[Bob] Hello, this is Dr. Bob Wildin.
And today's lecture is on Genetic Testing for Clinicians.
First question we want to ask is, why do testing?
Why do geneticists or other clinicians
do testing in the first place?
I break these kind of into two groups.
The first is indication-based testing,
where we suspect a health problem
or an increased risk for an inherited problem,
or we want some guidance on treatment
for which genetic testing is the appropriate means
of gathering that information.
The second approach is preemptive testing,
and that is to say there is no clinical symptoms
or abnormal lab values but we want to test
to see if there's a genetic variation present
that has health impacts or health predictive value.
And that group can be broken into two parts,
focus testing, which comes about
as the result of additional clinical information
such as family history
or family history of a particular disorder
or a family history of someone who has tested positive
for a genetic variant of importance.
And a broad net.
So a wide screen for genetic variations
that may have clinical importance
even though you don't necessarily suspect
any of those things being present
or active at this time in the individual being tested.
So what is variation?
Variation is defined by reference to a standard.
The standard may or may not be normal.
But in the current versions of sequence standards
are typically tweaked in order
to be as free as possible
of known disease-associated genetic variation.
Variation occurs naturally without regard
to functional consequence.
It's part of what drives evolution.
Variation is subject to selective pressures.
So certain variations may be sort of bred out
if they result in the inability
to reproduce unless they are able to be carried
by other people and passed on by other people
without an impact on their genetic fitness.
Variation typically originates in one member
of a population, and some other descendants
will inherit that and pass it on.
And that results in frequency differences
in different population lineages,
which we call founder effect,
as the result of what we call founder mutations.
All right, we talked previously about the word mutation.
We tend to talk about a variant,
a variation in genetic sequence
that we can observe as a variant instead of a mutation.
A mutation is the process which leads to the variation.
However, that term is frequently used synonymous
to genetic variations.
So, what are the different kinds of variations
or mutations that can occur?
One is a deletion happening where a single base
or more than one base is sort of subtracted
from the string of letters.
A second is an insertion,
an added base along with its complement,
when that added base strand is replicated.
A substitution.
And any base can be substituted
with any of the three other bases.
And then on replication,
it is fixed by copying the substituted base
in place of the complement of the original base.
That's on a DNA sequence base scale.
On a macro scale,
there is a deletion shown here,
where a segment of a chromosome goes missing.
A duplication, where a segment
of a chromosome becomes duplicated.
In this case, in a tandem head to tail fashion.
And inversion, where a segment of a chromosome
gets flipped in its own location.
A substitution, where instead of
something being duplicated on-site,
it is simply moved from one location
to another in the genome,
in this case from chromosome 4 to chromosome 20.
And that results in this substitution
or interstitial translocation.
A translocation involving the ends of the chromosomes
is involved here,
and this is a reciprocal translocation
where a segment from one chromosome
is exchanged for a segment from another chromosome.
Those result in hybrid chromosomes.
These last two result in hybrid chromosomes,
which in and of themselves are balanced,
meaning they have no additional
or deficit of genetic information
or of the copy number of each of the genes.
That's in contrast to the duplication
or the deletion over here, which are unbalanced.
So this is just reviewing some of the stuff
that we've talked about in other lectures.
The de novo mutation is recognized
because there is no family history
in the case of a dominant condition.
It is not present in the DNA of either parent.
It is evidence supporting variant pathogenicity.
And we'll talk a little bit more about that later
in the lecture.
And there's one little caveat with this feature
of it not being present in the DNA of either parent,
and that is the question of alternate paternity.
So let's just review some of the basics
in genetics so we can move forward with the concept
of genetic testing and some of the technologies
that are frequently employed.
So of course, the genome is your DNA.
It is all of the genetic material in the nucleus
plus the mitochondrial genome.
I'll say that in the case of testing,
often the mitochondrial genome is left out
unless you specifically specify
that you need to include that in the analysis.
Molecules of DNA that contain coded instructions
for how to build, maintain, and replicate a human being.
It is not identical,
your genome in anyone but identical twins.
It always contains benign variation
and variation that can cause or contribute to diseases.
That is to say we all have some variance
that may predispose to disease
or we are carriers of recessive diseases,
usually we are.
And the genome is quite big,
3,3 billion base pairs from mom
and the same number of base pairs roughly from dad.
Okay, let's review chromosomes.
So chromosomes are the packets of genetic material
which make up the genome and its sequence.
It's the DNA sequence packaged together by histones,
rolled together to nucleosomes
and tightly coiled to make a chromosome, say,
in this metaphase chromosome picture.
Prophase or metaphase.
The chromosomes are visualized under the microscope,
and then cut out and stuck onto a piece of paper
based on their banding pattern and their length.
And that's called a karyotype.
Chromosomes in humans have 23 pairs.
That's 22 pairs of autosomes
and one pair of sex chromosomes.
There are the packages of DNA,
they have a consistent structure.
All right, DNA replication happens during cell division
and during the production of the germ cells.
The replication occurs in a 5 prime
to 3 prime direction on both strands.
All you really need is a template DNA strand.
So these single-stranded ones that are being unwound
from the double-stranded template.
You need nucleotides and you need a DNA polymerase molecule,
which will march along and add those nucleotides
to the growing nascent chain to make the copy.
Information is preserved, but errors are made.
Errors result in sequence variation
when they are not corrected, okay?
Let's review the Basic Gene Structure: Exons and Introns.
In that chromosome,
there are thousands of genes,
or hundreds to thousands of genes
in a particular chromosome.
And the gene is sort of the entire segment
that incorporates the element
which when inherited together
encodes a protein in its regulatory elements
as well as additional DNA.
The exons, which contain the coding information
of the DNA are those that will code for proteins,
are divided by introns,
which will be spliced out after the DNA
is transcribed into RNA.
And once spliced out,
that becomes a messenger RNA.
So exons are the segments of the gene
that contain code for proteins.
Introns are the spacers.
And the gene coding regions,
or the exons are about 1 to 2% of the entire genome.
That's the exome.
All right, again, a little bit of review.
We've talked about the transcription,
splicing occurs at this point.
The RNA, and now a messenger RNA after splicing
is used as an information source for the ribosome
to start and extend a growing amino acid chain,
which becomes a protein.
Okay.
Genes and the proteins they encode
can break for a number of reason.
