Laboratory Research
This section includes Class of 2024 Embark Projects within the Laboratory & Bench research disciplines. These projects include traditional bench research in a variety of basic and clinical science areas.
High Carbohydrate Intake Correlates With Elevated White Blood Cell Count in Obese Black Women Relative to Non-Obese Counterparts: An Examination of NHANES Data from 1999-2010
Kimberly Anyadike, B.S.1, Jacob Keeley, M.S.1, Virginia Uhley2,3,
Anna Bruins, M.D.4, Kyeorda Kemp, Ph.D.2
1Oakland University William Beaumont School of Medicine, Rochester, MI
2Department of Foundational Medical Studies, Oakland University William Beaumont School of Medicine, Rochester, MI
3Family Medicine and Community Health, Oakland University William Beaumont School Of Medicine, Rochester, MI
4Trinity Health Grand Rapids Family Medicine Residency, Grand Rapids, MI
INTRODUCTION
The metabolic homeostasis of adipose tissue is dysregulated in obesity, corresponding to excess pro-inflammatory molecules such as C-reactive protein (CRP) and white blood cells (WBC). A low-carbohydrate diet may help mitigate inflammatory sequelae in obesity-related diseases. However, less is known about the impact of diet on inflammation levels in women of color compared to white women. The primary goal of this study is to investigate the association between adherence to a low-carbohydrate dietary pattern and inflammation levels in obese and non-obese non-Hispanic black women compared to obese and non-obese non-Hispanic white women.
METHODS
National Health and Nutrition Examination Survey (NHANES), years 1999-2010, was accessed to extract data related to CRP and WBC expression for non-obese/ obese non-Hispanic black women and non-Hispanic white women. Adherence to low/ moderate/ high-carbohydrate dietary patterns was assessed based on one 24-hour recall interview. Dietary strata were established based on %Kcal per day (% Macronutrients per day). Participants were assigned to groups based on BMI and diet adherence. Analysis was done as a two-way ANOVA with an interaction effect.
RESULTS
CRP levels were similar when comparing black and white women who are non-obese or black and white women who are obese. There were also no differences within racial groups when comparing obese and non-obese women for CRP. WBC levels were also similar when comparing black and white women who are non-obese or black and white women who are obese, regardless of dietary pattern. However, obese black women had higher WBC than non-obese black women when reporting adherence to a high carbohydrate dietary pattern (pWBC=0.0001).
CONCLUSIONS
The results indicate that diet influences inflammation differently in non-obese compared to obese black women consuming a high-carbohydrate diet. Further research should be done investigating the reason behind this difference and to inform optimal diet for obese non-Hispanic black women.
Genome-wide screening reveals the genetic basis of mammalian embryonic eye development
Justine M. Chee, B.S.1, Louise Lanoue, Ph.D.2, Dave Clary, B.S.2, Brian Brooks, M.D./Ph.D.3, K. C. Kent Lloyd4, Ala Moshiri, M.D./Ph.D.5
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Mouse Biology Program, University of California Davis, Davis, California
3Ophthalmic Genetics and Visual Function Branch, National Eye Institute, NIH, Bethesda, Maryland
4Department of Surgery, School of Medicine, University of California Davis, Sacramento, California
Medical Research Council Harwell (Mammalian Genetics Unit and Mary Lyon Centre), Harwell, Oxfordshire
5Department of Ophthalmology & Vision Science, School of Medicine, University of California Davis, Sacramento, California
INTRODUCTION
Microphthalmia, anophthalmia, and coloboma (MAC) spectrum disease encompasses a group of eye malformations which play a role in childhood visual impairment. Although the predominant cause of eye malformations is known to be heritable in nature, with 80% of cases displaying loss-of-function mutations in OTX2 or SOX2, the genetic abnormalities underlying the remaining cases of MAC are incompletely understood. This study aims to identify the novel genes and pathways required for early eye development through systematic forward screening of the mammalian genome.
