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Basic Concepts in Genetics and
Intervertebral Disc Degeneration
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8
CHAPTER
Introduction to Genetics
Genetics has a growing role in the eld of clinical medicine.
Genes are the fundamental make-up of any organism; thus,
understanding what genes and environmental factors contribute to certain disease traits/phenotypes are of interest. By
identifying the mutations in the genes that lead to disease,
focused management such as early detection, targeted therapies, or even disease prevention can be developed.
With genome sequencing and public access to the electronically compiled genome data, the Human Genome Project
allows scientists to identify genes of interest at particular
locations in the genome. is can allow researchers to use a
certain DNA sequence of a particular gene as an initial reference sequence to carry out mutation screening and permit
rapid identication of genetic markers within or adjacent to
genes of interest. is database of DNA reference sequences
can also help design primers that bind to a certain sequence
of the genome. e available data allow studies regarding
expression patterns in dierent tissues and sequence variations between subjects such as single nucleotide polymorphisms (SNPs).
Many genetic disorders have already been identied and
stored in the Online Mendelian Inheritance in Man (OMIM)
database. e pattern of how genetic information is passed
through subsequent generations is based on the Mendelian
laws of inheritance. Using modern technology, potential
genetic variants that predispose to disease can be determined.
For spine surgeons, disc degeneration and scoliosis are two
of the most sought aer conditions in terms of genetic background. Both conditions are likely inuenced by multiple
genes and various environmental factors. is chapter aims
to provide clinicians with a basic concept of genetics with
regard to the terminology and principles, with a specic focus
on disc degeneration and scoliosis. Readers may be able to
equip themselves with a general understanding and thereby
the ability to follow novel literature regarding advances in
genetics.
Chromosomes and DNA
Before discussing genetics and how to determine genetic variations, concepts of chromosomes, DNA, and polymorphisms
and Scoliosis
Jason Pui Yin Cheung
Kenneth M.C. Cheung
need to be understood. Any single human genome is comprised
of 22 pairs of homologous chromosomes with an additional
pair of sex chromosomes inherited from parents. A full set
of chromosomes is also known as diploid and a half set is
known as haploid. Chromosomes are made up of DNA, which
is a nucleic acid that carries genetic material. DNA contains
a super-phosphate with nitrogenous bases. e nitrogenous
bases include base pairs of adenine (A) and guanine (G)—
purines—and cytosine (C) and thymine (T)—pyrimidines.
Each molecule of DNA is comprised of two nucleotide chains
that are coiled in a clockwise fashion to form the double helix.
ese chains have two ends, called the 5′ and 3′ ends, and the
two nucleotide chains run in opposite directions, either from
5′ to 3′ or from 3′ to 5′. Base pairings in these chains are
obligatory A:T and G:C.
e main function of these nucleic acids is coding protein
synthesis. e basic architecture of proteins is comprised of
amino acids, and the order of sequence in which amino acids
are encoded judge the way the resulting protein is formed and
functions. ese proteins are encoded by DNA, and therefore
by its gene. DNA is double stranded, whereas ribonucleic acid
(RNA) is single stranded. In addition to this dierence, uracil
(U) replaces T in RNA sequences. Exons are sequences of
DNA that contain coding information, whereas introns are
noncoding sequences. DNA is used as a template to create
RNA in a process known as transcription. As any set of three
base pairs form the codes for an amino acid, the transcribed
messenger RNA (mRNA) encodes the information for a
certain protein with a specic amino acid sequence that is
imprinted by a certain gene. rough the transcription process,
the DNA strands separate and act as the template for the
enzyme RNA polymerase II to synthesize nucleotides in the
opposite direction of the DNA template (5′ to 3′ matched to
3′ to 5′). is process will include all introns and exons.
Despite being a noncoding region, introns have regulatory
functions during transcription. Introns are eventually removed
aer splicing; the remaining exons form mature mRNA.
e next stage in protein synthesis is known as translation, which occurs in the cell cytoplasm. e mRNA attaches
to a ribosome; each ribosome moves along the mRNA to
form a matching transfer RNA (tRNA), which will contribute
its specic amino acid to a growing protein chain until it
reaches a stop codon. Proteins are important for normal
I
133
134 BASIC SCIENCE
bodily functions, as they regulate dierent mechanisms both
intracellularly and extracellularly.
Genetic Variations
Despite having pairs of chromosomes, there are variations in
sequencing that distinguish between chromosomes. Variants
on the same location of paired chromosomes are known as
alleles. ey may be homozygous, meaning the same, or het-
erozygous, meaning dierent. Small variations can exist in
individual nucleotides. Larger variations include microsatellites, deletions, insertions, and copy number variations
(CNVs). All variations can cause signicant alteration of the
protein structure, thus gene expression and possibly disease
manifestation. However, these genetic variants can be manipulated into a genetic marker for identifying their location on
the DNA sequence. ese markers are important for studying
the causative relationship between a genetic variant and an
inherited disease.
