mRNA will go to the cytoplasm and be translated into the Facto
IX protein, this point mutation has the same effect as a frameshi
mutation (an insertion or deletion of 1 or 2 base pairs). Every amin
acid is changed in the Factor IX protein after the mutation. Peopl
with this mutation make normal Factor VIII protein. Their Factor I
i
protein contains a normal version of the part of the protein code
for by the first three exons. Eleven amino acids of the fourth exo
are produced, but they are completely different from the norma
Blood clotting is a multistep
mRNA will go to the cytoplasm and be translated into s
the
Factor
eque
nce. After these 11 amino acids, there is a stop codon, so mos
process,IX prote
requiring
a tion
cascade
of
in, this point muta
has the same e
ffect as a frameshift
of
the
fourth exon aswell asthefifth, sixth, seventh, and eighth exon
mutation (an insertion or deletion of 1 or 2 base pairs). Every amino
chemical
reactions. An enzyme
acid is changed in the Factor IX protein after the mutation.
eople translated into protein at all (Figure 3). This truncated Facto
arePnot
with this
mutation
ke normal Fareactions,
ctor VIII protein. Their Factor IX
catalyzes
each
ofmathese
IX
prote
protein contains a normal version of the part of the prote
in code
d in is not functional, resulting in severe hemophilia. Ther
and a gene
codes
for. Eleeach
ofacidsthese
for by the
first three exons
ven amino
of the fourth exon
a
re
6
billion
base pairs in a diploid human genome of 46 chromo
are produced, but they are completely different from the normal
enzymes.
Genes
the
X, thereis astop codon,
Figure
1: ble
Alexi
sequence
. After theseon
11 amino
acids
so mos
some
s.t It is
remarka
that a single-base-pair change, in a genom
of thefourth exon aswell asthefifth, sixth, seventh, and eighth exons
chromosome
code
two
of
the
enzymes
of
6
billion
ba
s
e
pa
irs
ca
n result in such a devastating disease. Eve
arenot translated into protein at all (Figure3). This truncated Factor
IX prote
in is
not functiona
l, resulting in severe hemophilia
Thereroya
in .this
l family, no
hemophilia
c malelived past histhirties. A dr
required
for
the
blood-clotting
cascade,
Factor
VIII
and Factor
are 6 billion base pairs in a diploid human genome of 46 chromola
b
illus
tra
ting
this
cha
nge
is
pre
s
e
nted in theAppendix.
s
ome
s
.
It
is
re
ma
rka
ble
tha
t
a
s
ingle
-ba
s
e
-pa
ir
cha
nge
,
in
a
ge
nome
IX.
of 6 billion base pairs can result in such a devastating disease. Even
This
work
a
ls
o
te
lls
us
which
of the Tsar’s daughters were car
in this royal family, no hemophiliac male lived past his thirties. A dry
lab illustrating this change is pr esented in the Appendix. riers for hemophilia. Alexei, as predicted, had one mutated Facto
Factor VIII
is a large protein, coded for by a gene on the X
This work also tells us which of the Tsar’s daughters were carge
ne on his single X chromosome. Alexandra, his mother an
rie
rs
for
he
mophilia
. Alexei, asIX
predicte
one mutaIX
ted Fa
ctor
chromosome.
Factor
isd,ahadsmaller
protein,
also coded for by a
IX gene on his single X chromosome. Alexandra, his mothe
r and ’s granddaughter, had one normal Factor IX gene and on
Victoria
ughter, had one normal Fa(Figure
ctor IX gene and1).
one Mutations in either of
gene onVictoria
the’s gra
Xndda
chromosome
muta
mutated Factor IX gene. The three oldest daughters – Olga
, Tatiate
na,d Factor IX gene. The three oldest daughters – Olga, Tatiana
these genes
hemophilia
a dsex-linked
and Maria cause
– had two norma
l Factor IX genes and inherited
were not carriers,
a
nd
Ma
riain
– ha
two normal Factor IX genes and were not carriers
whereas Anastasia had one normal Factor IX gene and one mutated
manner.
The
Factor
VIIIwhe
contains
26
or coding
reas Anasta
siaexons,
had one norma
l Factor IX gene and one mutate
Factor
IX genegene
and was afor
carrier (Figur
e 2).
Thismutation can bevery useful when teaching about sex-linked
regions,inhewhereas
the
gene
for
Factor
IX
contains
8
exons.
Fa
ctor
IX
ge
ne
a
nd
wa
s
a
ca
rrie
r
(Figur
e2).
ritance, as well as about introns and their role in the genome.