They can cause disease for a number of reason.
One is too much signal.
There's too much of the gene,
there's too much of the protein activity,
such as a gene duplication or triplication,
and that will change potentially the developmental program.
There can be too little signal.
Let's say a gene is somewhat disabled,
such as in metabolic disorders
where there is less enzyme activity.
And if you get well below 10% of enzyme activity,
you may actually have a disease.
A loss of function is when there's no function left.
Those are important, for example,
in tumor suppressor genes.
And gain of function is important in cancer as well,
when certain mutations result
in intracellular growth signaling molecules
becoming constitutively active
and giving their growth signal
even when their upstream signaling pathway
is not being activated.
Finally, genes can simply not be expressed,
meaning that the gene has been deleted,
the RNA is damaged in such a way that it's eliminated
by the cell, or the promoter and regulatory elements
are not present.
Okay, so what about genetic testing?
What are the categories of genetic
and genomic variation testing technologies?
So I tend to sort of break this down into groups
based on my intent when I'm thinking about the testing.
The first is a scanning technology,
and I use that when I'm not sure exactly
what I'm looking for.
I don't know, I can't sort of shine the light
in a direction where a sound is coming from
and know really what I'm looking for.
So I've really gotta turn on the light in the whole room
and look at everything that's there,
or everything that I can see
with that particular technology.
The second is a focused.
So I want to look for phenotype-associated variations.
I have a phenotype, I have a pretty good idea
of which genes or which variations may be important
in that particular phenotype,
and so I'm going to focus my evaluations
into those areas of the genome or those variations.
A targeted assessment is when there is specific information
about where in the genome I need to look.
Is there a specific variant that's already been identified
in a family member?
Is there a specific gene?
Is the only gene that's variant
in this particular condition?
An example is achondroplasia,
where there's essentially one location
in the gene, in the FGFR3 gene,
where mutations occur that cause that specific phenotype.
So those are examples where I would do a targeted test
and look very narrowly.
So examples of scanning technology are a karyotype.
We're looking at all the chromosomes
at a certain resolution.
Chromosome microarray, where you're looking
at greater resolution.
We're gonna talk more about that.
Exome or genome sequencing,
where you're really sort of scanning
the entire genome sequence
And a genotyping array,
that covers many, many known variants all over the genome.
In the focus group,
we might do a targeted gene sequencing panel,
multiple genes, for example,
that might be associated with long QT syndrome.
So we know a handful of genes that are important there.
We'll look just at those genes in a gene panel
rather than looking at the entire exome or genome.
And then, we might use a gene-specific chromosome microarray
to look for copy number variants in the exons
of that gene only.
So two reasons why you might inactivate a gene.
You created a mutation and a promoter or within the gene,
so the protein doesn't work.
Another reason is that you've deleted the gene entirely,
or at least one copy of the gene.
So there are so-called deletion duplication assays,
or del/dup assays,
which we used to use a technique called MLPA,
and now we just use a targeted chromosome microarray.
The targeted technologies are single gene sequencing,
single exon sequencing,
if you know exactly where in the gene you wanna look.
And then there are technologies that can type
a particular variant.
Is this variant present? Is it not?
Is this G to A variant present,
or is the G to A variant absent?
So those kinds of genotyping technologies exist
and can be used to look at, for example,
common tumor mutations like the KRAS G12V,
which is a mutation in the 12th codon,
which changes the glycine to a valine.
in a RAS signaling protein called K-Ras,
and it's frequently observed, for example,
in non-small cell lung cancer.
All right, let's talk about the sort
of development and the history
of clinical variation detecting technologies in genomics.
From older to newer,
there's the karyotype chromosome number we could identify
by the late 50s, 1950s.
And then, as we developed better techniques
to band the chromosomes with stains,
we could identify their structure.
So the karyotype is useful for looking for aneuploidy,
for large deletions and duplications,
for inversions because the band pattern
is changed so that you can recognize an inversion.
And a balanced translocation, for the same reason.
The band patterns and the links
to the chromosomes may change.
RFLP, which stands
for restriction fragment length polymorphisms,
and Southern blots,
you may have learned about in classes in high school
or undergraduate college.
We don't use them very much anymore.
They still are occasionally used
with a lab technique called Southern blot
in order to follow diseases as they run through
a large family to see whether we can figure out
which part of the genome the putative gene is in.
Sanger sequencing is sort of the gold standard sequencing,
and I'll talk about it a little bit more.
It is for sequencing anywhere from single exons
to single genes or maybe small gene panels
of two or three genes.
It is essentially a blinded, base sequence variation.
So you're looking for any base sequences in there,
not for very specific ones,
until you get the sequence and you can see what's there.
Oligonucleotide probe hybridization.
So you've probably heard of FISH,
or fluorescence in situ hybridization,
where probes that are actually much larger
than oligonucleotides, they're a couple of megabases
in length usually, are used on chromosomes in a karyotype
to detect the presence or absence
of a deletion or duplication.
Small probes, oligonucleotide probes
can be used to detect single nucleotide variants, CNVs,
SNVs, or a copy number variation
in a technique called a chromosome microarray
or a SNP microarray.
And I'm gonna talk more about those in a moment.
And then there are these genotyping platforms
which, as I mentioned,
are looking only in a binary fashion
to say is a particular spelling change present or absent?
These can be very widespread
and look all over the genome for lots of different variants.
That's what you find in, for example,
23andMe and other direct to consumer types
of genetic testing.
And those are also sometimes useful,
although they're more important in research.
Finally, there's next-generation sequencing,
or NGS for short.
It is basically a multiple modification
of Sanger-type sequencing, that uses automation,
miniaturization, and is very high throughput.
The length of the sequence reads are relatively short,
on the order to of 100 to 150 bases.
And as such, it requires a great deal
of computation to assemble those into a genome
and it's really done by matching up each read
to its putative position on a reference sequence,
and then assembling it all together.
Next-generation sequencing can process entire genome
or a selected subset such as an exome or a gene panel.
In addition, we can use non-DNA-based technologies
to identify genetic variation in individuals.
We can look at protein sequence.
We can look to do electrophoresis
to identify shifts in electrophoretic mobility,
which usually relates to charge of the protein.
We can use immuno detection and a lot of other techniques.
We can also test for the metabolic
or functional consequences.