SNPs are particularly of interest, as they are commonly
used for genetic analysis. ey are also commonly found
throughout the genome, comprising up to 90% of the genetic
variants. ey are 1-base pair (bp) substitutions of DNA
sequences that can occur anywhere in the genome. On average,
a SNP may be observed in every set of 300 nucleotides; thus,
there are approximately 10 million SNPs found in the entire
human genome.
Microsatellites or variable number of tandem repeats refer
to repeating short nucleotide sequences. Due to the variable
number of repeats in dierent chromosomes, it is a useful
marker for identifying dierent people. Deletion or insertion
of one or more base changes may lead to signicant changes
in DNA sequences, thus causing missing or extra amino acids
in any protein chain. A complete change in protein sequence
may result from more than 3 bp of sequence addition or loss.
ese usually cause serious genetic diseases. CNV refers to
large structural variations in DNA sequences. e “copy
number” refers to the number of duplications that occur. CNV
involves duplications of large segments of a certain chromosome that alters one or more genes. Inversions may also occur
as a result of reversal in a segment of chromosome, which may
cause complex genetic diseases.
Mutations and Polymorphisms
Mutations result from permanent damages to DNA or replication errors. e eects are variable: from fatal to only mildly
detrimental. If fatal, these mutations will not be inherited,
thus are very rarely found in the population. For mild cases,
individuals are likely to reproduce and encourage the mutation to be inherited, leading to an increase in prevalence in
the general population. Rare variants are more commonly
researched, as they are more likely to have positive ndings in
genetic research and usually are associated with more clinically
relevant diseases. ey are dened as variants with a minor
allele frequency (MAF) of less than 1%. Polymorphisms are
dened as MAF greater than 1%; among these, MAF greater
than 5% are common variants and those from 1% to 5% are
low-frequency variants. Polymorphisms rarely cause signicant external eects despite changes in the gene sequence.
Terminology and Types of Disease
Genotype is the denition of the status of two alleles or actual
denotation of the genetic data. In contrast, phenotype is the
observable expression of the subjects’ traits. Dierences in
genotypes contributed by polymorphisms lead to phenotypical variations between individuals. Expression can exist at the
molecular level in the form of protein expression or can be
more clinically notable, such as height dierences and symptoms. As the clinical expression of the disease relies heavily
on the genotype, being able to phenotype a disease carefully
is very important for any genetic analysis. Not all genotypical
dierences may manifest clinically, however, as this relies on
penetrance. Incomplete penetrance may not result in disease,
as the genotype is not fully expressed clinically. ese cases
are considered to be complex genetic disorders in which environmental factors must interact with the susceptibility genes
before developing disease. Many orthopedic conditions—such
as osteoarthritis, disc degeneration, and even scoliosis—are
types of complex genetic disorders. is is in contrast to Mendelian diseases, which are simpler but rarer forms of disease
caused by single gene mutations.
1,2
Usually, these are severe
diseases, such as osteogenesis imperfecta. Mendelian diseases
are usually predictable in inheritance patterns.
For complex disorders, meticulous qualitative and quantitative phenotyping is important to classify disease severity.3 For
example, in disc degeneration, most phenotyping relies on
magnetic resonance imaging (MRI) which is used to assess
the disc’s hydration status, any bulging discs or herniation, and
Schmorl’s nodes or endplate irregularities. Qualitatively, whether
there is disc degeneration is evaluated. However, quantitative
evaluation is more important in these complex disorders, as
the phenotype may alter in severity and over time, such as
with the degenerative process. On the MRI, Schneiderman’s
and Prrmann’s grading describe the signal intensity of the
nucleus pulposus on T2-weighted MRI. Schneiderman’s grading
describes the signal intensity of the nucleus pulposus by four
grades (grade 0 indicating a normal disc and grade 3 indicating
hypointensity with disc space narrowing).4 Prrmann’s classication evaluates the homogeneity of disc structure, signal intensity,
distinction of nucleus pulposus and anulus brosus, and disc
height by ve grades (grade 0 indicating a homogeneous disc
structure, hyperintense signal, and normal disc height with
grade 5 indicating inhomogeneous disc structure, hypointense
signal, loss of distinction between nucleus pulposus and anulus
brosus, and collapsed disc space).
