic programming occurs. Twenty-five mg/kg/day is half the No Observable Adverse Effect Level (NOAEL) exposure of 50 mg/kg/day. Glyphosate has a rapid metabolism turnover of 5-10 hour half-life, such that the concentration will decrease approximately 75-90% daily during the transient exposure period. The 2-4 half-lives that occur each 24-hour period indicates that after 7 days of exposure less than 50 mg/kg/daily, the exposures NOAEL, would be present. The offspring F1 generation rats (directly exposed in utero) were aged to 90 days of age and bred within the lineage to generate the grand-offspring F2 generation (directly exposed through the F1 generation germline), which were then bred at 90 days of age to generate the F3 generation (not directly exposed so transgenerational). The control lineage used F0 gestating rats exposed to the vehicle control dimethyl sulfoxide (DMSO) or phosphate buffered saline (PBS). All the lineages were aged to 1 year and euthanized for pathology and sperm epigenetic analysis. At each generation five litters were obtained with no sibling or cousin breeding to prevent any inbreeding artifacts in the control or glyphosate lineages. Due to the lack of any inbreeding, the potential frequency of genetic segregation is minimal in the F3 generation and was not observed with sibling comparisons. As previously described and to prevent litter bias, at each generation between 6 and 8 unrelated founder gestating females from different litters were bred, and 5 litters were obtained and culled early in postnatal development to 10 pups per litter with animals of each sex from each litter used to generate 25-50 individuals of each sex for each generation for analysis. Similar numbers of males and females were used from each litter to avoid any litter bias.
Pathology calls were made by assessing histology sections of testis, kidney, prostate, and gonadal adipocytes. For the testis pathology, the abnormalities quantified were atrophy of seminiferous tubules, vacuoles within seminiferous tubules, and sloughing of cellular debris into the tubular lumen (maturation arrest). For prostate pathology, the abnormalities quantified were atrophy of prostatic epithelium, vacuoles within the prostatic epithelium, and prostatic epithelial hyperplasia. For kidney pathology, the abnormalities quantified were reduced glomerular size, thickening of the Bowman's capsule, and renal cysts. The age of puberty onset was determined. Obesity was assessed with an increase in adipocyte size (area), body mass index (BMI) and abdominal adiposity. In all cases the number of abnormalities in an animal's tissue was compared with the mean number of abnormalities in the control group to determine if that tissue was diseased. For the F3 generation glyphosate lineage male pathology, the individual animals are listed and a (+) indicates presence of disease and (−) absence of disease (Table 1). Table 1 shows the F3 generation glyphosate lineage male pathology. The individual animals for the glyphosate lineage males are listed and a (+) indicates presence of disease and (−) absence of disease. The animals with shaded (+) were used for the epigenetic analysis due to the presence of only one disease, except the multiple (≥2) disease.
The F3 generation control lineage male pathology, the individuals are listed similarly in Table 2. As shown in Table 2, individual animals for the control lineage males are listed and a (+) indicates presence of disease and (−) absence of disease.
Only the individuals with a single disease for a specific pathology were used for that pathology, except in the case of the multiple category disease when animals with two of more diseases were used. This allows a more accurate association with disease and eliminates the confounding presence of other disease or co-morbidities. The control lineage did not have sufficient numbers of animals with a specific disease (see Table 2), so were not analyzed further. Although the F3 generation males had a testis disease group, two individuals had only testis disease, so too few for further analysis. The other disease had n=4 individuals for kidney disease, n=7 with prostate disease, n=13 for obesity, and n=10 for multiple 2) disease, see Table 1. The disease animals were compared to animals with no disease n=8, see Table 1. The limited number of individuals needs to be considered in data interpretation and statistical analysis. In contrast to previous analyses using DDT or vinclozolin sperm biomarkers for disease, when all animals with a specific disease were analyzed independent of co-morbidities, the instant example sought to optimize disease specific biomarker assessment.