METHODS
Bioinformatics
We queried the IMPC database for phenotypes associated with significant eye defects in mouse embryos using mammalian phenotype annotation terms. The final literature search looked for review articles and case reports that documented genes in humans related to MAC spectrum disorder, so that a gold standard list of published human genes (n = 114) could be created for comparison to the 74 IMPC mouse genes. These two groups of genes were analyzed using the STRING [1] biological database software to predict protein-protein interactions within and between the groups, and molecular functions were generated using the Panther tool [2] within Gene Ontology [1, 2, 3].
Animals
All institutions involved in this study operated under the regulation and accreditation from their national animal welfare committee [Institutional Animal Care and Use Committee (IACUC) or Animal Care Committee (ACC)].
RESULTS
Query of the International Mouse Phenotyping Consortium (IMPC) database identified 40 previously unimplicated genes linked to mammalian eye development. Of note, our analysis suggests that the serine-glycine pathway producing glycine, a mitochondrial one-carbon donator to folate one-carbon metabolism (FOCM), is essential for eye formation.
CONCLUSIONS
Using genome-wide phenotype screening and bioinformatic methods, this study identified genes and pathways heretofore unassociated with MAC phenotypes. These findings have the potential to hasten the diagnosis and treatment of this congenital blinding disease.
Evaluation of a Novel Targeted-Sequencing Panel for Detection of FZD4 Gene Variants in Subjects with Familial Exudative Vitreo-Retinopathy.
Drekh, Mary, B.S.1, Dailey, Wendy, B.S.2, Le, Victor, B.A.1, Krikor, Savyo2, Drenser, Kimberly, M.D./Ph.D.3, Mitton, Kenneth, Ph.D.2
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Oakland University, Eye Research Institute, Rochester, Michigan
3Associated Retinal Consultants PC, Royal Oak Michigan
INTRODUCTION
Identify potential pathogenic mutations in the Frizzled 4 gene (FZD4) in patients with clinically diagnosed Familial Exudative Vitreo-Retinopathy (FEVR). FEVR is caused by variants impacting several genes including the FZD4 gene. FZD4 is the central cognate receptor for Norrin WNT signaling in the human retinal endothelium. An initial cohort of 76 subjects diagnosed with FEVR, and near relatives, were sequenced using a custom Ampliseq panel that includes eight genes related to FEVR/ND and Retinoschisis (NDP, CTNNB1, TSPAN12, KIF11, FZD4, LRP5, ZNF408, RS1). An additional 34 subjects were sequenced more recently.
METHODS
A custom Ampliseq targeted panel (180 amplicons) for 8 genes was designed with Illumina's Design Studio. The targeted panel used three pools (PCR reactions) per patient sample for complete coverage of 83 exons with 25 bp of adjacent intron sequence. Targeted Genes were: NDP (ChrX), RS1 (Chr10); CTNNB1 (Chr3); TSPAN12 (Chr7); KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), and ZNF408 (Chr11). Ampliseq libraries were sequenced on the Illumina iSeq-100 platform. Variant impacts and allele frequency data were obtained from ClinVar and The Genome Aggregation Databases (gnomAD).
RESULTS
Results: 44 protein-altering variants were found in six FEVR/Norrie Disease related genes. Of note, 9/44 (20.4%) of the variants were present in the FZD4 gene. The 9 variants included 1 pathogenic, 1 novel pathogenic variant (Cys450Ter) and 3 of uncertain significance. 95.5% of the base reads were > Q30 quality, the percent on-target bases passing filer was 92.2%, and the average sequencing depth coverage for the entire panel was 978.
CONCLUSIONS
Our custom targeted-sequencing panel was developed to detect gene variants associated with FEVR, Norrie Disease, and Retinoschisis. The panel’s sequencing coverage was of sufficient depth to detect protein-altering variants in the FZD4 gene and to determine variant zygosity. To date, disease-causing variants of FZD4 were found in 5 subjects representing 4 different families.