5
Gene Mapping
Gene mapping must be performed prior to claiming that a
disease is caused by a certain gene. Accurate mapping helps
us understand the etiology and pathogenesis, and identies
genes that may be manipulated into targeted therapies. e
method for gene mapping works dierently for Mendelian
versus complex diseases. In Mendelian diseases, due to the
Chapter 8 Basic Concepts in Genetics and Intervertebral Disc Degeneration and Scoliosis 135
rarity of the contributing gene variations, most are identied
by studying aected families. Family genetics allow visualization of the mode of inheritance, and can help locate the
position of the genetic variant. Although there are limitations
in this analysis because family members are exposed to similar
environmental factors and may mask some real genetic factors,
examining monozygotic (identical) twins may help analysis
for purely genetic disorders since they should both have
disease. However, if both monozygotic and dizygotic (nonidentical) twins both have similar disease rate (concordance
rate), then it is likely that shared environmental factors are the
major factor instead. In complex genetic diseases, there is
contribution from multiple genes; with their complex interactions with the environment, special mapping designs are
required. For family-based designs, linkage analysis will be
used. For population-based designs, association analysis or
biologically relevant candidate gene analysis are used.
Linkage Analysis
Preferably, linkage analysis has the greatest yield with large
families and multiple aected members. Genotyping for
microsatellite markers is performed to locate disease genes
that are nearby (Fig. 8.1). e premise of linkage analysis is
that recombination is less likely to occur between the disease
gene and the designated genetic marker since their positions
are nearby, allowing “linkage” of the disease gene with the
marker. In other words, aected members of the same family
are likely to pass on this region of the genome with the disease
gene. If sucient microsatellite markers are available to cover
the whole genome, linkage analysis can help locate the diseased gene even if it is unknown at the outset. However, this
is dependent on the distance between the disease gene and
marker. With a large distance, it is likely that recombination
may have occurred in between. us, the rate of recombination can help calculate the distance between the diseased gene
and marker used. In general, a 1% recombination rate (θ) is
represented as 1 centimorgan (cM) apart or approximately 1
million bp distance.
One of the main tests used is the parametric linkage analy-
sis, in which the test hypothesis (true linkage of the diseased
6,7
gene to the marker) is tested against the null hypothesis (no
linkage). Sequential recombination rates are performed to
compare the likelihood of either hypothesis. is is known as
likelihood ratio or odds. Using a logarithm to base 10 of this
ratio, the logarithm of odds (LOD) score can be calculated.
e highest LOD score represents the likely distance between
the disease gene and marker.8 LOD is an important parameter
to decide whether a nding is signicant or not; a score of 3.3
is required to achieve genome-wide signicance.9 is LOD
score can be strengthened by combining results from dierent
studies with the same disease model.
With linkage analysis, prior knowledge of the position of
the disease gene is unnecessary since it can be determined by
linkage with microsatellite markers. us, it is most useful in
diseases in which only a small number of genes are involved.
e main limitation is its inability to detect common alleles
without a strong inuence on the disease. erefore, in
common conditions with multiple gene and environmental
contributions, such as disc degeneration, association studies
are preferred.
Association Studies
Population-based gene association studies aim to identify
alleles associated with a single trait across the population.
Although it is similar to linkage analysis, in that it identies a
disease gene in subjects with a common ancestry (population
based instead of family based), it assumes that the “linkage”
distance between the marker and disease gene of interest is
extremely close so that recombination over generations would
not aect its position. us, positive association represents
that a particular disease gene is overexpressed in diseased
individuals as well as underexpressed in normal subjects.
Association studies can be classied as either direct or
indirect.10 Direct association studies target the variants that
have functional eects, which leads to disease. Although successful identication of a predisposing allele shows powerful
association, the chance of positive identication is low. In
indirect association studies, the association between the
marker and disease gene is targeted. is association relies on
the concept of linkage disequilibrium (LD), that is, because
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I
Disease-causing variant(s)
Can be SNP4
SNP4
chr22 (q11.21) q11.21 q12.1 12.2 22q12.3 22q13.31q13.1 q13.222p13 22p12 p11.2
Or adjacent
FIG. 8.1 SNP4 is the disease-causing variant. In linkage analysis, markers nearby (or adjacent in this gure) to
the disease variant can be easily picked up, as recombination is unlikely to have occurred in between.
136 BASIC SCIENCE
B
A
markers are near the disease gene, they are associated and
thus lead to higher frequency of disease than expected.
Identifying this marker will suggest that a disease-causing
variant is nearby, helping to narrow down the search for the
disease gene.
Association studies can be conducted via a candidate-gene
or genome-wide association type of approach. Candidate-gene
approaches utilize possible disease genes that are previously
identied or are in relation to the disease pathway and directly
screen the individuals for these disease genes using markers,
which are usually selected SNPs. Genome-wide association
studies (Fig. 8.2) adopt a similar principle as candidate-gene
studies, but, rather than testing for possible single genes, a
SNP mapping of the entire genome is performed. All possible
SNPs are tested for association with the disease by comparing
the frequencies of occurrence between cases and controls.