Most eukaryotic genes are made of exons and introns. The eThis
xons mutation can bevery useful when teaching about sex-linke
code for the order of amino acids in the protein. Introns are DNA
inhe
rita
sequences that are translated into messenger RNA and the
n splice
d nce, as well as about introns and their role in the genome
out before the final mRNA is sent to the cytoplasm. It isMos
estima
dukaryotic genes are made of exons and introns. The exon
ttee
that 40% of the DNA in the human genome consists of introns,
code
for
whereas only ~1.5% of the DNA in the human genome is found in the order of amino acids in the protein. Introns are DN
exons and actuaof
lly code
s for proteins.married
In general, intronAlexandra,
sequence is
Tsar Nicholas
Russia
aregranddaughter
of
sequences that a
translated into messe
nger RNA and then splice
not highly conserved, becausemost mutations in intronsdo not affect
the order of amino aThey
cids in a prote
in and, five
therefore,children,
do not
affectbe
the
Queen
Victoria.
had
four
phenotypically
out
fore
the
fina
l
mR
NA
is
s
e
nt
to
the
cytoplasm. It is estimate
Figure 1. Partial map of the human X chromosome (idiogram
folding or properties of proteins. As aresult, mutations in introns are
2: Partial
copyright 1994Figure
by David Adler,
http://www.pathology.
tha
t
40%
of
the
DNA
in
the
huma
n
ge
nome
consists of intron
normal
ainsboy,
who
had
hemophilia (Figure 2).
usgirls
ually not sand
elected aga
t by naturaAlexei,
l selection. Howe
ver, thenucle
washington.edu/research/cytopages/idiograms/human;
used
map of the human X
otide sequences at the ends of introns arerecognized by the
e
nzyme
s
wheduring
reas only ~1.5%
of the DNA in the human genome is found i
with permission).
The entire
family
was
1918
the Bolshevik
that splice
out the introns
. Theskilled
e sequences in
are highly
conserved
chromosome.
e
xons
a
nd
a
ctua
lly
code
for prote
ins. In general, intron sequence
because mutations in them change the
proteins
Revolution. Recent
discovery of their graves madestheir
tissue
coded for by the genes. As seen in this real case,
not
highly
cons
e
rve
d,
be
ca
us
e
mos
t
mutationsin intronsdo not affec
a muta
tion at the
endRogaev
of an intron canet
cause
a (2009), was technically
available for sequencing. This work,
done
by
al.
the
orde
faulty splice, resulting in an abnorma
l prote
in r of amino acids in a protein and, therefore, do not affect th
Figure 1.because
Partial maponly
of the
human
X chromosome
(idiogram
and
ry serious
diswere
ease.
difficult
tiny
amounts
ofavetissue
available.
This
made
itins
possible,
folding or prope
rtie
s of prote
. Asaresult, mutations in intronsar
In studying a mutation that causes hemocopyright
1994
by
David
Adler,
http://www.pathology.
but difficult, to obtain enough DNAphilia
for
sequencing.
, youaccurate
would expect the muta
tion
occur
ustoua
lly not seSophisticated
lected against by natural selection. However, thenucle
washington.edu/research/cytopages/idiograms/human;
in an exon, a coding
part of the gene, and you
methods,
such as using multiple primers
forused
polymerase
chain reaction and
otide
would expect themutation to changethe
order s
ofequences at the ends of introns are recognized by the enzyme
with permission).
a
mino
a
cids
in
the
prote
in.
This
muta
tion,
howthat splice out the introns. These sequences are highly conserve
massively parallel sequencing methods,
were required.
ever, occurs at the end of an intron and changes
because mutations in them change the protein
the splice site for that intron. This results in a
two-base insertion in the final mRNA, leading
coded for by the genes. As seen in this real case
to the truncated protein discussed here. This
Figure 2. The Russian royal family. Alexandra was a granddaughter of Queen
mutation provides a novel way of discussing the
Victoria. Alexei had hemophilia, so it was assumed that his mother, Alexandra,
a mutation at the end of an intron can cause
nature of introns, how they are spliced out, and
was a carrier. The work of Rogaev et al. (2009) confirmed that Alexandra was a
faulty splice, resulting in an abnormal protei
why the DNA sequences at the ends of introns
carrier and showed that Olga, Tatiana, and Maria were normal, while Anastasia
are critical and highly conserved.
was a carrier.
and avery serious disease.
In studying a mutation that causes hemo
THE AMERICAN BIOLOGY TEACHER
ROYAL HEMOPHILIA
653
philia, you would expect the mutation to occu
in an exon, a coding part of the gene, and yo
This content downloaded from 158.123.151.254 on Tue, 19 Nov 2013 13:17:33 PM
would expect themutation to changetheorder o
All use subject to JSTOR Terms and Conditions
amino acids in the protein. This mutation, how
ever, occurs at the end of an intron and change
the splice site for that intron. This results in
two-base insertion in the final mRNA, leadin
to the truncated protein discussed here. Th
Figure 2. The Russian royal family. Alexandra was a granddaughter of Queen
mutation provides a novel way of discussing th
Victoria.
Alexei
had
hemophilia,
so
it
was
assumed
that
his
mother,
Alexandra,
Figure 3: The Russian royal family. Alexandra was a granddaughter of Queen
nature of introns, how they are spliced out, an
was
a carrier.
Thehad
workhemophilia,
of Rogaev et
confirmed
was a
Victoria.
Alexi
soal.
it (2009)
was assumed
thatthat
his Alexandra
mother, Alexandra,
was aand
carrier.
