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Functional genomic variant patterns in Caucasian patients diagnosed with idiopathic scoliosis: a controlled, observational study

Authors:

Abstract

Previous studies have identified various genetic variants associated with idiopathic scoliosis. However, only individual single nucleotide polymorphisms (SNPs) were described, making application to clinical practice difficult. The current study examined functional SNP groups, where individual SNPs were collectively categorized based upon a common metabolic function or pathway. The number and types of functional SNP groups were identified and compared between a group of Caucasian idiopathic scoliosis patients, and a group of Caucasian, non-scoliosis patients. Intergroup comparison showed a significant difference in 19 functional SNP groups out of 131 reported. Intragroup comparison of the scoliosis group found a statistically significantly higher average number of the 19 significant SNP groups in the scoliosis patients with curves above surgery threshold compared to those below. Within the scoliosis patient group, those patients above the recommended surgery threshold of 50° were found to have a higher average number of positive SNP categories when compared to patients below the recommended surgery threshold. It remains to be determined if these functional SNP group differences may explain some of the metabolic abnormalities reported in patients with idiopathic scoliosis. Further research should explore means of treating or supporting these SNP group impacts on metabolic function.
Mark W. Morningstar et al. Medical Research Archives vol 7 issue 9. Medical Research Archives
Copyright 2019 KEI Journals. All Rights Reserved
The
Functional genomic variant patterns in Caucasian patients diagnosed with idiopathic
scoliosis: a controlled, observational study.
Authors
Mark W. Morningstar,1 Clayton J. Stitzel,2 Megan Strauchman,3
Affiliations
1 Chiropractic Medicine, Natural Wellness & Pain Relief, Grand Blanc, MI 48439, USA.
2 Director, Lancaster Spinal Health Center, Lititz, PA 17543, USA.
3 Medical Director, Natural Wellness & Pain Relief, Grand Blanc, MI 48439, USA
Correspondence:
Mark W. Morningstar; drmorningstar@nwprc.com
Abstract
Previous studies have identified various genetic variants associated with idiopathic scoliosis.
However, only individual single nucleotide polymorphisms (SNPs) were described, making
application to clinical practice difficult. The current study examined functional SNP groups,
where individual SNPs were collectively categorized based upon a common metabolic function
or pathway. The number and types of functional SNP groups were identified and compared
between a group of Caucasian idiopathic scoliosis patients, and a group of Caucasian, non-
scoliosis patients. Intergroup comparison showed a significant difference in 19 functional SNP
groups out of 131 reported. Intragroup comparison of the scoliosis group found a statistically
significantly higher average number of the 19 significant SNP groups in the scoliosis patients
with curves above surgery threshold compared to those below. Within the scoliosis patient group,
those patients above the recommended surgery threshold of 50° were found to have a higher
average number of positive SNP categories when compared to patients below the recommended
surgery threshold. It remains to be determined if these functional SNP group differences may
explain some of the metabolic abnormalities reported in patients with idiopathic scoliosis.
Further research should explore means of treating or supporting these SNP group impacts on
metabolic function.
Keywords: Genomics; Idiopathic; Scoliosis
RESEARCH ARTICLE
Mark W. Morningstar et al. Medical Research Archives vol 7 issue 9. September 2019 Page 2 of 9
Copyright 2019 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/mra
Introduction
The etiopathogenesis of idiopathic scoliosis has
been the focus of many recent genetic studies.
Many of these studies only focus on one singular
genetic variation. Although the incidence of
scoliosis in adolescence is 2-3%,1 its occurrence
in relatives who have idiopathic scoliosis may
range from 9% in males to 29% in females,
suggesting a distinct genetic influence.2 For
example, studies have examined the LBX1
locus,3 which is responsible for spinal cord
differentiation and somatosensory signaling.4
Genomic variants causing abnormal spinal
ossification (GPR126),5 craniofacial
osteocartilaginous development (BNC2),6
vitamin D receptor polymorphism (VDR),7 and
leptin receptor polymorphism (LEPR)8 have also
been confirmed. However, methods or treatments
to minimize the impact of these genetic variants
have not been developed. Zaydman et al9
identified genomic variants relating to delayed
chondrocyte activation on the concave side of
vertebral growth plates in scoliosis, leading to
asymmetrical growth. Despite this information,
proposed treatments or solutions for these
genomic variants is lacking.
When metabolic or hormonal signalling is
examined in idiopathic scoliosis, many authors
have previously observed distinct variations in
such functions when compared with non-
scoliotics. Estradiol, for instance, has been
shown to inhibit chrondrocytes in vertebral
growth plates.10 In addition to these direct effects
on chondrogenesis, estrogen receptors seem to be
related to circulating levels of leptin, which in
turn up-regulates certain estrogen receptors, and
down-regulates collagen production.11 Abnormal
leptin bioavailability,12 and central leptin
resistance,13 have also been implicated in the
onset of scoliosis.
While many genetic variants such as those
mentioned above have been identified, little is
presently known about how to create medical
interventions that reduce or eliminate their
impact on the development or progression of
idiopathic scoliosis. Advances in genomic testing
have provided unique insights into many
diseases, and its increased accessibility to
clinicians has allowed them to apply this
information to their management strategies.