Conventionally, the threshold for genomewide signicance
is 5 × 10−8. Technological advancements with the addition
of DNA genechips allow for this scale of study.11 e cost
and time spent to perform these studies are now much more
reasonable.
Newer Technologies
With the advancement in sequencing technologies,12 whole
human genome or whole exon (1%–2% of the human genome)
FIG. 8.2 (A) Genome-wide association studies pick up all single nucleotide polymorphisms (SNPs) between
cases and controls. (B) Manhattan plots are used to dene which SNPs are signicant by logarithm to base 10
of the observed P value.
Chapter 8 Basic Concepts in Genetics and Intervertebral Disc Degeneration and Scoliosis 137
sequencing is becoming more aordable and feasible. is
allows assessing of all DNA variations in the genome, such
as CNVs instead of only SNPs or microsatellites. Exome
sequencing is used for sequencing the protein-coding genes
in a genome. us, only the DNA that encodes for proteins
is sequenced. is technique is a simpler approach to rare
variants. Since diseases are caused by these rare variants,
targeting the protein coding sequence usually has a high
yield for identifying the causative variants. Because of this, an
understanding of the clinical implication of the disease and
the protein sequence that is defective is required. is is in
contrast to whole-genome sequencing, which determines the
entire DNA sequence of an individual’s genome. More than
95% of the genome is thus genotyped, allowing information
on the individual’s genetic susceptibility to diseases to be
generated. Despite its wide coverage and reduced cost with
technological advancements, it is still comparably the most
expensive sequencing technique.
Interpretation of Results
Using association studies, possible signicant results may be
generated in the form of direct or indirect association or falsepositive results. Direct association indicates that the genotyped
polymorphisms are the true causal genetic variant leading to
disease. Indirect association indicates that the polymorphisms
are in LD with the true variant. A false-positive result is usually
due to population stratication, suggesting that dierences in
allele frequency exist between subpopulations of the subject
population, indicating that these individuals are of a dierent
ancestry.
As with all statistical analysis, genetic testing to determine
whether variants are associated with disease susceptibility
requires certain P values. e null hypothesis with regard to
P value is the possibility of no association in genotype distribution between cases and controls. Similarly, this hypothesis
of no association is rejected with a P < .05. Special tests may
be adopted in candidate-gene or genome-wide association
studies since all variants undergo study. Increased falsepositive rates exist with increased hypothesis testing. Bonferroni correction is one of the most common approaches13 in
these studies. e corrected statistical signicance level is 1/n
times what is expected from only one variant testing. erefore, the signicance threshold is 0.05 divided by the number
of markers under testing.
Disc Degeneration Genetics
ere has been a gradual shi in understanding of disc degeneration from purely a reaction to aging and prolonged
mechanical load to a more complex interaction between
genetics (Table 8.1) and environmental factors.14 Earlier
studies suggest contributions of age, gender, occupation, cigarette smoking, and increased height and weight.15 Using
familial studies, several genetic variants have suggested this
relationship, as young patients may also develop disc degeneration.
16,17
In addition, a familial link can be generated due to
similarities (26%–72%) found between identical twin pairs.18
With analysis of monozygotic twins, up to 61% of the genetic
variance can be explained by familial aggregation with only
limited (16%) contribution by age and mechanical loading.19
Subsequent twin studies suggest that 74% of disc degeneration
is heritable aer adjustment for age, body weight and height,
smoking, occupation, and degree of physical exercise.
As compared to spine deformity, in which the phenotype
is clear, disc degeneration is overall more subjective for diagnosis (Fig. 8.3). ere is a wide range of symptoms, severity,
and presentations, with particularly complex MRI features
including loss of nucleus pulposus signal intensity, disc herniation and bulging, endplate irregularities and Schmorl’s
nodes, osteophyte formation, and disc space narrowing, Modic
changes, and high-intensity zones. Some very young individuals may develop severe disc degeneration, while some of the
elderly may have normal discs. Clinical presentation is especially variable as not all individuals with “black” discs develop
back pain.
Despite evidence of heritability suggested by studies
of twins, limited validation studies exist for the identied
common variants. Considering the substantial population
with disc degeneration, causality is still unclear with many
unknown common variants still requiring identication. Most
of the current studies focus around candidate-gene analysis
with common SNPs. However, there is an increasing interest
in rare variants due to their role in complex diseases.
Via linkage study, a novel susceptible variant in carbohydrate sulfotransferase 3 (CHST3) is found to be associated
with early-onset disc degeneration.21 is is accomplished by
genotyping candidate regions on chromosomes 1, 5, 8, 10,
and 20. A follow-up epigenetics study detected a reduction
in expression of CHST3 mRNA in intervertebral disc cells
of individuals carrying the A allele of the SNP rs4148941.