So, example, look in urine by mass spectrometry
for metabolites that shouldn't be there
or that give a clue to a particular metabolic disorder.
Okay, I'm gonna throw out some clinical scenarios,
I'm gonna go through this really quick.
And if you want, you can pause and and think
about this a little bit further.
First, you examine a 2 week old infant at his first visit.
He's small for dates, has microcephaly,
brachycephaly, small ears with overturned superior helix,
up-slanting palpebral fissures,
and a deep vertical plantar crease directly
between rays 1 and 2.
That means if you look at the bottom of his foot,
there's a vertical crease,
the top of which is between his big toe and his next toe.
You suspect blank syndrome.
So I'm pausing to see if you want to go forward with that.
But we'll go forward.
Which genetic test technology would be best
to confirm your diagnosis and provide information
on recurrence risk?
Single gene sequencing of MECP2,
karyotype, chromosome microarray,
or exome sequencing?
So you see, in this case you have to have
some idea of what kind of disorder you're looking for
and what type of genetic variation you would expect
to find, type or types, in that disorder.
In this case, we're looking at a case
of Down syndrome, trisomy 21.
And the answer is-
Going backwards.
Is not A, because in single gene sequencing of MECP2
won't confirm Down syndrome.
A karyotype would be a really great choice
because it is good at picking up aneuploidy,
and that's what you're expecting in Down syndrome.
A chromosome microarray is an okay choice.
It would pick up Down syndrome,
it would come back with a report of Trisomy 21,
but it is overkill if you're really suspecting Down syndrome
in this child.
Exome sequencing isn't a good choice
because exome sequencing is not that sensitive
to copy number variations.
In this case, it might really tell you
that Down syndrome is present,
but exome sequencing is really designed
to pick up variations in the DNA sequence itself,
not variations in the copy number.
Okay, so here's another little quiz.
What disorder has tall statures,
small testes, delayed puberty,
infertility, and gynecomastia?
That is a disorder of copy number.
How about Klinefelter syndrome, XXY?
All right, so karyotypes are good for aneuploidy,
entire chromosome is missing or duplicated.
The most common ones are Trisomy 21, 18, 13,
Monosomy X, or Turner syndrome,
and Klinefelter syndrome.
Large deletions and duplications,
such as you might see in
a classical cri-du-chat syndrome, 5p-.
If you haven't heard of cri-du-chat,
walk into a big newborn nursery
and listen for the baby who's tiny,
who's cry sounds like a cat.
Translocations and rearrangements,
even when they're balanced,
can be seen in karyotypes.
And the balanced ones can predispose
to unbalanced offspring with copy number variations.
Those will not be detected by technologies
that look only for copy number variations.
So let's talk about those technologies.
The chromosome microarray, or CMA,
which uses a technology
called comparative genomic hybridization
or array comparative genomic hybridization.
But CMA or chromosome microarray
is much easier to say and it indicates
that we're looking at the chromosome.
So it's useful for looking for copy number variants,
deletions, duplications, triplication, etc.
And it has a higher resolution than a karyotype,
meaning we can find smaller deletions,
duplications than we can with a karyotype.
A variant on the chromosome microarray
is a SNP array, which also has probes
that will detect normal sequence variants.
And as a result,
show you when there are regions of the genome
where there is only one pattern
of polymorphism in an individual.
And those are regions of homozygosity.
They point to either loss of a copy,
but you can tell that on the chromosome microarray.
So if it's called a region of homozygosity,
it's not a copy number variation,
it is a region which is predicted to be inherited
from a common ancestor.
Small regions of homozygosity
and small numbers of homo percentages,
homozygosity across the genome,
are fairly common,
especially in regions where people don't move around a lot.
If you detect large percentages of homozygosity
in the genome, you suspect some degree
of consanguinity or close relatedness of the parents.
Chromosome microarray is not sensitive
for balanced translocations,
no copy number variation.
For simple inversions, no copy number variations.
And for small CNVs,
say on the order of an exome or smaller,
because it depends on having two
or three or five contiguous probes
that are in line with each other,
showing the signal for a deletion or duplication
at the same time.
So, I have two little pictures over here on the right.
One is of a person with visual impairment using a cane
to sample his environment in a selective way.
So the cane allows this person
to see not every single surface
in every single part of the sidewalk,
but selections along that way.
And that's essentially what a chromosome microarray does.
It has anywhere from 180,000
to 800,000 small oligonucleotide probes
that are designed to hybridize all over the genome.
And the technique tells us whether they hybridize
at the same rate more often or less often
than a reference DNA.
And so, in the picture here,
this person has what looks like a duplication,
or 1.5 times the signal in fluorescence
in this red segment from a large number of probes in there.
And this is 15q12,
so this is probably the Prader-Willi-Angelman syndrome area.
Okay, here's a clinical scenario.
You see a 24 month old for a well child visit.
She started walking at 18 months,
most kids start walking around 12 to 14 months,
and she has not had her first word yet.
Again, around 12 to 14 months
is when you should expect that.
She has borderline short stature,
a short up-turned nose,
and possibly low-set ears.
What are the reasonable possibilities for next steps?
Refer to genetics for dysmorphology evaluation,
get a karyotype,
do a chromosome microarray,
or do hearing testing.
Hearing testing's not a bad idea.
If she's not had any speech,
it's possible she's not developing speech
because she has a hearing impairment.
Referring to genetics for dysmorphology evaluation
seems reasonable because she has some soft features
and is developmentally delayed,
and maybe those two things go together.
And someone will recognize a syndrome.
A karyotype probably isn't gonna tell you very much.
The yield on karyotypes for a patient
with sort of relatively subtle presentation like this
is not very high.
And you're not suspecting an aneuploidy
because the features don't add up to an aneuploidy disorder.
Chromosome microarray turns out to be a good choice too,
because we know from experience
that in the context of developmental delay
not due to a single gene syndrome,
the chromosome microarray will be positive
in somewhere between 15 and 30% of patients.
So, let's switch from the copy number variation approach
to thinking about disorders caused by sequence variation.
So, changes in the actual genetic code.
So here's one.
An individual with a copper-colored ring
around the edge of the cornea,
that's an almost pathognomonic sign
for Wilson's disease, a copper storage disorder
that is a progressive neurodegenerative disorder as well.