Ultimately, the ability to apply genomic
information into daily clinical practice seems
entirely dependent upon the genomic variants
examined. For example, Morningstar et al14
found that nearly 50% of idiopathic scoliosis
patients were either A1298C homozygous or
C677T/A1298C heterozygous positive for the
methylenetetrahydrofolate receptor (MTHFR)
variant, compared to about 20% of non-
scoliotics. The novelty of identifying this type of
genomic variant in idiopathic scoliosis patients is
that there have been functional medicine
strategies developed in efforts to minimize the
impact of this variant on downstream
physiology.15
In the present study, we evaluated the genomic
test results of a cohort of idiopathic scoliosis
patients and compared them to the same results
in a cohort of non-scoliotics. This genomic
testing looked at a series of variants for which
functional medicine treatments have previously
been created and published.16
Materials and Methods
All subjects whose provided their raw data had
previously provided online informed consent for
the evaluation of their data. This study was
granted IRB exemption through IntegReview
IRB. Subjects data were collected according to
the following inclusion criteria: 1) Subject had a
Mark W. Morningstar et al. Medical Research Archives vol 7 issue 9. September 2019 Page 3 of 9
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history of idiopathic scoliosis, 2) Subject had
previously completed a salivary DNA genomic
test, and 3) a copy of the subject’s raw DNA file
was forwarded to the authors for analysis. All
subjects had been diagnosed with idiopathic
scoliosis by current accepted radiographic
standards.17 An additional set of DNA files were
compiled from subjects who did not have a
history of idiopathic scoliosis. Many of these
DNA files were provided by non-scoliosis family
members of the scoliosis group, while the
remainder were non-scoliosis patients of the
authors (MNS, MWM). These subjects served as
the control group. All of the patients in the study
were Caucasian, and resided in the United States
Midwest and East Coast regions, comprising a
total of 6 states. The average of the scoliosis
group was 31 years, while the non-scoliosis
group was an average of 27 years. Patient
samples were collected beginning in 2018
through early 2019. All DNA files were analyzed
by an online genomic website, the NutriGenetics
Research Institute
(http://www.nutrigeneticresearch.org). The
software evaluated up to 7000 genes, and
produced a subject single nucleotide
polymorphism (SNP) report on specific genomic
variants associated with specific metabolic
pathways, such as methylation, transsulfuration,
detoxification, and mitochondrial activity. The
DNA samples were analysed using the Axiom
myDesign Genotyping Array (ThermoFisher
Scientific). These resultant SNPs were divided
into specific functional genomic groups based on
common metabolic function. Patterns of positive
homozygous or heterozygous genomic variants
were identified and recorded according to their
function and loci. These were recorded for both
the scoliosis group as well as the control group.
Once the analysis was completed, and the report
generated, various genetic polymorphisms within
specific functional genomic groupings were
identified. As an example, rather than to look at
just the MTHFR C677T or A1298C SNPs, 3
additional SNPs were included in the same
functional grouping. All 5 of these SNPs
collectively code for different functional aspects
of the folate methylation pathway. A total of 131
such genomic groups were analyzed for both
patient groups. Figure 1 illustrates an example of
two different SNP groupings, where two are
considered positive and the other negative.
Mark W. Morningstar et al. Medical Research Archives vol 7 issue 9. September 2019 Page 4 of 9
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Figure 1: These are examples of a functional genomic grouping that are positive (top, middle) and negative
(bottom).
Results
From the inclusion criteria listed, a total of 93
scoliosis subject DNA files were selected. The
non-scoliosis control group was comprised of
115 DNA files. The scoliosis group was
comprised of 12 males and 81 females. The
control group had 29 males and 86 females.
The average number of individual genes in the
131 functional genomic groups was 7. Since at
least 50% of the genes in a given functional
genomic group needed to have a positive variant
to consider the genomic group positive as a
whole, we conducted a power analysis to
determine if a sample size of 93 was enough to
establish a statistically significant effect size of
14% difference. This difference required a
Mark W. Morningstar et al. Medical Research Archives vol 7 issue 9. September 2019 Page 5 of 9
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minimum sample size of 51 patients in each
group. Among the 131 total genomic groups
evaluated, 19 total groups were significantly
more likely to be found in the idiopathic scoliosis
group than the control group. All genomic
groupings were analyzed in Microsoft Excel
using independent t-tests with unequal variance.
With a 95% confidence interval, each of the 19
groups attained a P value of <.05. Table 1 shows
the 19 genomic groups, along with their
respective P value.
Notable SNP differences on Intergroup comparison
SNP Group
Scoliosis (n=198)
P Value
FUT2
132
0.002331
HNMT
223
0.017452
PANK1
197
9.83094E-07
PANK2
134
0.000333
PANK3
PANK4
FADS1
FADS2
FADS3
SOD1
SOD2
SOD3
PON1
MTHFR
B12F
MAOA
COMT
BH4
VDR
202
37
117
111
159
85
142
166
103
143
153
161
144
144
133
0.047161
3.91E-05
0.003992
0.033005
0.032982
0.001746
0.033396
0.030746
0.0003344
0.035155
0.002058
0.013888
0.039539
3.189E-12
0.009082
Table 1: The SNP Groups that reached a statistically significant difference at P<.05.