Transgenerational inheritance of pathology and disease requires the germline (sperm or egg) to transmit epigenetic information between generations. Purified sperm were collected from the control and glyphosate lineage F3 generation males for epigenetic analysis. Potential differential DNA methylation regions (DMRs) in the sperm were identified using a comparison between the control and the glyphosate lineage (
DNA from the sonicated purified sperm was isolated, fragmented and the methylated DNA immunoprecipitated using a methyl-cytosine antibody (MeDIP). The MeDIP DNA fragments were then sequenced for an MeDIP-Seq analysis. The MeDIP-Seq analysis was used since >90% of the genome has low CpG density regions, so can assess >90% of the genome-wide DNA methylation. The sperm DMR numbers are presented in
The previously reported transgenerational F3 generation sperm glyphosate versus control lineage DMRs used three pools of different animals to identify the glyphosate induced sperm DNA alterations. The current study used individual animal sperm analysis to identify the glyphosate induced transgenerational F3 generation DMRs (
The animals with a specific disease were compared to non-disease animals to identify the disease specific sperm DMRs. The group of animals with prostate disease had 242 DMRs at p<1e−04 with two multiple windows (i.e., 1 kb each) detected, (
Therefore, the different diseases were found to have altered DNA methylation in the F3 generation sperm. Interestingly, negligible overlap was observed between these different DMRs at a statistical threshold of p<1e−04, (
The DMRs chromosomal locations are presented in
Differential histone retention in sperm also appears to have a role in epigenetic transgenerational inheritance. Similarly to the DMRs, the differential histone retention regions (DHRs) in the sperm were identified using a comparison between the control and the glyphosate lineage (
Unexpectedly, a high number of DHRs (836) was found at edgeR p<1e−05 in the glyphosate versus control comparison (see
The chromosomal locations of these DHRs are presented in
The DHRs CpG density and length of DHRs are presented in
A relatively stringent edgeR p-value for the DMRs or DHRs is used for the identification of these disease specific epimutations. A reduced statistical threshold of p<0.05 was used to compare and further evaluate the potential overlap of the DMRs or DHRs between the glyphosate versus control and the different disease biomarkers when compared to the higher edgeR p<1e−04 statistical threshold DMRs. By lowering the stringency to a p-value of <0.05 for the comparison (i.e. extended overlap) the procedure allows for the identification of more potential overlaps between the glyphosate versus control, and the prostate, kidney, obesity and multiple disease comparisons. The relatively high statistical threshold is used as the epigenetic site definition. A comparison of the p<1e−04 for the DMRs, with each potential comparison at p<0.05 (see
The same reduced statistical edgeR threshold extended overlap was used for the DHRs in
The lists of DMR and DHR in common with all pathologies identify those with associated genes. Interestingly, when the common DMR and DHR sites for a specific disease comparison were identified and then compared between all the diseases, negligible overlap was observed (
The list of DMRs and DHRs for all the epigenetic alterations identified are presented in Tables 3-12. Epimutation gene associations used DMR or DHR identified within 10 kb of a gene so as to include proximal and distal promoter elements. The minority of DMR or DHR, less than 20%, have epimutations associated with genes. Therefore, the majority are intergenic and not within 10 kb of a gene. The DMR and DHR associated genes found were categorized into relevant functional categories for the glyphosate versus control, and for each set of disease biomarkers (
The disease specific DMR associated genes were analyzed using a Pathway Studio gene database and network tool to identify associated gene processes, (
Surprisingly, sperm differential histone retention regions (DHRs) were observed with different pathologies. The number of DHRs were less than the number of DMRs and the DHRs were also found to have disease specificity (
In the instant example, atrazine is utilized as an exemplary toxicant. Rats were used as the exemplary animal.
Female and male rats of an outbred strain Hsd:Sprague Dawley SD (Harlan) at 70 to 100 days of age were fed ad lib with a standard rat diet and ad lib tap water. Timed-pregnant females on days 8 through 14 of gestation were administered daily intraperitoneal injections of atrazine (25 mg/kg BW/day dissolved in PBS) (Chem Service, Westchester Pa.) or dimethyl sulfoxide (DMSO) or as previously described. Twenty-five mg/kg for atrazine is 4% of rat oral LD50 and 50% of NOAEL.