Here's a disorder with thick, sticky mucus
that blocks the airways and also blocking
the pancreatic and bile ducts
and causes pancreatic insufficiency
and eventually type 1 diabetes.
That would be what?
Yes, cystic fibrosis.
Here's a group of disorders,
there's a weakened heart muscle
and an enlarged ventricle and a thin ventricular wall.
That would be dilated cardiomyopathy.
And what about a disorder that predisposes to cancer?
In this case, familial adenomatous polyposis.
This is a histology section of a classical FAP polyp
removed from a colon of an affected person.
A single polyp, not a big deal.
Lots of polyps, big deal.
All right, and what about the technologies?
Sanger sequencing I mentioned earlier.
It's considered the gold standard,
although that's rapidly being replaced
by next-generation sequencing as the quality
and our understanding of how the sequencing technology
can work and how it can fail improves.
And so, Sanger sequencing is still considered the way to go.
In the early part of my career,
I used Sanger sequencing that was quite manual process.
The picture on the left shows an autoradiogram,
that's a piece of X-ray film
on which I've laid a dried gel
on which I had run sequencing reactions
using radioactive P32 labeled DNA in four lanes.
One lane for A, one lane for T,
one lane for G, and one lane for C.
And you simply sort of read down from the top down.
And in this case,
you see a T-A-G-C pattern,
as shown in that picture.
On the right is a more modern form
of Sanger sequencing or a capillary sequencing,
where instead of using radioactivity,
there are fluorescent tags that are used.
And those are run on
a capillary electrophoresis sequencing system.
And at the bottom of the capillary,
as the DNA fragments fly out,
their fluorescence is measured
and you get this tracing,
and then you can read the DNA sequence off of that.
It's a relatively low-throughput technology,
it is highly reliable,
and has a relatively high cost per base.
So Sanger sequencing would never have got us through
the entire human genome project,
although that's what we started with
when the project began.
Sanger sequencing is still used mostly
for targeted mutation testing of single exons,
single gene, or sequential gene testing.
So I'm gonna test this gene first,
if that's negative,
then I'm gonna test the next most likely gene,
sequence that one,
and then if that's negative,
I'll sequence the next most likely gene.
Small gene panels.
Two or three genes, depending on their size,
can be put into a panel
and sequenced efficiently with Sanger.
Sanger is also used for sequencing parts of the genome
that next-generation sequencing is not very good at.
So, filling in for next-generation sequencing gaps
or low-depth regions.
And finally, choose for confirmation
of important variants detected by other methods
such as next-generation sequencing.
So what about next-generation sequencing, or NGS?
As I mentioned, it's short read,
150 to 200 bases per read.
It's massively parallel,
so this is going on in a very micro-scale.
As you see in the center picture,
there's what's called a flow cell
and there are thousands and thousands and thousands
of sequencing reactions going on
in that one little slide apparatus.
It is really good for high throughput,
for lowest cost per base,
for high general reliability,
and it has, at present,
the highest clinical market penetration
in both research and clinical sequencing laboratories.
It has an advantage in that it's pretty good
at detecting mosaicism if you are sequencing depth
and your bioinformatics pathway are tuned for that.
All right?
So in the left-hand picture,
there is a picture of an Illumina MySeq machine.
We have two of these in our laboratory here,
along with a NextSeq machine
which has several-fold increased capacity,
sequencing capacity over that.
That pales in comparison
to the most recent Illumina sequencing machine,
which is a NovaSeq which can sequence huge amounts
of DNA simultaneously.
All right, Gene Panel Sequencing.
So I've mentioned that a few times.
What does that really mean?
So with Sanger sequencing,
maybe 1, 2, 3, 4, 5 genes.
It is guaranteed coverage of exons
and their flanking sequence.
So you have high confidence
that the entire region you were targeting
is being sequenced well.
Next generation sequencing can sequence anywhere
from 2 to 100's of genes at once.
And the cost of adding additional genes
to that next generation sequencing is not very high,
although you need to decide in advance
which groups of genes you want to sequence together.
Next generation sequencing often
has some low-coverage areas,
particularly in the first exons
and in other GC-rich regions.
So first exons tend to be GC-rich
because they often have regulatory elements
in them and are areas where methylation occurs
that help regulate the gene turning on and off.
Those GC-rich regions are substantially harder
for next generation sequencing technologies
to sequence through,
as are sequences with long repeats of the same base
or lots of repeats of small numbers of bases.
All right, what are the uses for next-generation sequencing?
The gene panel, I mentioned.
Multiple genes associated with disorders
with similar presentation.
For example, cardiomyopathy, hereditary neuropathy,
early infantile epileptic encephalopathy,
and tumor somatic mutations for therapy,
as we talked about in the cancer module.
So what all these have in panel,
the reasons I'm sort of giving you a landscape
of this is for you to get the idea
that a gene panel is helpful when you don't have
other tools to help you differentiate down to the level
of a single gene what is most likely
to be changed and to allow you
to do targeted genetic testing.
So when you can't quite target it,
but you know what the phenotype is,
you can group the genes and target those in a gene panel.
Clinical Scenario.
Your patient has an early onset cardiomyopathy.
Her father and her father's brother,
her paternal uncle,
and her brother also have cardiomyopathy,
but some of them have a dilated cardiomyopathy,
and others have a hypertrophic cardiomyopathy.
What rationales support doing genetic testing
in your patient?
A, she has a 21 year old daughter who wants
to know whether she's at risk,
and whether she should use reproductive technology
to avoid passing on a disease
like the cardiomyopathies in her family.
B, you're unable to differentiate
which of the molecular genetically distinct forms
of cardiomyopathy your patient might have.
So you can not formulate the best treatment plan
when it matters what type of cardiomyopathy is present.
The prognosis may be different.
The response to medications.
The urgency for finding a transplantation transplant donor,
for example, may all differ depending
on the type of cardiomyopathy.
Her affected paternal uncle lacks health insurance.
D, you would use a different pacemaker depending
on what mutation she has.
So the true answers here are A,
we're talking about reproductive risks.
And B, refining the prognosis and predicting
which medical approaches are going
to be most effective for this patient.
All right, exome sequencing compared to genome sequencing.
We're gonna start first by comparing
the entire exome to the clinical exome.
So the entire exome is about 20,000 genes.
It is coding and non-coding exons,
so just the exons.