By understanding the biologic pathways leading to the disc
degeneration phenotype, probable candidate genes can be
used to identify possible disease variants. e extracellular
matrix is an integral part of the disc architecture; thus, genes
encoding structural proteins including collagen and aggrecan are good candidate genes to analyze with regard to disc
degeneration.
e vitamin-D receptor (VDR) has been most commonly
replicated in dierent population cohorts, thus is the most
robust of all associated genes under study. A Finnish twin
study22 rst identied TaqI and FokI polymorphisms manifesting as reduced signal intensity of the disc on MRI. TaqI
has been replicated in a Japanese cohort23 and a Chinese
population-based study.24 e next stage of functional validation has yet to be performed, however. e expression of this
variant is likely extracellular matrix alterations.
e gene ACAN encodes aggrecan, which is responsible
for maintaining disc hydration as it is the major proteoglycan
contributing to the cartilage and nucleus pulposus structure
of the intervertebral disc. Variable number tandem repeats
in ACAN is associated with disc degeneration in a young
Japanese cohort,26 which has been replicated in the Han
Chinese,27 Korean,28 and Turkish29 populations. A greater risk
of symptoms has been observed in smokers (odds ratio [OR]
20
25
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138 BASIC SCIENCE
TABLE 8.1 Genetic Variants for Disc Degeneration
Gene Protein Cohort/Study Population Size (N) Variant Reference
ACAN Aggrecan Japanese (64) Variable number tandem repeats 26
Chinese (132) 27
Korean (104) 28
Turkish (100) 29
ASPN Asporin Japanese (1353) D14 allele 3
Chinese (1055)
CHST3 Carbohydrate sulfotransferase 3 Japanese (23,136) Chinese (6088) rs4148941 21
Finnish (6069)
CILP Cartilage intermediate layer
protein
COL11A1 Type XI collagen Japanese (1852) rs1676486 30
COL1A1, COL1A2 Type I collagen Dutch (517) rs1800012 35
COL9A2 Type IX collagen Finnish (966) Trp2 31
COL9A3 Type IX collagen Finnish (492) Trp3 33
GDF5 Growth dierentiation factor Northern European (5259) rs143383 38
IL6 Interleukin 6 Finnish (538) rs1800795
MMP1 Matrix metalloproteinase-1 Chinese (691) –1607 promoter (G to D allele) 40
MMP2 Matrix metalloproteinase-2 Chinese (480) –1306 promoter (T to C allele) 41
MMP3 Matrix metalloproteinase-3 Japanese (109) MMP-3 promoter (5A5A and 5A6A) 42
MMP9 Matrix metalloproteinase-9 Chinese (859) –1562 promoter (C to T allele) 43
PARK2 Parkin (E3 ubiquitin ligase) Northern European (4600) rs926849 47
SKT Sickle tail Japanese (1758) rs16924573 45
THB2 Thrombospondin-2 Japanese (1743) rs9406328 44
VDR Vitamin D receptor Finnish (85 twins) TaqI, FokI 22
Finnish (538) rs2073711 46
Chinese (804) 32
46
rs1800797
Finnish (538) 46
Japanese (205) TaqI 23
Chinese (804) TaqI 24
= 4.5), suggesting further interactions with environmental
factors.
Collagen is another structural protein that has been commonly studied. Type XI collagen encoded by COL11A1 (SNP:
rs1676486; T-allele) has been suggested in a Japanese study to
be associated with disc herniation and sciatica due to destabilizing mRNA.30 Trp2 allele is a rare mutation of COL9A2 (type
IX collagen) and is suggested to be a disease-causing mutation
in a Finnish family linkage study.31 is association is replicated in the Chinese population, in which its frequency is even
higher.32 Trp3 is another variant suggested to be causative in
a Finnish population,33 but is not replicated in the Chinese32
or Southern European34 cohorts. COL1A1 and COL1A2 are
two genes encoding collagen type I, and a SNP (rs1800012) is
identied to be associated with disc degeneration.35 However,
no large-scale study has replicated these results.
Other degeneration phenotypes, such as osteoarthritis,
may have similar variants as disc degeneration. For example,
associations with the gene ASPN (Asporin) have been observed
in Asian cohorts.3 Asporin is an extracellular matrix protein
that contributes to knee osteoarthritis. Growth dierentiation
factor (GDF5) is also a commonly used candidate gene for
osteoarthritis36 as it is important for joint formation,37 but
has also been shown to lead to disc degeneration. A SNP
(rs143383) is related to disc space narrowing and osteophyte
formation.38 In a meta-analysis with regard to rs143383, a
signicant association can be detected among women for this
phenotype.