Thethat
work
of Rogaev
al. Maria
(2009)
confirmed
Alexandra
carrier
showed
Olga,
Tatiana,et
and
were
normal,that
while
Anastasiawas a why the DNA sequences at the ends of intron
carrier,
and showed that Olga, Tataina, and Maria were normal, while Anastasia are critical and highly conserved.
was
a carrier.
Royal Hemophilia
The Russian Royal Family
was a carrier.
THEAMERICAN BIOLOGY TEACHER
ROYAL HEMOPHILIA
Sequencing
Rogaev et al. (2009) compared the sequences of the Factor VIII and Factor IX
genes with the normal human genes, known from the Human Genome Project.
All the exons in both genes were completely normal. They then looked at the
introns, the noncoding parts of the gene. All introns in the Factor VIII gene were
normal. However, the gene for Factor IX contained a point mutation from A to G
in the third base before the end of the intron between exons 3 and 4 (Figure 4).
Figure 4: The mutation that caused hemophilia in Queen Victoria and her descendants. Figure 3A
shows the sequence of DNA in the normal Factor IX gene, with the associated mRNA transcription
and protein translation. Figure 3B shows the sequence of DNA in the mutated Factor IX gene. With
the point mutation (a change of the nucleic acid A to nucleic acid G) is highlighted. Note that this
mutation alters splicing, so that the last two bases of the intron (AG) are now included in the mRNA.
Note that every amino acid after the faulty splice is different, and that after 11 altered amino acids
there is a stop codon, so that the last part of the protein is not produced. The changes in amino acids
are shown in bold. The single-letter abbreviations for the amino acids are as follows: A = alanine, C =
cysteine, D = aspartic acid, E = glutamic acid,
F = phenylalanine, G = glycine, H = histidine, I =
isoleucine, K = lysine, L = leucine, M = methionine, N = asparagine, P = proline, Q = gluamine, R =
arginine, S = serine, T = threonine, V = valine, W = tryptophan, and Y = tyrosine.
Most mutations in introns do not affect either proteins or phenotype. But due to
the location of this mutation, the two bases that are normally the end of the
intron are treated like the beginning of exon 4 instead of being spliced out.
Because this mRNA will go to the cytoplasm and be translated into the Factor IX
protein, this point mutation has the same effect as a frameshift mutation (an
insertion or deletion of 1 or 2 base pairs): every amino acid is changed in the
Factor IX protein after the mutation. People with this mutation make normal
Factor VIII protein. Their Factor IX protein contains a normal version of the part
of the protein coded for by the first three exons. Eleven amino acids of the fourth
exon are produced, but they are completely different from the normal sequence.
After these 11 amino acids, there is a stop codon, so most of the fourth exon as
well as the fifth, sixth, seventh, and eighth exons are not translated into protein at
all (Figure 4). This truncated Factor IX protein is not functional, resulting in
severe hemophilia.
There are 6 billion base pairs in a diploid human genome of 46 chromosomes. It
is remarkable that a single-base-pair change, in a genome of 6 billion base pairs
can result in such a devastating disease. Even in this royal family, no hemophiliac
male lived past his thirties.
This mutation can be very useful in examining sex-linked inheritance, as well as
about introns and their role in the genome. Most eukaryotic genes are made of
exons and introns. The exons code for the order of amino acids in the protein.
Introns are DNA sequences that are translated into messenger RNA and then
spliced out before the final mRNA is sent to the cytoplasm. It is estimated that
of the DNA in the human genome consists of introns, whereas only
~1.5% of the DNA in the human genome is found in exons and actually codes for
proteins. In general, intron sequence is not highly conserved, because most
mutations in introns do not affect the order of amino acids in a protein and,
therefore, do not affect the folding or properties of proteins. As a result,
mutations in introns are usually not selected against by natural selection.
However, the nucleotide sequences at the ends of introns are recognized by the
enzymes that splice out the introns. These sequences are highly conserved
because mutations in them change the proteins coded for by the genes. As seen in
this real case, a mutation at the end of an intron can cause a faulty splice,
resulting in an abnormal protein and a very serious disease.
Figure 5: The Romanov royal family, killed by Bolshevik revolutionists in 1918.
Figure 6: A partial pedigree of Queen Victoria and her descendents.
Figure 5 shows a partial pedigree of Queen Victoria and her descendants. This
family tree can lead to the following interesting questions:
1. Why is there no hemophilia in the present British royal family? Explain using a
Punnett square. Make sure to be clear how the Punnett Square helps your
explanation. 2. Three of Queen Victoria’s daughters had no descendants with hemophilia.
Could any of them have been a carrier? Explain your answer; specifically
explain what the chances are that Helena was a carrier.
3. The Bolsheviks killed the Russian royal family –Nicholas and Alexandra and
their five children, Olga, Tatiana, Marie, Anastasia, and Alexis. Their
graves were unknown until one of the people involved told where they
were in a deathbed confession. At this time, their bodies were found. They
were identified because they had the same mitochondrial DNA as Prince
Philip, the husband of the present Queen Elizabeth. Why was this an
appropriate method of identification, and why didn’t they use DNA from
Queen Elizabeth?
Adapted from: Royal Hemophilia Susan Offner
. The American Biology Teacher, Vol. 75, No. 9 (November/December 2013), pp.
652-656
i