And all the non-exons, the introns,
the DNA that's between genes,
the regulatory regions and so forth are not included.
The clinical exome is about 6,000 genes
that are known to be associated with disease.
Now, mind you, that the number of genes associated
with the disease keeps increasing every month
so it's hard to keep up with that,
but the clinical exome basically says,
well, there's a bunch of genes we know are out there,
but we haven't associated them with diseases yet
so sequencing them doesn't give us
any clinically important information.
So you may find that the clinical exome
is what you're going to get out
of a commercial testing laboratory.
Furthermore, when you look at the clinical exome,
you always are asked about what is the clinical phenotype.
And in fact, rather than looking at all 6,000 genes,
there's a computer that looks at them for things
that might be suspicious.
But for the most part,
based on the clinical presentation
that you outline in the paperwork
that you submit with the test,
the laboratory will look at a subset
of the clinical exome,
that is the genes that are known to overlap
with the clinical phenotype that you have described.
So it's really, really important
that you fully describe the clinical phenotype well
when you order a clinical exome test
because otherwise, you're gonna get garbage.
The other tweak on the exome,
which is often done,
is to also sequence the exome of both parents.
And the reason for doing this is twofold.
First, if there is a new genetic change
from one generation to the next,
so the patient has a genetic change in a gene
which neither of their parents has,
that would be a de nova mutation
and that's one of those levels of evidence
that we talked about that pushes you toward
that genetic variation being pathogenic.
The second reason is because if you find two variations
in a gene that you know to be associated
with recessive disease,
you don't know whether they're on the same copy.
In other words, those two genetic changes
both came from mom or both came from dad
or one of them came from mom and one of them came from dad.
The latter is what you would see
when both copies of the gene are disrupted
and you are expected to have clinical disease.
So having that so-called trio sequencing,
the patient, the mother, and the father makes up the trio,
helps those who are interpreting your exome sequence
do a much better job in the interpretation.
So the clinical exome is then mostly coding exons
plus 10 or 15 bases into the flanking introns.
Now why do they do about those flanking sequences
just into the introns?
That's because sequence variations there
can disrupt splicing of the RNA transcript into the mRNA,
and then you have disrupted exons
in your messenger RNA and the protein
will come out different.
All right, a clinical scenario.
Your patient has multi-system disease,
all non-genetic testing has failed
to come up with a unifying underlying process to explain it,
and the specialists involved
are uneasy giving advanced treatment
since they aren't sure what the cause is.
So this is something that comes up fairly frequently
in genetics and probably not infrequently
in primary care as well.
And it's just a picture where it's fairly clear
you've tried all the usual avenues,
you've consulted all the usual specialists,
and it's still you have sort of descriptive diagnoses,
but you've really never come up
with an underlying process that is explaining all of them.
So, what rationales support doing exome testing
in your patient?
A, continued reliance on traditional testing
and repeat specialist visits
will only consume more healthcare dollars
without advancing her management.
B, she has a 3% to 5% chance of having more
than one rare disorder,
explaining the difficulty
in understanding her complex phenotype.
C, she can have an extremely rare disorder
that will never be diagnosed clinically.
And D, there are specific treatments available
for almost all genetic disorders.
So which rationales would you pick?
So I would pick A,
that you've kind of exhausted what traditional testing
and specialist evaluation can do,
and just doing more of the same
isn't going to get you any further.
Interestingly, in the last few years,
we've recognized that B is also true.
So one of the reasons we have a hard time recognizing
what's going on in a complex patient
is that they have more than one disease
going on simultaneously.
And both of those diseases can be rare,
and our chances of figuring that out
and teasing out those different phenotypes is pretty low.
She could have an extremely rare disorder
that will never be diagnosed clinically.
That's also true.
D, not so much.
There are not specific treatments available
for almost all genetic disorders.
All right, so when do you
wanna use next generation sequencing?
The exome, all the exomes in genome plus
the protein coding segments
and those little bits of flanking sequence next
to the exomes in the introns.
When the presentation is rare
and you suspect a genetic cause,
it's an unrecognized disorder or area.
Other testing failed to diagnose.
You suspect more than one disease.
And sometimes, you just wanna end the diagnostic odyssey.
This has been going on for a long time,
the patient's upset we're expending more
and more healthcare dollars.
And sometimes it's enormously relieving
for the patient and their family
to come to an answer,
even if the answer means there's nothing they can do.
The whole genome is basically the exome
plus everything else,
and we really don't use that much in clinical care yet.
But as the price for genome testing comes down,
and as our understanding of how to interpret
the sequence variations in between the axons
and in between the genes, we will want to be doing
this more and more because we
are missing structural variations
and variations in regulatory regions
that may explain some of the areas where we expect
to find something in a gene panel or an exome,
and we don't.
So the whole genome is an exome plus the non-coding regions.
It's about 75 times larger than the exome
and probably costs about 30 times more,
maybe less than that, to sequence,
because it's just that much larger.
The potential to detect clinically important defects
in gene regulation is there,
and it can be used when it's really, really important
to diagnose when loss of function
and coding regions are normal
and when you can convince insurance to pay for it.
I would not waste my time on that at this point in time,
but fast forward a couple of years,
three years, four years,
we may be seeing that coming down the pike.
Remember that when you sequence the germline DNA,
you are sequencing DNA that essentially represents
your genetic instructions.
So when you sequence that,
the information that you get for that
is good for the patient's life, right?
So it's not gonna change over time
like other kinds of tests,
like cholesterol levels or sodium levels,
things that you test repeatedly.
So once you have a really good sequence
of your exome or, hopefully in the future, your genome,
that test won't have to be done again.
So the cost of those tests can be amortized over time
as additional queries are made on the information
that was generated in the initial sequencing test.
Okay, so when we get a report back from a genetic test,
it will contain information about variants
that are felt to be of significance, all right?
We have a standardized process now
that we use to classify genetic variants
in terms of their clinical importance.
And that classification assumes at the beginning
that it's a variant of uncertain unknown significance.
A VUS, and some people will call that a VUS, right?
I call it a V-U-S, variant of uncertain significance.
So our classification scheme
is based on two axis of information.
One is pathogenicity,
how likely is this variant to be the pathogenic source
of this disease, and what's the level of evidence
for that pathogenicity or lack thereof.