The gestating female rats treated were designated as the F0 generation. F1-F3 generation control and atrazine lineages were housed in the same room and racks with lighting, food and water.
Rats were euthanized at 12 months of age by CO2 inhalation and cervical dislocation for tissue harvest. Testis, prostate, ovary, kidney, and gonadal fat pads were fixed in Bouin's solution (Sigma) followed by 70% ethanol, then processed for paraffin embedding and hematoxylin, and eosin (H & E) staining by standard procedures for histopathological examination. Paraffin five micron sections were processed and stained (Nationwide Histology, Spokane Wash., USA).
Full necropsies were performed as required on animals that died prior to the time of scheduled sacrifice at one year and tumor classifications were performed.
Upon dissection a brief examination of abdominal and thoracic organs was performed to look for obvious abnormalities. The tissues evaluated histologically were selected from previous literature showing them to have pathology in transgenerational models, with an emphasis on reproductive organs. Histopathology readers were trained to recognize the specific abnormalities evaluated for this study in rat testis, ventral prostate and kidney (see below). Two different readers initially evaluated the tissues. If there was disagreement on whether an animal's tissue showed disease, then a third pathology reader was used. Readers were blinded to the exposure groups. A set of quality control (QC) slides was generated for each tissue and was read by each reader prior to evaluating any set of experimental slides. These QC slide results are monitored for reader accuracy and concordance.
Testis histopathology criteria included the presence of vacuoles in the seminiferous tubules, azoospermic atretic seminiferous tubules, and ‘other’ abnormalities including sloughed spermatogenic cells in the center of the tubule and a lack of a tubule lumen. Prostate histopathology criteria included the presence of vacuoles in the glandular epithelium, atrophic glandular epithelium and hyperplasia of prostatic gland epithelium (
Lean phenotype was assessed with a decrease in adipocyte size (area), body mass index (BMI) and abdominal adiposity. BMI was calculated with weight (g)/length (cm)2 with the length of the animal measured from the nose to the base of the tail. Gonadal fat pad slides were imaged using a Nikon Eclipse E800 microscope (10×) with an AVT Prosilica GE1050C Color GigE camera. Five field of view image captures were taken per slide in varying parts of the fat pad. Adipocyte size was measured converting pixels into microns using Adiposoft. Measurements of the 20 largest cells from each image for a total of 100 were averaged as hypertrophic cells are the most metabolically relevant and susceptible to cell death. Obesity and lean phenotypes were determined utilizing the mean of the control population males and females, and a cut-off of 1.5 standard deviations above and below the mean.
The individual animals are listed and a (+) indicates presence of disease and (−) absence of disease for the current F3 generation atrazine lineage male pathology (Table 3). Table 3 shows F3 generation atrazine lineage males pathology. The individual animals for the atrazine lineage males are listed and a (+) indicates presence of disease and (−) absence of disease. The boxes marked with an (*) represent animals with a single disease (+) or no disease (0) that were used for the molecular analysis. The number of disease animals/total animals is presented.
The F3 generation control lineage male pathology is listed in
Briefly, the epididymis was dissected free of fat and connective tissue, then, after cutting open the cauda, placed into 6 ml of phosphate buffer saline (PBS) for 20 minutes at room temperature. Further incubation at 4° C. will immobilize the sperm. The tissue was then minced, the released sperm pelleted at 4° C. 3,000×g for 10 min, then resuspended in NIM buffer and stored at −80° C. for further processing.