38
Matrix metalloproteinases (MMPs) are important proteins
that are expressed in intervertebral discs. us, MMPs have
increased enzymatic activity and increased expression in
degenerated disc cells.39 MMP1, MMP2, MMP3, and MMP9
have been linked to disc degeneration. In a cohort of Southern
Chinese subjects, the signicance of the MMP1 variant is
found only in subjects older than 40 years.40 A signicant
SNP located at the promoter region of MMP2 is associated
with severe disc degeneration.41 Polymorphisms of MMP3
may lead to the onset and progression of disc degeneration.42
Finally, for MMP9, a SNP at the promoter region may also
be associated with disc degeneration.43 All of these ndings
are in Asian cohorts and have yet to be replicated in other
ethnicities.
rombospondin-2 genes (THBS2) have also been studied
in Japanese cohorts as possible candidate genes. A signicant
Chapter 8 Basic Concepts in Genetics and Intervertebral Disc Degeneration and Scoliosis 139
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FIG. 8.3 Importance and variations in phenotypes observed in disc degeneration. The left T2-weighted
magnetic resonance image (MRI) shows a relatively normal phenotype with signal intensity of the nucleus
pulposus. The center MRI shows multiple discs with loss of signal intensity and disc height, bulging and
high-intensity zone anteriorly at L4–L5. The right MRI shows discs with normal signal intensities but multiple
endplate irregularities and Schmorl’s nodes.
SNP (rs9406328) has been proposed44 to be related to regulation of MMP expression in the disc. e combined eects of
THBS2 and MMP9 variants amounts to an OR of 3.3 for disc
degeneration.
44
Sickle tail (SKT) gene polymorphisms have been analyzed
in a Japanese cohort.45 Here, a signicant SNP (rs16924573)
observed has been replicated in the Finnish population.46 e
risk of lost signal intensity at the nucleus pulposus and the
SKT SNP has been established (OR = 0.27 [95% CI, 0.07–0.96],
P = .024).46 However, further functional studies of this rela-
tionship are required.
A recent large-scale (4600 subjects) genome-wide association study of a Northern European cohort suggests that the
SNP (rs926849) of the PARK2 gene (P = 2.8 × 10−8) is also
associated with disc degeneration.47 Polymorphisms have also
been found for CILP (rs2073711) and IL6 (rs1800795 and
rs1800797) genes via association study.
46
From the summary of genetic studies with regard to lumbar
disc degeneration, many genes have been reported, but just a
few have been replicated with dierent cohorts and ethnicities.
A systematic review of disc degeneration association studies14
suggests that most of the reported studies have only a weak
level of evidence. It is thus likely that cross-cohort validations
are required in subsequent studies to raise the signicance of
reported results.
Scoliosis Genetics
Early-Onset Scoliosis and Congenital Scoliosis
Early-onset scoliosis (EOS), according to the Scoliosis
Research Society, is a lateral curvature of the spine that is
diagnosed before the age of 10. us, any diagnoses can fall
under this category including neuromuscular, syndromic,
congenital, and idiopathic scoliosis. With regard to idiopathic
scoliosis, both infantile (ages 0–3 years) and juvenile (ages
4–10 years) idiopathic scoliosis are considered EOS. Due to
the variable presentations of EOS, little evidence is available
in regard to their inheritance. For idiopathic scoliosis, subjects
that are younger commonly are boys as compared to older age
groups, in which girls are more commonly aected.48 Olderonset scoliosis, in particular, has a higher incidence among
relatives.
e occurrence of congenital scoliosis is usually sporadic,
with an incidence of 0.5 to 1 per 1000 live births.