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Once the 19 statistically significant functional
genomic groups were identified, the scoliosis
group was further broken down to see if there
was a difference between those with scoliosis
below 50°, compared to those with scoliosis
between 10-49 degrees. Among the 93 scoliosis
patients, 40 had curves below 50°, while the
remaining 53 had curves above 50°. Of those
above 50°, 29 had a positive past surgical history
of spinal fusion for their idiopathic scoliosis.
Figure 2 shows an intragroup comparison of the
scoliosis group and their respective number of
positive functional genomic groups when
categorized by curve severity.
Figure 2: Scoliosis intragroup comparison based on curve magnitude. Difference was statistically different with
a P value at 1.558 e-12
Patients with curves below 50° had an average of
9.7 positive functional genomic groups out of the
total 19 (range 6-14). Patients whose curves
exceeded 50° had an average of 14 positive
groups, ranging from 9-19. This intragroup
difference was statistically different at P<.001.
Discussion
Although previous studies have identified several
genetic variants in scoliosis, it is not clear how
these genetic variants directly influence the
spinal curvature.17 Although newer studies have
begun investigating the epigenetic influences of
certain environmental factors on the DNA
methylation of some of these genetic
variants,18,19 it has not yet resulted in a
translation to clinical scoliosis management.
The main difference between the present study
and previously published studies on scoliosis
genetics is that researchers have attempted to
create associations or causal relationships to
single gene variants. This has led to the
discovery of several isolated gene variants in the
literature that were singularly linked to scoliosis.
0
2
4
6
8
10
12
14
16
18
20
Above 50° Below 50°
High
Low
Avg
Mark W. Morningstar et al. Medical Research Archives vol 7 issue 9. September 2019 Page 7 of 9
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Some of these studies showed different singular
genes based on different subject ethnicities.20
This is the first study in which specific functional
groups of genetic SNPs have been investigated in
scoliosis patients. As such, it is unknown if or
how the present findings can be compared to
previously published scoliosis genetic variant
data. The present data is approached from a
functional basis. The goal of future studies will
be to evaluate the impact on scoliosis occurrence
and/or progression in those patients where these
functional genomic groups are identified, and
subsequent treatment interventions are utilized in
the hopes of minimizing the impact of the
functional genomic variants on some of the
downstream metabolic functions that may or may
not govern scoliosis etiopathogenesis.
Conclusions
Caucasian patients with a history of idiopathic
scoliosis, when compared with a non-scoliosis
Caucasian patient group, displayed a significant
number of functional genomic groups with a
majority of their respective genes having a single
nucleotide polymorphism (SNP). When
examining and subcategorizing the scoliosis
patient group based upon their curve severity,
those with curves below surgery threshold had a
lower average number of functional genomic
SNPs compared to those above surgery
threshold. This research may have implications
for predictive genomic testing and/or early
metabolic interventions for the identified
functional genomic groups.
Acknowledgements: None Declared
Author Contributions: MWM prepared the
manuscript, aggregated and analysed the data,
and submitted it for peer review. CJS contributed
data, performed a literature search, and assisted
in manuscript preparation. MNS contributed
data, assisted with statistical analysis, and
approved the final version of the article.
Conflicting Interests: None Declared
Funding: No Funding was received for study
Ethical Approval: This study was granted IRB
Exemption by IntegReview IRB (Protocol
number 2019-03-01)
Mark W. Morningstar et al. Medical Research Archives vol 7 issue 9. September 2019 Page 8 of 9
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Background: There is considerable discordance in the curve progression of adolescent idiopathic scoliosis (AIS) patients between monozygotic (MZ) twins, indicating that nongenetic factors must be involved in the curve progression of AIS patients. Epigenetic processes may constitute one of these factors and have not yet been investigated in relation to curve progression in AIS patients. Methods: The genome and methylome of peripheral monocytes were compared between MZ twins discordant for curve progression. Sets of differentially methylated sites were validated using the MassARRAY platform of Sequenome on additional samples. Results: In the discovery study, we found evidence suggesting a lack of differences at the genome sequence level and the presence of epigenetic differences related to the curve progression of AIS patients. The top 4 differentially methylated CpG sites associated with curve severity were tested, and only site cg01374129 (CpG site located at chr8:122583383, Hg19) was confirmed in two replication cohorts. The methylation levels of site cg01374129 were significantly lower in the progression group than in the nonprogression group. Cox regression analysis demonstrated that hypo-methylation of site cg01374129 was an independent prognostic factor for curve severity. Site cg01374129 methylation as a marker achieved a sensitivity of 76.4% and a specificity of 85.6% in differentiating between samples from patients with and without curve progression (AUC = 0.827; 95% CI: 0.780 to 0.876). Conclusion: Increased curvature is associated with decreased methylation at site cg01374129. Our results indicate that methylation of site cg01374129 may therefore serve as a promising biomarker in differing between patients with and without curve progression.
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