Lipoprotein Particle Size and Function Predict New Cardiovascular Events in Patients with Chronic Kidney Disease
Alexander George, B.S.1, Anna Mathew, M.B.B.S.2
1Oakland University Wiliam Beaumont School of Medicine, Rochester, Michigan
2University of Michigan, Ann Arbor, Michigan
INTRODUCTION
Chronic Kidney Disease (CKD) is a risk factor for cardiovascular disease (CVD), and 40% of patients with CKD have CVD with greater than 10-fold mortality compared to healthy controls. Lipoprotein profiles predict CVD and CV events in the general population. However, the relationship between specific lipoprotein profiles and new CV events in patients with advanced CKD and cardiovascular burden is unknown.
METHODS
We profiled the distribution of lipoprotein size, particle concentration, and cholesterol and triglyceride content of the baseline plasma of 325 subjects with moderate CKD followed for 2.5 years using nuclear magnetic resonance (NMR) spectroscopy. We used Cox regression models controlled for various clinical factors to characterize the role of specific lipoprotein profiles in predicting CV events in this high risk population. The cholesterol uptake capacity of high-density lipoproteins (HDL) from peripheral tissues- cholesterol efflux capacity (CEC) and HDL oxidation were also quantified using standardized assays.
RESULTS
Patients with new CV events demonstrated increased HDL size, large HDL particle numbers, and CEC, while total LDL and VLDL particle numbers (in particular small VLDL particles) decreased. Increased HDL particle size [HR=2.56, p=0.002], large HDL particle numbers [HR=1.41, p=0.001], HDL-cholesterol levels [HR=1.03, p=0.008], and CEC [HR=1.46, p=0.03] predicted CV events. In contrast, an increase in small VLDL particles [HR=0.97, p=0.02] and LDL particles [HR=0.99, p=0.03] decreased the risk of CV events after adjusting for clinical factors.
CONCLUSIONS
Our study is the first to demonstrate that increased HDL particle size predicts new CV events in the CKD population with a high cardiovascular burden independent of CEC and HDL cholesterol. Collectively, the data strongly associates altered lipoprotein metabolism, in particular, HDL metabolism and new CV events in patients with established CKD and CVD, allowing us to risk stratify and reduce mortality and morbidity in this high-risk population.
Evaluation of a Novel Targeted-Sequencing Panel for Orphan Pediatric Retinal Diseases for detection of LRP5 Gene Variants
Daeun Jeong, B.S.1, Kenneth Mitton, Ph.D.1,2, Wendy Dailey, B.S.2, Kimberly A. Drenser, M.D./Ph.D.2,3
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Eye Research Institute, Rochester, Michigan
3Associated Retinal Consultants LLC, Royal Oak, Michigan
INTRODUCTION
Orphan pediatric retinal diseases which encompass Familial exudative vitreoretinopathy (FEVR), Norrie Disease, and Retinoschisis are associated with mutations in genes involved in the Wnt signaling network, including FZD4, LRP5, TSPAN12, and NDP. FEVR is a retinal disease that lacks normal vascularization and angiogenesis to the peripheral retina. LRP5 variants account for 10-25% of cases of autosomal dominant FEVR. However, not all cases have been linked to these existing variants. Next generation sequencing was used to sequence 76 subjects for novel variants to expand the spectrum of LRP5 variants and better understand the mechanism of pathogenicity.
METHODS
8 genes were run through a custom Ampliseq targeted panel (180 amplicons) which was designed with the Illumina’s DesignStudio Sequencing Assays Designer. The targeted panel was put into three pools (PCR reactions) per patient sample for complete coverage of 83 exons with 25 bp adjacent intron sequence. The targeted genes included: NDP (ChrX), RS1 (Chr10); CTNNB1 (Chr3); TSPAN12 (Chr7); KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), and ZNF408 (Chr11). Then the Ampliseq libraries were sequenced on the Illumina iSeq-100 platform. Through the use of ClinVar and The Genome Aggregation Databases (gnomAD), the variant impacts and allele frequency data was collected.