An appropriate amount of rat sperm suspension was used for DNA extraction. Mammalian sperm heads may be resistant to sonication unlike somatic cells. Somatic cells and debris were therefore removed by brief sonication (Fisher Sonic Dismembrator, model 300, power 25), then centrifugation and washing 1-2 times in 1×PBS. The resulting pellet was resuspended in 820 μL DNA extraction buffer and 80 μl 0.1M DTT added, then incubated at 65° C. for 15 minutes. Then, 80 μl proteinase K (20 mg/ml) was added and the sample was incubated at 55° C. for 2-4 hours under constant rotation. Protein was removed by addition of protein precipitation solution (300 μl, Promega A795A), incubation for 15 minutes on ice, then centrifugation at 13,500 rpm for 30 minutes at 4° C. One ml of the supernatant was precipitated with 2 μl of Glycoblue (Invitrogen, AM9516) and 1 ml of cold 100% isopropanol. After incubation, the sample was spun at 13,500×g for 30 min at 4° C., then washed with 70% cold ethanol. The pellet was air-dried for about 5 minutes then resuspended in 100 μl of nuclease free water. For all generations, equal amounts of DNA from each individual's sample was used to produce 6 different DNA pools per lineage and the pooled DNA used for methylated DNA immunoprecipitation (MeDIP).
Genomic DNA was sonicated and run on 1.5% agarose gel for fragment size verification. The sonicated DNA was then diluted with TE buffer to 400 μl, then heat-denatured for 10 min at 95° C., and immediately cooled on ice for 10 min to create single-stranded DNA fragments. Then 100 μl of 5×IP buffer and 5 μg of antibody (monoclonal mouse anti 5-methyl cytidine; Diagenode #C15200006) were added, and the mixture was incubated overnight on a rotator at 4° C. The following day magnetic beads (Dynabeads M-280 Sheep anti-Mouse IgG; Life Technologies 11201D) were pre-washed per manufacturer's instructions, and 50 μl of beads were added to the 500 μl of DNA-antibody mixture from the overnight incubation, then incubated for 2 h on a rotator at 4° C. After this incubation, the samples were washed three times with 1×IP buffer using a magnetic rack. The washed samples were then resuspended in 250 μl digestion buffer (5 mM Tris PH 8, 10 mM EDTA, 0.5% SDS) with 3.5 μl Proteinase K (20 mg/ml), and incubated for 2-3 hours on a rotator at 55°. DNA clean-up was performed using a Phenol-Chloroform-Isoamyalcohol extraction, and the supernatant precipitated with 2 μl of Glycoblue (20 mg/ml), 20 μl of 5M NaCl and 500 μl ethanol in −20° C. freezer for one to several hours. The DNA precipitate was pelleted, washed with 70% ethanol, then dried and resuspended in 20 μl H2O or TE. DNA concentration was measured in Qubit (Life Technologies) with the ssDNA kit (Molecular Probes Q10212).
Histone chromatin immunoprecipitation with genomic DNA was performed. Individual rat sperm collections were generated, and the sperm counts were determined for each individual. Equal numbers of sperm were added from each individual for a total of 1.5 million sperm. To remove any somatic cell contamination sperm samples from each animal were sonicated for 10 seconds using a Sonic Dismembrator Model 300 (Thermo Scientific Fisher, USA) then centrifuged 1800×g for 5 min at 4° C. then resuspended and counted individually on a Neubauer counting chamber (Propper manufacturing Co., Inc., New York, USA) prior to pooling. The sperm pools were reconstituted up to 1 ml with PBS (phosphate buffered saline). To reduce disulfide bonds, 50 μl of 1 M DTT was added to each pool and the pools were then incubated for 2 hours at room temperature under constant rotation. To quench any residual DTT (dithiothreitol, Fisher Scientific, NY USA) in the reaction, 120 μl of fresh 1 M NEM (N-Ethylmaleimide, Thermo Scientific, Rockford, USA) was then added and the samples were incubated for 30 min at room temperature under constant rotation. The sperm cells were pelleted at 450×g for 5 min at room temperature and the supernatant was discarded. Pellets were resuspended in PBS and then spun again at 450×g for 5 min at room temperature. The supernatant was discarded and resuspended in 130 μl of complete buffer supplemented with tergitol 0.5% and DOC 1%. The samples were then sonicated using the Covaris M220. Covaris was set to a 10 min “Chromatin shearing” program and the program was run for each tube in the experiment.