ogy is likely to be multifactorial, with contributions from both
genetic and environmental factors. Vertebral anomalies may
arise from the fetal development with mothers exposed to
environmental factors such as hypoxia, hyperthermia, carbon
monoxide, and alcohol exposure.51 Gestational hypoxia, in
particular, has been shown to potentiate abnormal broblast
49,50
Its etiol-
140 BASIC SCIENCE
TABLE 8.2 Genetic Variants for Congenital Scoliosis
Gene Function Cohort/Study Population (N) Variant/Location Reference
JAG1 Notch signaling pathway United States (4 families) Chromosome 20p12 58
PAX 1 United States (48) Chromosomes 20, 21 64
DLL3 United States (50) Chromosome 19 63
United Kingdom (3 families) Chromosome 19q13.1-q13.3 57
TBX6 T-box 6 China (254) rs2289292 rs3809624 62
TABLE 8.3 Genetic Variants for Adolescent Idiopathic Scoliosis
Linkage Cohort/Study Population (N) Reference
Chromosomes 6p,10q,18q United States (1 family) 72
Chromosome 19p13.3 Chinese (7 families) 73
Chromosome X United States (202 families) 75
Chromosomes 5p13, 13q13, 13q32 United States (7 families) 76
Chromosome 17p11 Italy (1 family) 77
Chromosomes 9q31.2-q34.2, 17q25.3-qtel United States (10 families) 78
Gene Protein Cohort/Study Population (N) Variant/Location Reference
CHD7 Chromodomain-helicase-DNA-binding
protein 7 (also associated with CHARGE
syndrome)
MATN1 Matrilin 1, cartilage matrix protein Italy (81 families) Chromosome 1p35 81
MTNR1b Melatonin receptor 1B Chinese (1465) rs4753426 82
Xbal Estrogen receptor Japanese (304) Chromosome X 85
CHL1 Neural cell adhesion molecule L1-like protein
(Robo3 related)
LBX1 Ladybird homeobox 1 Japanese (1376 families) rs11190870 90
GPR126 G protein–coupled receptor 126 Japanese (1819 cases) rs6570507 94
France (10) Intron 2 79
United States (52 families) Exons 2–4
Chromosome 8q12
Chinese (376) 86
United States (419 families) rs1400180
rs10510181
ICSG (6 Asian, 3 non-Asian cohorts) 93
80
89
ICSG, International Consortium for Scoliosis Genetics.
growth factor (FGF) signaling in mice, which thus develop
congenital scoliosis.
52
e phenotype of congenital scoliosis includes vertebral
and rib malformation caused by failure of segmentation or
formation in utero. Overall, the genetic understanding (Table
8.2) of this condition is still limited, with only a few signicant
association studies performed. Complex interactions between
the signaling pathways such as FGF, Wnt, and Notch, occur in
the embryo to form vertebral bodies from somites.53 Various
notch pathway genes. including MESP2,54 LFNG,55 and HES7,56
have been identied to trigger normal somite segmentation
and vertebral development in mice. Any mutation in these
genes alters the pathway of vertebral development and may
lead to anomalies. e association is more complex in humans
due to the less predictable vertebral and rib malformations.
Several gene variants of the Notch pathway have been identied in spondylocostal dysostosis57 and Alagille syndrome.
58,59
However, the actual gene and protein mechanisms responsible
for the phenotype representation are still unknown; thus,
these mutations may not be the sole contributory element. Via
animal studies, several human candidate genes from the Wnt ,
FGF, and Notch signaling pathways have been identied.
PAX 1, DLL3, and TBX6 are candidate genes that have been
studied using association analysis.
61–65
One study on Han
Chinese subjects comparing patients with congenital scoliosis
with normal spines yielded two SNPs of the TBX6 gene
(rs2289292 and rs3809624) to be in strong LD (LOD = 57.48),
suggesting that these rare variants play important roles in the
development of congenital scoliosis.
62
Adolescent Idiopathic Scoliosis
As compared to EOS, adolescent idiopathic scoliosis (AIS)
involves patients older than 10 years of age. AIS usually
involves girls and with right-sided thoracic involvement
instead of the opposite in early-onset idiopathic scoliosis.
It is the most common pediatric spinal deformity, aecting
2% to 3% of children.66 ere is an adequate sampling for
genetic studies (Table 8.3) as shown by more research in this
area as compared to EOS. Original twin studies supported a
genetic etiology in AIS.
characteristic may dier and may not aect every generation.
48,67
Within AIS families, the disease
49,60
Chapter 8 Basic Concepts in Genetics and Intervertebral Disc Degeneration and Scoliosis 141
Similar to other spinal deformities, it is a complex trait that
likely involves more than one gene.
In a further assessment of AIS family history, one study
found that 97% of AIS patients have familial origins.68 e
genes that contribute to AIS are likely to exist with dierent
types of expression and penetrance, which explains why some
subjects have more orid phenotypes than others despite
sharing a similar gene pool. is suggests that only up to 30%
male and 50% female carriers of the disease variant develop
more pronounced scoliosis.
69
Based on family studies, linkage analysis and association
studies are conducted to identify the disease susceptibility
genes. Previous reviews on familial AIS genetics have been
published
70,71
with identication of signicant linkage regions
specically located on chromosomes 6, 10, and 18. Large
family genetics showed the highest LOD score on chromosome 18.72 is prompted further study into AIS families,
which found a plethora of susceptibility areas in the genome
that might give rise to scoliosis. Some found linkage areas with
a LOD score of 3.63 on chromosome 19p13.3.73 is region
was veried in a subset of families with probands having Cobb
angles of 30 degrees or greater.74 Others reported the X chromosome with a maximum LOD score of 1.6975 and specically
kyphoscoliosis to be associated with linkage at chromosomes
5 and 13.76 Other positive ndings were observed with marker
D17S799 (LOD 3.20) in a three-generation Italian family,77
and linkage at marker D9S2157 of chromosome 9q (LOD
3.64) and at marker AAT095 of chromosome 17q (LOD 4.08)
in the British population.