RESULTS
33 protein-altering variants were found in six FEVR/ND related genes. Of note, 13/33 (39.4%) of the variants were present in the LRP5 gene. The LRP5 gene is involved in the Wnt-signaling pathway associated with FEVR with mutations showing a lack of vascularization to the retina. For the variants, two are found to be pathogenic, 1 novel likely pathogenic and 1 novel variant of unknown significance.
CONCLUSIONS
Our custom targeted-sequencing panel was developed in order to detect variants in seven FEVR/Norrie Disease genes. We conclude that this panel provides sufficient depth of sequencing variants in the LRP5 gene and to determine zygosity of variants.
Combination of Protein-Altering Variants of FZD4 May Contribute to Severity of FEVR
Lance Jones, B.S.1, Mary Drekh, B.S.1, Kenneth P. Mitton, Ph.D.1,2
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Eye Research Institute, Oakland University, Rochester, MI
INTRODUCTION
Orphan inherited retinal diseases account for a significant portion of currently untreatable vision loss and blindness. The Norrin/beta-catenin signaling pathway, which is involved in the development of the neural retinal vasculature, has been implicated as a target for understanding the genetic origin of these diseases. This continuing study sequences a targeted panel of genes to identify protein-altering variants that contribute to these diseases. One specific family group is highlighted here involving FZD4 variants.
METHODS
Genomic DNA was extracted from frozen whole blood collected from subjects with familial exudative vitreoretinopathy (FEVR). Ampliseq targeted panels for 8 genes were prepared and sequenced using the Illumina iSeq-100 platform. Variant call files were analyzed, aided by the web-based Variant Effect Predictor (at Ensembl) and ClinVar database, to categorize the consequence of each protein-altering variant.
RESULTS
In the proband (female, age 3, FEVR grade 5) and mother (age 30, FEVR grade 1) of one family, a novel protein-altering variant of FZD4 (single nucleotide substitution NM_012193.4:c.1350T>A leading to the nonsense variant NP_036325.2:p.Cys450Ter) was detected that truncates the protein upstream of the motif involved in intracellular signaling. Additionally, a previously described pathogenic variant (single nucleotide substitution NM_012193.4:c.313A>G leading to the missense variant NP_036325.2:p.Met105Val) was detected in the proband. This causes a single amino acid substitution in the cysteine-rich domain required for Norrin binding.
CONCLUSIONS
Due to truncation upstream of the motif involved in intracellular signaling and the fact that this is the only known variant in the mother of this family, we have categorized the novel FZD4 variant as likely pathogenic. Due to the presence of two protein-altering variants within the proband and the proband's more severe presentation, we conclude that a combination of protein-altering variants of FZD4 or variable expressivity of one or both of these variants may contribute to the severity of FEVR.
Utility of UW-based solutions for reducing apoptotic signaling and retinal ganglionic cell death in a whole eye transplantation rodent model
Neil J. Khatter, B.S.1, Charles R. Owens, B.S.2, Bing Li, M.D.2, Yong Wang, M.D.2, An-Jey A. Su, Ph.D.2, Kia M. Washington, M.D.2
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2University of Colorado Anschutz Medical Campus
INTRODUCTION
Overcoming retinal ganglionic cell (RGC) death induced by ischemia reperfusion injury and optic nerve transection is necessary for whole eye transplantation (WET). Current organ transplants use preservatives like University of Wisconsin (UW) solution. We tested UW and a modified version including BaCl2 and valproic acid (mUW) as RGC preservation solutions in an orthotopic rodent WET.
METHODS
Untreated naïve eyes from Brown Norway rats (n=32) were harvested and retinas stained for Brn3a and phospho-c-Jun (PNJ) to quantify RGC preservation and apoptotic signaling, respectively. Donor eyes were injected with 10μL of saline (n=3), UW (n=5), mUW (n=5), or not injected (n=3) and hemifacial flaps with donor eyes were harvested for WET. Data presented as mean±standard error compared using one-way ANOVA and two-sample Z tests.