After the Covaris sonication, 10 μl of each sample was run on a 1.5% agarose gel to verify fragment size. Samples were then centrifuged at 12,500×g for 10 min at room temperature. The supernatant was transferred to a fresh microfuge tube. 65 μl of protease inhibitor cocktail (1 tablet dissolved in 500 μl, 20× concentrated) (Roche, cat. no. 11 873 580 001) were added in each sample as well as 3 μl of antibody (anti-histone H3 pan-monoclonal antibody, cat no. 05-928, or anti-trimethyl-histone H3 (Lys27) polyclonal antibody, cat no. 07-449, both with broad spectrum species specifically form Millipore Corp, Temecula Calif. USA). The DNA-antibody mixture was incubated overnight on a rotator at 4° C. The following day, magnetic beads (ChIP-Grade protein G magnetic beads, Cell Signaling 9006) were pre-washed as follows: the beads were resuspended in the vial, then 30 μl per sample was transferred to a microfuge tube. The same volume of Washing Buffer (at least 1 ml) was added and the bead sample was resuspended. The tube was then placed into a magnetic rack for 1-2 min and the supernatant was discarded. The tube was removed from the magnetic rack and the beads were washed once. The washed beads were resuspended in the same volume of IP buffer as the initial volume of beads. 30 μl of beads were added to each DNA-antibody mixture from the overnight incubation, then incubated for 2 h on a rotator at 4° C. After the incubation, the bead-antibody-DNA complex was washed three times with IP buffer as follows: the tube was placed into a magnetic rack for 1-2 min and the supernatant was discarded, then washed with IP buffer 3 times. The washed bead-antibody-DNA solution was then resuspended in 300 μl of digestion buffer (1 M Tris HCl, pH 8.0, 0.5 M EDTA, 10% SDS) and 3 μl proteinase K (20 mg/ml). The sample was incubated for 3 h on a rotator at 56° C. After incubation the samples were extracted with Phenol-Chloroform-Isoamylalcohol and precipitated with 2 μl of Glycoblue (20 mg/ml), a one-tenth volume of 3 M sodium acetate and two volumes of ethanol overnight at −20° C.
The precipitate was centrifuged at 18,000×g for 30 min at 4° C. and the supernatant was removed, while not disturbing the pellet. The pellet was washed with 500 μl cold 70% ethanol, then centrifuged again at 18,000×g for 10 min at 4° C. and the supernatant was discarded. The tube was spun briefly to collect residual ethanol to bottom of tube and as much liquid as possible was removed with a gel loading tip. Pellet was air-dried at RT until it looked dry (about 5 min), then resuspended in 20 μl H2O. DNA concentration was measured in the Qubit (Life Technologies) with the BR dsDNA kit (Molecular Probes Q32853).
MeDIP DNA was used to create libraries for next generation sequencing (NGS) using the NEBNext Ultra RNA Library Prep Kit for Illumina (San Diego, Calif.) starting at step 1.4 of the manufacturer's protocol to generate double stranded DNA from the single-stranded DNA resulting from MeDIP. After this step the MeDIP DNA, and starting with the ChIP DNA, the manufacturer's protocol was followed indexing each sample individually with NEBNext Multiplex Oligos for Illumina. The WSU Spokane Genomics Core sequenced the samples on the Illumina HiSeq 2500 at PESO, with a read size of approximately 50 bp and approximately 20 million reads per pool. Ten libraries were run in one lane.