78
Overlap of genes associated with congenital abnormalities
is also observed in AIS subjects. e CHD7 gene has been
found to be associated with CHARGE syndrome during gene
mapping.79 e CHD7 gene is known to be expressed in undif-
ferentiated neuroepithelium and in neural crest mesenchymal
cells. It is found in the dorsal root ganglia, cranial nerves, auditory area, pituitary area, nasal tissues, and neural retina near
the end of the rst trimester. Gao et al.80 identied an A to G
SNP in intron 2 of the CHD7 gene that disrupted a transcription factor binding site associated with late-onset idiopathic
scoliosis. Resequencing of the CHD7 gene veried potential
functional polymorphisms that may disrupt this transcription
factor binding site, suggesting an etiologic overlap between
CHARGE syndrome and idiopathic scoliosis.
80
Using genome-wide association studies, several genes have
been identied as potential susceptibility genes. An allele of a
microsatellite marker in the MATN1 gene is overtransmitted
from parents to aected probands, suggesting a link to familial
idiopathic scoliosis.81 Using a chicken pinealectomy model,
melatonin deciency is observed as a possible disease mechanism for AIS. e melatonin receptor 1B (MTNR1B) has also
been identied as a possible candidate gene in a study on
Chinese AIS subjects.82 A “C-C” genotype with a promoter
SNP (rs4753426) signicantly increases the risk of AIS (OR,
1.29). ese results, however, are not veried in Japanese and
Hungarian cohorts.
83,84
e Xbal polymorphism of the gene
encoding the estrogen receptor has been linked to curve severit y.85 Although this association is observed in a Chinese
dataset,86 this has not been replicated in other studies.
87
Several contributing variants have been detected by linkage
analysis. However, detecting disease genes related to AIS
remains limited. Complex genetic disorders such as AIS are
expected to be associated with multiple gene variants with
only moderate eects of each. In view of this, linkage analysis
may be limited in detection of all genes, and association
studies may have better success.88 In addition to larger sample
sizes to obtain signicant ndings, identied disease genes
require verication in other populations and ethnicities.
Using genome-wide association studies, several recent
ndings are worth mentioning. e SNPs (rs10510181) near
the CHL1 gene89 and (rs11190870) near the LBX1 gene90 have
been identied and replicated in the Chinese population.
91,92
Several Asian and non-Asian cohorts have also veried
rs11190870 in a meta-analysis and yielded P values of 1.22 ×
–43
10
for both genders and 2.94 × 10
–48
for females.93 is is
the rst susceptibility locus for AIS that is replicated in several
populations. A third signicant SNP (rs6570507) has also
been detected in a Japanese population to exist in the GPR126
(encoding G protein–coupled receptor 126) gene, which has
been replicated in Chinese and European cohorts.94 ese
SNPs are suggested to have ORs of 1.2 to 1.4 for AIS susceptibility. ese association studies are now the go-to method
for identifying gene variants; further studies are expected to
be generated in a similar manner.
Conclusions and the Future
is chapter is an introduction into the eld of genetics. It acts
as a basic guideline for clinicians to understand the genetic
jargon and available evidence with regard to genetic susceptibility genes identied for disc degeneration and scoliosis.
Numerous genetic studies have been carried out to locate
susceptibility genes responsible for development of lumbar
disc degeneration and scoliosis. As both are complex diseases,
studies have identied multiple gene interactions as well as
dierent risk patterns according to exposure to environmental
factors such as aging and smoking for disc degeneration. With
increasing demand and interest for higher-level genetic
studies, the advancement of genotyping and sequencing
technologies have followed suit, with the more complex and
stronger association studies and exome-sequencing methods.
Due to these advances, better understanding of complex
disorders is possible.95 However, there are still signicant limitations in our current understanding. Very few of the susceptible genes are replicated, and many of their functions are
unknown. Before functional studies can be performed by
testing gene knockout in animal models, larger sample sizes
with cross-validation of dierent cohorts are necessary to have
a more accurate evaluation of possible genetic variants to
avoid testing incorrect and false-positive polymorphisms.
us, multicenter and multiethnic genetic studies with international collaborations are inevitable to produce results of
higher signicance. To ensure standardization among research
groups, phenotypes should be dened properly with good
reliability of assessment. In addition to the study phenotypes,
gene–gene and gene–environmental interactions must be
SECTION
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142 BASIC SCIENCE
assessed and accounted for during analysis. is is a necessary
process for all complex genetic diseases.
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