RESULTS
Naïve retinas expressed 102.4±3.2k Brn3a+ RGCs with 12% background expression of PNJ+ signal. At POD2, native recipient retinas show 107.8±4.0k Brn3a+ RGCs with 9% co-staining for PNJ+. In the untreated WET condition, retinas show 114.6k±5.6k Brn3a+ RGCs with 39% co-staining for PNJ+, a significant increase from naïve (P<0.05). In the saline condition, retinas show 90.4±8.8k Brn3a+ RGCs with 21% co-staining for PNJ+, a significant increase from naïve (P<0.05). UW treated retinas show 78.7±4.5k Brn3a+ RGCs with 1% co-staining for PNJ+. mUW treated retinas show 88.9±4.9k Brn3a+ RGCs with 2% co-staining for PNJ+. UW and mUW are not statistically different (P>0.05) and both provided a significant reduction in RGC apoptosis in the setting of WET at POD2 (P<0.05).
CONCLUSIONS
We have established a novel system to assess RGC preservation and apoptosis post-WET. Injecting UW-based preservation solutions represents a promising strategy for RGC survival post WET. UW and mUW reduced apoptotic signaling compared to no treatment and saline in donor eyes at POD2 after WET. Future research will include monitoring Brn3a+ and PNJ+ expression at other timepoints and analyzing other apoptotic markers.
Normative Percentile Ranking Best Reveals Sensorimotor Impairments of Postural Sway in Type 2 Diabetes
Michael Ko, B.A.1, Trevor Lopatin, M.S.2, Elise Brown, Ph.D.2, Daniel Goble, Ph.D.2, Joshua Haworth, Ph.D.2
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2School of Health Science, Oakland University, Rochester, MI, USA
INTRODUCTION
Type 2 diabetes mellitus (T2D) is a condition that is associated with higher magnitudes of postural sway often through long term complications of uncontrolled blood sugar such as diabetic peripheral neuropathy, diabetic retinopathy, and diabetic vestibulopathy. The primary goal of this investigation is to compare three statistical methods to explore the role of sensory modalities contributions to posture in T2D. A secondary goal is to compare these measures of postural stability to normative data matched to age and sex to determine if there is a significant deficit.
METHODS
10 participants with T2D (age 54.6 ± 11.09 years) where selected to undergo the modified Clinical Test of Sensory Integration in Balance (mCTSIB) consisting of four 20-s trials on a balance plate with manipulations of vision and support surface to target the contributions of proprioceptive, visual, and vestibular senses. These scores were compared to the normative data of 10 age/sex matched healthy participants (age 53.18 ± 9.89 years).
RESULTS
The two-way ANOVA used for the group wise analysis of path length and percentile rank showed significant differences between groups scores (p < .05), but no significant interactions between group and condition. The frequency distribution of percentile rank of the T2D group revealed unimodal distributions for all conditions except for vestibular, which was found to have the highest and lowest percentile ranks of any condition.
CONCLUSIONS
The results show that the individualized normative analysis revealed aspects of individual impairments that would have otherwise been missed using a group-wise method. Though limited, our findings also suggest that impairments to the vestibular system may be more pronounced but less frequent compared to proprioceptive and visual impairments.
Molecular characterization of KIF11 variants in FEVR
Konstantinos Koustas, B.S.1, Kenneth P. Mitton, Ph.D.1,2, Wendy Dailey, B.S.2, Kimberly A. Drenser, M.D./Ph.D.3
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Eye Research Institute, Rochester, Michigan
3Associated Retinal Consultants LLC, Royal Oak, Michigan, United States
INTRODUCTION
Detected potential pathogenic mutations in the KIF11gene among individuals clinically diagnosed Familial Exudative Vitreo-Retinopathy (FEVR). FEVR displays to genetic variations affecting multiple genes, including KIF11. KIF11, a motor protein, aids retinal vasculature growth. Loss of KIF11 function is linked to peripheral avascular neural retina. A specialized AmpliSeq panel, covering eight genes related to FEVR, Norrie disease, and Retinoschisis was utilized to sequence individuals diagnosed with FEVR probands and some relatives.