Data quality was assessed using the FastQC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and reads were cleaned and filtered to remove adapters and low quality bases using Trimmomatic. The reads for each MeDIP and ChIP sample were mapped to the Rnor 6.0 rat genome using Bowtie2 with default parameter options. The mapped read files were then converted to sorted BAM files using SAMtools. The MEDIPS R package was used to calculate differential coverage between control and exposure sample groups. The reference genome was broken into 1000 bp windows. Only genomic windows with at least an average of 10 reads per sample were kept for subsequent analysis. The edgeR p-value was used to determine the relative difference between the two groups for each genomic window. Windows with an edgeR p-value less than an arbitrarily selected threshold were considered DMRs or DHRs. The DMR/DHR edges were extended until no genomic window with an edgeR p-value less than 0.1 remained within 1000 bp of the DMR/DHR.
DMRs and DHRs were annotated using the biomaRt R package to access the Ensembl database. The genes that associated with DMRs/DHRs were then input into the KEGG pathway search to identify associated pathways. The DMR/DHR associated genes were then automatically sorted into functional groups using information provided by the DAVID and Panther databases incorporated into an internal curated database.
Outbred Sprague Dawley gestating female rats (F0 generation) were administered an intraperitoneal dose of 25 mg/kg body weight of atrazine (4% of rat oral LD50 and 50% of NOAEL). These doses were administered at 90 days of age, during embryonic days 8-14 (E8-E14) of fetal gonadal sex determination. The F1 generation offspring was directly exposed as a fetus and F2 generation grand-offspring exposed as the germline in the F1 generation. These were each bred at 90 days of age within the lineage. The F3 generation great-grand-offspring is required to establish the transgenerational inheritance generation of ancestral exposure. This transgenerational generation was the focus of the current study. A control lineage was established that used F0 gestating rats exposed to the vehicle control dimethyl sulfoxide (DMSO). Disease pathology was evaluated in atrazine exposure and control lineages at 1 year of age. The atrazine exposure lineage transgenerational individuals with specific disease or pathology were grouped as representatives of the pathology exhibited. The remaining individuals were grouped as “no disease.” Comparisons between these two groups were made during analysis of sperm DNA methylation and histone retention. The differentially methylated regions and differential histone retention site allows the identification of specific disease associated epigenetic biomarkers.
Pathology analysis was assessed with histology sections of testis, kidney, prostate, and gonadal fat pads. The complete histological sections were analyzed by two different observers blinded to the exposure, unless they disagreed, and then an additional different third observer was used. Briefly, each counter records the incidence of abnormalities in each tissue. In testis, atrophy of a seminiferous tubule, the arrest of maturation of sperm (indicated by sloughed cells in the center of the tubule), and the presence of vacuoles were indicated disease pathologies. The abnormalities counted in kidney include a reduction in size of glomeruli, a thickening of the Bowman's capsule, and the presence of cysts. Prostate abnormalities counted include atrophy of the epithelial cells, hyperplasia in the epithelial layer, and the presence of vacuoles within the epithelial layer of the prostatic glands. Obese and lean phenotypes were assigned following assessment of adipocyte size (area), body mass index (BMI) and abdominal adiposity. Late puberty was noted during development. The individual animals are listed in Table 3. A (+) indicates presence of disease and (−) indicates absence of disease for the current F3 generation atrazine lineage male pathology. The control lineage males were analyzed in a similar manner to allow a comparison to assess atrazine induced disease in the atrazine lineage (Table 4). Only the individuals with a single disease for a specific pathology were used for that pathology molecular analysis. Animals exhibiting more than one disease are all listed under the category “Multiple Disease.” Due to low prevalence of disease in the control animal groups (Table 4), those animals were not used in the identification of epigenetic biomarkers.