So this creates sort of a graph,
which we're doing not as a graph
but in sort of stepwise bins.
And so, as you get some information
that this variant may be pathogenic,
you can move it up from VUS to likely pathogenic,
you get more information, more evidence,
you can move it up to a classification of pathogenic.
If you get evidence that it is probably not pathogenic
but you're not sure, you can move it to likely benign,
and then you can move it further,
with more evidence, to benign.
The types of evidence that are used
include a population frequency,
so variants that are fairly frequent
in the population in individuals who are healthy
are unlikely to be the cause of a rare genetic disease,
unless we're talking about a recessive disorder,
in which case a significant fraction
of the population could be carrying it
and it could be a recessive pathogenic mutation.
If this variant is reported in this disease
and is not reported in people who don't have the disease,
that's a mark on the plus column for pathogenicity.
On the other hand,
if people that's reported in this disease
but it's also reported in an equal frequency
of people who don't have the disease,
then it moves more toward the benign column.
If it tracks with the disease in family,
a so-called co-segregation of the disease
and the variant in large families,
or especially if that happens in multiple families,
that is evidence to support pathogenicity of the disease.
If introducing the genetic variant into cells
causes a biochemical or a protein function change
that you can measure in the laboratory,
a so-called functional assay,
that is further evidence that it may be pathogenic.
And as I mentioned twice already,
if it is new in an individual with the disease,
it wasn't present in their parents
and neither of their parents are affected,
that's another sort of element
that leads of evidence that can help you decide
that it might be pathogenic.
Okay, I've added this little clock
to remind me to tell you that the evidence space
for these ratings changes over time
as more information is gathered about populations,
about families with the disease,
and about protein function
and the impacts of the variation on protein function
as that's studied further.
So a classification at one point in time
may actually be different two or three years later.
So, for the most part,
that's going to be moving things from variants
of uncertain significance up to likely pathogenic
or pathogenic or to likely benign or benign.
But occasionally, things will have been classified
as likely pathogenic or pathogenic early on,
and new evidence arises that says,
eh, not so much, we really now think it's benign
or likely benign,
and so they get moved down
in the pathogenicity classification system.
The important point is that once you have
a result like this,
you may have to revisit its classification
at a later point in time in order to be certain
that you are interpreting it
with the current understanding
of its importance for disease.
So what strategies might you use for a variant
if you get a report back that has on it a variant
of uncertain significance?
One is to ignore it, and that's okay.
One is to wait and revisit.
One is to see if your patient wants to participate
in research studies that might help clarify
whether it is pathogenic or not.
You can get online and go to one of several websites
where you can put in your genetic VUS
and your disease phenotype
and try to find other individuals across the world
who have also been tested,
have been found to have the same variant,
and compare whether they have the same disease
or the same phenotype that you do.
So that's a new tool that's kind of interesting,
and then sort of crowdsourcing evidence base.
In general, we do not base therapy
on a variant of uncertain significance.
We do not test relatives for a variant
of uncertain significance
unless we're in the process of trying to gather evidence
to see whether it's significant or not.
And we don't generally tell a patient
that we know the cause of their disease.
So I'll tell a really quick story,
that there was a lawsuit in Oregon State
where a practitioner did a test
to look for lynch syndrome and found an MLH1 variant.
They made two mistakes.
First, they decided that the variant,
since it was found and reported,
was important and that meant that the patient
had lynch syndrome when in fact it was a VUS.
The second mistake that they made
was not understanding lynch syndrome
and the phenotype spectrum.
They believed that that resulted in a high risk
of breast cancer and performed bilateral mastectomy.
In fact, that was inappropriate.
Hundreds of thousands of dollars changed hands in court
as the result of that misinterpretation of genetic testing.
All right, here's a clinical scenario.
Your patient says he had genetic testing five years ago,
and nothing was found.
What questions do you wanna ask about the result?
What technology was used for the test,
what type of test was it?
That sounds reasonable.
Did they report any VUS,s?
That sounds reasonable because you might want
to look at those VUS's again
and see if their categorization has been changed.
Was the test a consumer-oriented one?
Yeah, that's important,
because the consumer-oriented tests
are generally not done in CLIA certified laboratories.
In other words, they're not done in medical laboratories,
and the results from those are not usable
in clinical medicine by law.
If you want to use that information,
you have to confirm by retesting for those variations
in a CLIA certified laboratory.
And also, where's the test result documented?
Because, as we'll talk about,
you'll get a lot more information
by looking at the documentation,
the actual report from the test,
and getting a copy of that than you will
from talking to the patient themselves.
And you will have that in your records
so you can validate what you did
on the basis of that result.
Okay, let's talk about those reports.
Understanding testing results
as they reported in clinical tests.
We'll talk about variant nomenclature,
what is the meaning of a reference sequence,
what it is that is varied from the reference,
what's the clinical significance of that variation,
what may have been missed,
and what are your next steps?
So describing sequence variation type
and location in the genome.
So we want to say, you have this variant.
How do we do that?
How do we say, this,
and how does everybody recognize what, this, means?
I'm gonna use here a postal address analogy
to describe roughly how that's done,
and then I'll show how it's actually done in genomics.
First of all, you have to have an agency
that defines the standard
of how you communicate that information.
And in this case,
for postal address in the United States,
it's the United States Postal Service.
There are actually two types of addresses.
To simplify it,
there's a street address and a P.O. Box, right?
So for the street address,
we might have 111 Colchester Avenue
in Burlington, Vermont with a zip code.
This is showing a segment of the country,
a region, so to speak,
that we wanna look at,
and then a more specific position in that region.
And in this case,
for a street address,
we put the number in front of the street name.
So there are conventions here
that help you differentiate what kind of address it is.
And for a P.O. Box address,
you might have an address like this.
So in the same way you have the same segment of the country,
it's a different segment 'cause I'm in South Burlington now,
and I have a different zip code,
and I have a P.O. Box with a number.
So how is that number different from the other number?
The position of the number follows the word P.O. Box.
So that's the convention, all right?
There are ways we can sort of avoid trying
to mix up these different types of addresses.
All right, so let's talk about
different nomenclature systems
for different types of test results.
So we're gonna talk about cytogenetics
and copy number variants on this slide.
The agency is HGVS,
or the Human Genome Variation Society.
And ISCN is the international system
for cytogenetic notation.
And you can look it up in books, basically.
