3 - ENABLING TECHNOLOGY
Genetics and Proteomics: Phenotype Issues and Methodological
Sleep behavior is
extremely variable across and within animal species, suggesting
the importance of genetically based differences. Limited genetic
epidemiological data indicate that many sleep disorders have
a strong genetic component. Advances in genetics and genomics
have been spectacular and include sequencing the genomes of
various organisms and high throughput studies using genetic
arrays and polymorphic markers. Animal models of sleep and circadian
disorders with selected genetic alterations are now being generated.
Similar developments in the area of protein characterization
and the more general field of proteomics are now rapidly developing.
The field of sleep disorders medicine is now well positioned
to take advantage of these new technologies.
A solid foundation
in the area of phenotyping sleep and its disorders in both animals
and humans is needed before proceeding with genetic analysis.
The discovery of new methods and improvements in existing sleep
recording techniques in humans are also needed. When performing
genetic studies, it is important to consider potential study
design limitations. The strength and location of linkage regions
identified, for example, depends on the strength and precise
phenotype selected. Thus, linkage regions may not be identified
if the power of the study is insufficient, and large numbers
may be required for such studies to be successfully accomplished.
Even if linkage regions are identified, these may be large and
contain many candidate genes. Sequencing of candidate genes
may not yield mutations or may identify mutations that are not
relevant to the phenotype. In this case, the use of complementary
approaches such as DNA expression arrays and proteomics to identify
novel genes of interest may be a powerful approach to identify
relevant candidate genes.
of sleep and diurnal rhythms would be important for a wide range
of clinical studies. Much human research relies on blood samples,
which are easily obtained, but often there is little knowledge
about chronobiologic variations in the parameters being measured,
and no regard for the time of day or the sleep history of the
subject when the sample is taken. The impact of sleep and diurnal
variation on other systems is exemplified by blood coagulation
and thrombotic tendencies. Myocardial infarctions or strokes
occur more often in the morning, and blood properties such as
platelet aggregation may change during the day. It would be
useful to have molecular markers to assess chronobiologic and
sleep history variability.
In The Last 5 Years
- There have been
advances in technology development in the area of sleep phenotyping
in mice. In the clinical arena, sleep recording devices have
been made more portable and easier to use. Similar progress
has been made in automated sleep scoring algorithms that utilize
concepts such as neural networks, fuzzy math, and wavelet fitting,
allowing for more rapid analysis and the possibility of high-throughput
- Eight genes that
significantly contribute to the generation of circadian periodicity
have been isolated in mammals. Recently, studies in humans have
shown, for the first time, a correspondence between human and
animal sleep phenotypes. Most strikingly, a mutation in the
gene HPER2, a gene known to be involved in the regulation of
circadian rhythmicity in mammals, was demonstrated to cause
Familial Advanced Sleep Phase Syndrome (FASPS) in a human family.
Additionally a polymorphism in CLOCK, another gene involved
in the generation of circadian rhythmicity, was found to influence
morningness-eveningness tendencies in humans. These studies
are likely to be extended, with the discovery of other human
mutations and polymorphisms affecting circadian regulation.
- Similar progress
has been made using a genetic approach in Narcolepsy (Section
V). Using a positional cloning approach, mutations in the hypocretin
receptor 2 gene) have been isolated in a canine model of Narcolepsy.
The knocking-out of preprohypocretin, a gene initially believed
to be involved in appetite regulation, led to the establishment
of a murine model of narcolepsy. These findings were found to
be directly applicable to human narcolepsy-cataplexy, as it
has been now shown that most patients have a hypocretin deficiency.
This last finding is remarkable as the disorder in humans is
genetically complex and HLA-associated. These results demonstrate
the importance of careful phenotyping of human sleep disorders
to reduce disease heterogeneity and the importance of animal
- Genome screening
and genetic association studies have been initiated in Sleep-Disordered
Breathing (SDB) and Restless Legs Syndrome (RLS) (Section V).
Significant linkage results have been reported and await confirmation.
In the candidate gene area, an association between APOE e4 and
sleep apnea has been reported and will need to be replicated.
- Develop new methods
to measure sleep, circadian physiology and sleepiness in large
numbers of animals and human subjects. One goal is to develop
and validate surrogate measures. Another goal is to define normal
sleep pattern variation in the general human population. Normative
data will also be critical to define and validate existing or
novel sleep disorder phenotypes. These data will be needed to
elucidate corresponding genetic factors.
- Continue the study
of animal models such as fruit fly, zebra fish and mice to enhance
our understanding of physiology and circadian biology. Use of
these models to study sleep or the regulation of rest/activity
should be a priority, as this may lead to the discovery of novel
sleep regulatory pathways. Powerful new genetic approaches,
such as those used to discover circadian clock genes (e.g. mutagenesis
screens), can be used to find new genes that are involved in
the homeostatic need to sleep and in interactions between the
circadian and sleep-wakefulness systems. Other approaches such
as "quantitative trait loci" should be considered
insofar as true "sleep knockouts" may be not viable
in mutagenesis screens.
- Identify new disease
phenotypes, including rare familial sleep disorders or subtypes
of current sleep disorders based on treatment response or other
characteristics. The study of multiplex families where sleep
disorders appear to be segregating as a single gene could lead
to the positional cloning of novel sleep disorder-related genes.
This may facilitate our understanding of other, more common
sleep disorders, as well as increase our understanding of the
normal physiology of sleep.
- Study inter-individual
differences in baseline sleep, circadian physiology and response
to sleep deprivation in a large number of subjects to better
define normal and pathological conditions. Recent results indicate
large inter- individual differences in how people react to sleep
deprivation. Additionally, subjective sleepiness varies significantly
in patients with equivalent degrees of SDB and sleep fragmentation.
The study of these inter-individual differences has both clinical
and basic research relevance.
- Twin prevalence
and segregation analysis studies need to be conducted for all
sleep disorders across various populations in order to estimate
heritability and environmental contributions for each sleep
disorder. This will help prioritization and design of further
- Genome screening
studies using classical family design and candidate gene-based
research should be continued and extended. As most sleep disorders
are genetically complex, large numbers will be needed and there
is a need to encourage the blending of epidemiological and genetic
designs. Ethnic variation in the expression and the genetic
basis of various sleep disorders has been identified and will
require further exploration. Studies of SDB, RLS, and other
disorders such as hypersomnia will benefit from this approach.
- Genetic array and
proteomic studies in selected tissue samples or protein-protein
interaction experiments should be encouraged. The use of these
new techniques can be extremely useful to discover novel components
within a molecular or a disease pathway.
- Mouse models are
increasingly used in genetic and behavioral studies, and have
been created but not yet widely utilized in Narcolepsy. A large
number of mice with various genetic alterations are being created
in multiple laboratories but are rarely tested for sleep abnormalities.
Finding sleep abnormalities in some of these models could lead
to the discovery of novel sleep regulating pathways that may
be involved in selected sleep disorders. To remedy this situation,
there is a need for developing and distributing genetically
determined animal models for sleep disorders. Collaborative
efforts should be explored to phenotype sleep in mice models
for investigators that are working outside of the field of sleep