The experimental design was focused on the identification of transgenerational DMRs and DHRs in sperm. Sperm were collected from the atrazine lineage F3 generation males for epigenetic analysis. DNA from the sperm was isolated and fragmented with sonication. The methylated DNA immunoprecipitation (MeDIP) using a methyl-cytosine antibody was used to identify alterations in DNA methylation. The methylated DNA fragments were then sequenced for an MeDIP-Seq analysis. The differential DNA methylation regions (DMRs) were identified between the disease versus non-disease within the atrazine lineage animals (
The previously reported transgenerational F3 generation sperm atrazine versus control lineage DMRs identified atrazine induced sperm differential DNA methylation. A bioinformatics reanalysis of these sperm samples used updated method parameters, including a wider 1000 bp window size and increased read depth required for each window. In the instant example, animals for each disease category were chosen only if they exhibit that single disease and no other. Any animals exhibiting multiple disease phenotypes were grouped in the multiple (≥2) disease category. A comparison of these two studies demonstrates a difference in the disease specific DMR sets, which were identified. For example, the lean phenotype was found to have 301 total DMRs at an edgeR p-value threshold of p<1e−4 with 2 of these having multiple neighboring windows (
Chromosomal locations of the DMRs are presented in
Sperm were collected from the atrazine lineage F3 generation males for analysis. Chromatin from the sperm was isolated and fragmented. A histone H3 antibody is used in a chromatin immunoprecipitation (ChIP) analysis. The retained fragments of DNA were then sequenced for a ChIP-Seq analysis, similar to the MeDIP-Seq analysis, as described in the Methods. This analysis yields the differential histone retention sites (DHRs), which were identified in the sperm using a comparison between the disease specific and non-disease atrazine exposure lineage males (
A similar genome wide response is seen in the widespread distribution of DHRs across chromosomes (
In addition to the atrazine lineage disease specific DHR analyses, control lineage F3 generation (Table 4) sperm was compared to the F3 generation atrazine lineage male sperm. The control versus atrazine lineage F3 generation sperm identified DHRs at a variety of statistical thresholds, with 786 DHRs at p<1e−04 (
A comparison of the different epigenetic data sets for each disease category among both DMRs and DHRs demonstrated only a handful of overlapping sites at the statistical threshold of p<1e−04 (see
A comparison of the p<1e−04 for the DMRs and DHRs between the different data sets at p<0.05 demonstrates a much higher overlap between the various DMRs and DHRs identified than the Venn diagrams in
Between 49% and 55% of the DMRs and between 41% and 54% of the DHRs from the specific disease prevalence have epimutations associated with genes. These epimutation associated genes are presented in
The final analysis used a Pathway Studio gene network approach to associate previously identified disease specific associated genes with the disease specific DMRs and DHRs identified. A large number of previously identified kidney disease linked genes were found to be within the DMR and DHR associated genes,
Surprisingly, the instant example demonstrates not only DNA methylation alterations but also provides one of the first observations of disease-specific alterations in histone retention sites. These transgenerational epigenetic shifts are associated with ancestral exposure to the toxicant atrazine, and potential disease specific biomarkers for pathologies were identified. The pathologies observed with sufficient numbers of animals include the lean phenotype, kidney disease, testis disease, late puberty, and multiple disease where individuals exhibited two or more different pathologies.
The frequency of epimutations tend to be much higher among individuals with disease. In the instant example, the number of differential DNA methylated regions (DMRs) occurring in the transgenerational males is between 300 and 600 at an edgeR p<1e−04 threshold (
There is also a sub-population of DMRs and DHRs with overlap between the different individual disease pathologies, suggesting some of the epimutations are less disease specific and indicative of multiple pathologies. This suggests some common epimutations may have a role in promoting generational disease susceptibility. Therefore, the majority of epigenome-wide association study (EWAS) associated epimutations were disease specific. The DMR and DHR associated genes suggest the most affected gene categories were signaling, metabolism, and transcription. The analysis of previously identified disease associated genes yields a number of links with the diseases examined in the current study (see
The atrazine induced transgenerational lean pathology was quite prevalent in the exposed population, with nearly a third of the animals presenting this pathology. The epigenetic signature was also strong, with 301 DMRs and 2,859 DHRs at an edgeR p<1e−04 threshold. A lean phenotype can be as significant an indicator of disease as an obese phenotype. More importantly, the early developmental effects of exposure to EDCs could have magnified effects in later development and this may be particularly important with regards to other metabolic disorders.
This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application Ser. No. 63/121,644, filed on Dec. 4, 2020, the entire disclosure of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63121644 | Dec 2020 | US |