METHODS
An AmpliSeq panel was created through the Illumina Design Studio, incorporating a three-pool PCR approach for a total of 180 amplicons covering 8 genes and 83 exons, with adjacent intron sequences of 25 base pairs. The panel included seven FEVR genes and one Retinoschisis gene: NDP (located on ChrX), CTNNB1 (Chr3), TSPAN12 (Chr7), KIF11 (Chr10), FZD4 (Chr11), LRP5 (Chr11), ZNF408 (Chr11), RS1 (located on ChrX). The cohort included 76 samples, comprising both FEVR patients and unaffected relatives. Variant impact and allele frequency data were derived from ClinVar and gnomAD databases.
RESULTS
A high-quality sequencing performance was observed, with 95.5% of base reads > Q30 quality. The on-target bases passing filter reached 92.2%, with average sequencing depth coverage of 978. Six genes related to FEVR/ND were examined, revealing a total of 33 protein altering variants. Notably, the KIF11 gene accounted for 3 out of the 33 variants (9%). Two were classified as benign, while one (Glu129Ala) was of uncertain significance, and not documented in ClinVar. This variant was detected in the teenage daughter diagnosed with stage-1 FEVR.
CONCLUSIONS
The custom AmpliSeq targeted-sequencing panel provided sequencing coverage of adequate depth to identify protein-altering variants in the KIF11 gene.
NDP Gene Variants Causing FEVR and Norrie Disease Detected with a Custom Targeted Sequencing Panel
Andrew Santos, B.S.1,2, Wendy Dailey, B.S.2, Krikor Savyo, B.S.1,2, Christopher Alexopoulos, B.S.1,2, Vincent Le, B.S.1,2, Kenneth Mitton, Ph.D.1,2
1Oakland University William Beaumont School of Medicine, Rochester, Michigan
2Eye Research Institute, Rochester, Michigan
INTRODUCTION
To confirm the functionality of a novel orphan pediatric retinal disease panel for detecting variants in the Norrie Disease Protein (NDP) gene. Familial Exudative Vitreo Retinopathy (FEVR) and Norrie Disease (ND) can be caused by variants of the NDP gene. A cohort of 85 subjects diagnosed with FEVR/ND and close relatives were sequenced using a custom Ampliseq panel that includes NDP and seven other genes linked to FEVR/ND and Retinoschisis (CTNNB1, TSPAN12, KIF11, FZD4, LRP5, ZNF408, RS1).
METHODS
A custom Ampliseq panel for 8 genes was designed with Illumina's DesignStudio Sequencing Assay Designer. The targeted panel was distributed into three pools per patient sample for complete coverage of 83 exons with 25 bp adjacent intron sequence. The target Genes were: NDP, CTNNB1; TSPAN12; KIF11, FZD4, LRP5, ZNF408, and RS1 (for Retinoschisis). Ampliseq libraries were quality-controlled by capillary electrophoresis and sequenced on the Illumina iSeq-100 platform at a scale of 30-50 samples per run. Variant impacts and allele frequency data were determined using the Variant Effect Predictor (VEP), ClinVar, and GnomAD Databases.
RESULTS
A total of 40 protein-altering variants were found, six in the FEVR cohort and five ND cohort. Of note, 4/40 (10%) pathogenic or likely pathogenic variants were present in the NDP gene: NP_000257.1:p.His42Arg, NP_000257.1:p.Arg37Ter, NP_000257.1:p.Met32TrpfsTer9, and NP_000257.1:p.Leu13_Met19del. The sequencing data revealed 95.5% of the base reads were > Q30 quality, the percent on-target bases passing filer was 92.2%, and the average sequencing depth coverage was 978.
CONCLUSIONS
The custom Ampliseq targeted-sequencing Orphan Pediatric Retinal Disease panel was developed to detect genes associated with FEVR/ND and Retinoschisis. The panel’s sequencing coverage was of sufficient depth to detect protein-altering variants in the NDP gene, the primary gene for ND. Variants were identified causing Norrie Disease for three families, including one novel variant: NDP:Met32TrpfsTer9.