A chromosome variation starts out with the count
of chromosomes per cell.
So, we look under the microscope at a cell prep,
and when we count the number of chromosomes there,
we're actually counting the number of centromeres.
And in a normal cell,
we would count 46, right?
Two pairs of 23.
We then look among those chromosomes
and identify the sex chromosomes,
and we say, what is this sex chromosome constitution
of that cell?
It's either an X + Y, an X and an X.
Or in the case of Turner syndrome,
an X without a second X.
Or in the case of Klinefelter syndrome,
two Xs and a Y, okay?
So those are listed subsequently.
And then, abnormal stuff,
like a translocation, or in this case an inversion,
because you're going from 2p to 2q,
are described after that initial part.
And that can get quite complicated,
now I'm not going to go into the details of that.
So chromosome microarray has a related,
but not similar to nomenclature.
It typically starts with the abbreviation arr in advance.
If you see FISH reports,
you might see FISH or something like that,
or ISH in there to show you
what kind of an technology it is.
So that is the array comparative genomic hybridization
on the chromosome microarray.
If there's a copy number variant,
it will tell you what the chromosome band position is,
what position on chromosome 22 it is,
and it's followed by a pair of numbers in parentheses.
There will be a lower number and a higher number.
And those tell you the sequence position on chromosome 22,
which is the start and end of the copy number variation.
That parenthesis group is followed by a times and a number.
And that basically says this segment of chromosome 22
only had one copy number instead of the expected two.
So x1 tells you it was a deletion, all right?
Or copy number of variation of a loss.
Deletion or loss.
If it was times 3, it would be also unexpected
and it would suggest that there was a duplication
or gain of copy number, all right?
So that's the basic nomenclature.
I do not expect you to remember or memorize all of this,
I just want you to have a vague concept
that you've seen this before
when you see these reports coming across your desk.
I'm gonna skip that.
All right, what about sequence variation?
We talked about cytogenetic and copy number variation,
let's talk about sequence variations.
HGVS is the agency that defines the nomenclature standards.
You can look them online,
they have a website, varnomen.hgvs.org.
Within their site, there is a quote unquote,
"simple description" of the nomenclature
for sequence variation.
It may be worth reading.
It's not something that you learn terribly quickly,
but I think it is helpful to begin to look at these
and try to parse them so that over time
you're gonna become more and more comfortable
with what they mean.
Okay, so we're gonna break this up into two parts.
I'm gonna talk first about the reference type
and the location.
So what is the reference sequence?
What are we saying has changed?
What are we saying is the standard
which we're noticing has changed
in this particular patient's sequence?
So what is the reference sequence that defined
what is expected at the specific genome location?
So we want to provide a unique identifier
that points to that reference sequence.
Otherwise, when we specify a position,
we won't be specifying anything
that's unique and re-identifiable, okay?
And then we want to specify whether the reference sequence
is in a genomic sequence, a chromosome,
in which case we prefix the sequence position
with a "g." for genome.
If it's in a cDNA sequence,
in which case we prefix it with a "c."
Or if it's in a protein sequence,
in which case we prefix it with a "p."
All of those are lowercase, all right?
So, and then we say what is the location in the sequence?
So that first position in the reference sequence
is location number 1.
So that's the 1st base in the nucleic acid sequences 1.
And with respect to chromosomes,
that is the first base that's recorded
in the reference sequence starting from the tip
of the short arm of the chromosome, or the P arm.
So we're kinda going from left to right
when you lay the chromosome on its side
with the P arm to its left.
An example then would be we're gonna say
it's a segment called chromosome 2,
it's a colon, it's a genome sequence,
that's what the g. tells us.
It tells us this a specific sequence position, which is-
Let's see, where's my commas?
Commas go there and there.
326,890,112 bases into the sequence from the P arm.
And the reference base at that position, is a g.
So there's a guanine at that position
in the reference sequence.
In the case of a cDNA sequence,
the position 1 is the "A" of ATG initiation codon
for the cDNA.
cDNA .167A tells you that there is an adenine,
again, a base, at position 167 in that cDNA sequence.
That may correlate with this place on the genome.
So a lotta times you're given both
a genome reference sequence and a cDNA reference position
that correlates with that.
In a protein sequence,
the 1st methionine,
the 1st amino acid position in the protein chain encoded
by that ATG is position number 1.
So here's an example.
Protein sequence glycine 55.
So there's a glycine at amino acid 55 in the sequence.
It can also be written as p.G55.
G is the single letter amino acid code for glycine, okay?
You can see how this can be confused,
because here's a G and here's a G,
so you need to be able to differentiate
what is a position in a DNA
and what is a position in a protein
so you can properly interpret the single amino acid codes.
All right, so here's an example of a sequence
for chromosome 11.
This is a reference sequence,
and you can get to it by going to the nucleotide core
or what's called RefSeq.
And you can recognize it
'cause it's a nucleotide sequence,
and anything that ends in 11 here is chromosome 11.
If it says 02, it's chromosome 2 et cetera.
And base position 1 here in that sequence is an N.
So in the case of the reference sequence
for chromosome 11, the tip of the P arm is very messy,
and so the sequence actually begins
with a whole bunch of Ns,
which means we don't know which base
or it could be any base.
At some point, a couple of pages down,
you'll enter actual ACGT sequence.
Okay, what about the reference sequence?
So, I just gave you information about that.
And this NCBI reference sequence
is what you might quote for that, okay?
So, a couple of things are important there.
One is that you need to get
the reference sequence version right.
So this is the genome build, hg19,
that's the British nomenclature.
And the US nomenclature, or the NCBI nomenclature,
is GRch38, or build 38.
And those may have suffixes like decimal suffixes
to them as well that you really don't have to worry about.
So that tells you that this number
you can look up with confidence in that genome build
in that genome build's sequence for chromosome 2
and you know exactly which base
you're talking about, always.
Okay, so that's the version
of the reference human genome sequence,
the segment of the human genome chromosome,
and the position in the reference sequence,
so thinking back to those US postal addresses.
And then, the base found at the reference
at that position is really for your convenience,
because once you have this other information,
you could go find that base
in that whole chromosome sequence
and you would be able to determine what the base was.
But it's written there for your convenience.
A cDNA sequence is also given a reference.
Those have the letter M in the reference sequence name
as an mRNA sequence.
You'll also see X in the first position there.
Those are alternative cDNA sequences,
they're old cDNA sequences.
You wanna pay attention to the N,
which is the standard.
And then you have c.345G.
Again, you can look up position 345
in that reference sequence
and see that it's a G,
but it's presented here for your convenience.
Notice that in nucleic acid sequences,
you have the position and then the reference base
at that position.
All right.
Okay.
And then the variation is added onto the end of those.
So you've told at where we're going,
which chromosome version we're looking at,
which chromosome we're looking at,
which sequence position,
what the base and the reference sequence is,
and now we wanna say, what did we observe in our patient?
So for nucleic acid sequences-
(alarm beeping)
For nucleic acid sequences we have
a system of adding a open, I'm sorry,
a greater than sign and the base that we observed,
or the bases that we observed.
So, example for a genomic would be a G to A change.
And the cDNA, an A to an T change, okay?
In the case of protein,
it's a little bit different.
We have the P, we have the Gly, the 55, the location.
And if it's changed glycine to proline,
then the variation is listed after
the sequence position here.
That's in a three letter amino acid code.
That's the same thing in a one letter amino acid code.
And you'll see both of them.
So, the difference here is that there's
a little bit of rearrangement in the order of things.
We have the position coming just before the variant
in the protein and just after the reference base in the DNA.
So there's a little bit of difference in the order.
You have your G, your C, and your P,
that helps you too.
And then there is also the greater than symbol,
which is only seen in the nucleic acid ones.
And again, this position is between
the reference amino acid and the variant amino acid
in your protein.
Variants come in different forms.
There's heterozygous variants found
on one of two copies.
Homozygous, the same variant was found on both copies.
There was no non-variant copy.
Compound heterozygous, two variants were found
in the same gene,
presumed to be on different copies in trans versus cis.
And hemizygous, the variant was found,
no normal copy was found,
and there appears to be only one copy of the segment.
That's typically in the Y chromosome in males.
I'm sorry, in the X chromosome in males that should be.
But it occurs in other scenarios too.
So this is important when you look at the test result
to know if you're dealing with a recessive disorder,
whether you've only found a carrier state,
or you found enough evidence
to say the patient may be affected.
So what does it mean?
What are the parts of a test result report?
In addition to the nomenclature that you'll see,
that we've just talked about,
you'll see a section which is an interpretation.
Now the interpretation will put in prose what was observed.
In other words, it will it will sort of replicate
what the nomenclature says, but in kinda real words.
It will tell you what was found or was not found.
For example, you may have been looking
for a specific variant that had been observed
in a family member,
and it will tell you that they looked for that variant
but they didn't observe it.
So that was a negative test for that familial variant.
What is the predictive effect on the cDNA
and on the protein if it's in a protein coding region?
What is the pathogenicity rating?
Again, a variant of unknown significance.
Benign, likely benign, likely pathogenic, and pathogenic.
What is the evidence supporting the rating
and the references for that evidence?
So you can go look it up yourself.
And what is the predicted clinical significance?
So the laboratory person has a landscape of information,
which is the clinical information you gave them
when you requested the test.
They don't have any other information,
unless it's a local laboratory
where they can look up in your EHR more information
about the patient if they have questions.
So the predicted clinical significance
may be circumscribed because they don't have
all the information that you do.
So you may have to take a look at that
and decide for yourself whether that's appropriate or not.
And then, it will usually include a recommended
or suggested clinical actions.
That may be genetic counseling,
that may be testing a family member,
that may be doing an additional test
which covers things that this test didn't cover
or which will help you clarify the result on this test.
Finally, it should tell you the methods that were used.
This can be a lot of fine print and detail,
but sometimes you have to read through it
to make sure you understand what you got.
It will include, for example, in a gene panel,
the genes that were covered and what coverage was obtained.
In other words, were there gaps
in the sequence that was obtained with this particular run?
It will tell you the reference sequence identification
which you need in order to figure out exactly
what the position part of the nomenclature means.
And it will tell you what the capabilities
and the limitations of the test are.
So, I wanna end by just sort of summarizing the process
that you might go through when using genetic testing.
First, you have to decide.
You have to decide how are you gonna use
the information that you can get out of the test.
You have to decide on the right technology
with the greatest sensitivity and specificity
for the types of genetic variants that you are looking for.
You have to use the most cost-effective steps
for detecting all the likely types of variants,
and that may be doing one small test first
then following up with a bigger test.
That might be starting out with a bigger test.
It really depends on the clinical situation.
You might be concerned about turnaround time.
So the turnaround time for a chromosome microarray
is a week or two.
Turnaround time for a small gene panel
might be three to six weeks.
A turnaround time for an exome or a trio exome
might be six to 18 weeks.
So you need to think about what is going to work
for your particular clinical situation.
And you have to have some sense that the laboratory
that you're going to is trusted.
And I won't talk about it being CLIA certified,
CAP certified, all of those things
that we look for in clinical laboratories,
but you have to have a sense
that this laboratory knows what it's doing.
For most genetic tests you need to preauthorize them
with the payer, the patient's payer,
or find some way to minimize
the out-of-pocket expense for the patient,
and a lotta the commercial laboratories
will have mechanisms for this.
You may need to write a letter of medical necessity
to the payer to justify getting this a test approved.
You need to organize the sampling
which happens after the preauthorization is obtained.
So you can't simply do this in the same visit
with your patient saying,
ah, we're gonna do this genetic test.
Order the genetic test, send him to the laboratory.
You can't do that.
You have to have 'em come back
for a blood or some kind of sampling.
And that's just the way things work
with insurance these days.
Then you have to review the report
when you get it back.
Review it carefully and go through the elements
that we talked about in this lecture.
And then, if you're not really clear about it,
consult a genetics professional.
You can either refer the patient
or you might be able to just call us up
and say, you know, can you look at this
and answer a couple of quick questions for me?
Usually we're willing to do that.
So, I'm gonna stop here,
because that's been a really lot of information.
As I said earlier,
I don't expect you to remember all of this,
I want you to be exposed to it
because this is all coming down the pike.
Geneticists are used to it.
It's gonna be seen more and more in primary care
and in other specialties.
And so, it's important to really have
this grounding in it so that you can develop
and learn further in the future.
I'm Dr. Bob Wildin, thank you for learning.