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Finding the Fault in Nic's Genome
Nic at 4-years age loved Batman. He was also dying of a mysterious and
painful disease. See how geneticists saved his life by sequencing all of his
protein-coding DNA to find the 1-in-billions fault. Learn about our genome,
human genetic variation and how mutations can have very different effects.
Click “Nic” to view video at Journal-Sentinel site
The case history
Nicholas was born October 2004, the fifth child in the
family. Before his 2nd birthday, an abscess formed near his
rectum. Over the next 3 years holes appeared in his colon
and large intestine, and stool leaked into his abdomen. The
symptoms resembled irritable bowel disease (IBD) or
Crohn's disease, but medical, surgical, and diet treatments
all failed to stop his illness. By fall of 2009, Nick had spent
more than 300 days (250 consecutive) at the hospital, and
had suffered through 100 operations. His doctors were
baffled, out of clues, and desperate.
Slide 2
Can sequencing Nic's DNA solve this mystery?
Nic's doctor and other specialists had already tested Nic for a
number of genetic mutations that could cause the observed
symptoms, and found nothing.
Now they wondered if they could sequence Nic's entire genome,
quickly enough to save him, at a reasonable cost?
Slide 3
The human genome project took 13 years and $3 billion to complete a
draft of the first human genome in 2003. However, recent advances in
DNA sequencing technology has dramatically lowered the cost. In
this chart from the NHGRI, Moore's Law is the observation that
computing power doubles every two years.
What was the cost of
sequencing a human
genome in 2009?
a) $100 million
b) $10 million
c) $1 million
d) $100,000
e) $10,000
Slide 4
Nic's doctors decided to sequence Nic's exome, consisting
of all the exons that are expressed as mRNA. The diagram
below shows the structure of eukaryotic genes and how
introns are removed during nuclear RNA processing
Illustration by Jung Choi, April 2015, CC-BY
Slide 5
Recall from the human genome video or your
readings: what percentage of Nic's genome would be
sequenced by exome sequencing?
A) 80%
B) 50%
C) 30%
D) 10%
E) 2%
Slide 6
Although exome-sequencing would save time and money,
Nic's doctors knew they would miss any mutations in non
protein-coding DNA. Mutations in which non-exomic regions
could cause severely reduced amounts of a normal protein to
be made?
a) a mutation in an intron
b) a mutation close to the transcription start site
c) a mutation in an exon
d) a mutation in the DNA after the stop codon
In groups with your neighbors, discuss how each of
these mutations could affect gene expression, or
cause disease.
Slide 7
Ethics of genome sequencing: small group discussion
What questions and concerns would Nic's parents have?
Nic has four older sisters. What stake do they have in Nic's DNA
sequence information? Consider that exome sequencing will
reveal information about all of his protein-coding genes, not
just the genetic basis of his disease. Information about other
genes are sometimes called “incidental findings.”
Slide 8
In your opinion, which of these hypothetical “incidental
findings” in a patient's genome sequence should be notified to
the family? You may select zero, one, or more.
1) a mutation in the BRCA1 gene, known to be
associated with early breast cancer
2) a novel mutation in the BRCA1 gene, of unknown
significance
3) a mutation in the ApoE gene, associated with earlyonset Alzheimer's
4) a mutation associated with high blood pressure and
early coronary disease, treatable with medication and
monitoring.
Slide 9
Over 15,000 variants were found in Nic's exome
sequence. How can we determine which variant is
causing his disease, if any?
Need a scheme to categorize each variant and to
filter them according to their likely impact on the
protein function.
Slide 10
Single base changes can have very different
consequences
With your neighbors, discuss the consequences of the following
mutations in a protein-coding sequence:
1) TCA codon  TCG
2) TCA codon  TGA
3) TCA codon  TCCA
4) TCA codon  TTA
Which of these will result in no
functional protein?
Which will most likely have no
phenotypic effect?
Which might cause the protein to
be misfolded?
Slide 11
Which of these criteria would be the least useful in
identifying the mutation (variant) responsible for Nic's
rare disease?
A) variants that are rare in the human population
B) variants that create in-frame stop codons
C) variants that create frameshifts
D) variants that affect both copies of autosomal genes
E) variants in genes that are known to cause common
human diseases
Slide 12
Summary of protein-coding variants
Protein-coding variants
substitutions
Total
Novel
14,886
1,223
insertions
147
65
deletions
239
119
7158
879
13
2
Nonsynonymous (changes
amino acid sequence)
Homozygous for early stop
codons
Adapted from Worthey et al., Table 1A
Liz Worthey looked for novel variants that crippled both copies of the gene. She
found 2 genes where both copies had early stop codons. But they were in genes
where stop codons are known to occur in healthy people.
Slide 13
Hypothesize that variant is recessive, focus on
damaging mutations
Homozygous or hemizygous*
70
Both alleles predicted to be damaging
17
Novel
8
Altering highly conserved positions
4
Not known to frequently contain deleterious
1
mutations
*hemizygous = having only one copy of the gene, as in X-linked genes in males
Adapted from Worthey et al., Table 1C
Worthey et al. narrowed the candidate mutations to just one, a
single nucleotide substitution in the XIAP gene, located on
the X chromosome.
Slide 14
Mutation in the XIAP gene
The XIAP mutation identified from exome sequencing was verified
using traditional targeted gene sequencing. The top is a sequencing
trace from a healthy control. The second is from Nic, and the
bottom is from Nic's mother. Nic's mother is heterozygous for the
mutation; one copy of her X chromosome has a normal G, but the
other has an A.
Slide 15
In Nic's XIAP gene, a TGT codon is changed to a TAT
codon. What is the amino acid change in the XIAP
protein?
A) T (Thr) to I (Ile)
B) C (Cys) to Y (Tyr)
C) W (Trp) to Y (Tyr)
D) T (Thr) to A (Ala)
E) No change
Notes: TGT is on the coding strand of DNA, which has the same sequence as the RNA; just
substitute U for T. The answer choices show both the single-letter code and the three-letter
abbreviation for amino acids.
Slide 16
What does the XIAP gene do?
It regulates programmed cell death (apoptosis) and the gut
immune system.
Just as Nic's doctors discovered his XIAP mutation, a new
paper reported another mutation in this gene that causes
an extremely rare disease called XLP, inability to fight
Epstein-Barr virus, and death by age 10. The only cure is a
bone marrow transplant.
Nic's copy of gene results in a single amino acid change.
Why is this particular change so harmful?
Slide 17
Nic’s mutation changes a highly conserved
amino acid
Alignment of XIAP amino acid sequences from different species, from Worthey et al.
2011.
Nic's XIAP sequence is the second row from the top (Var_XIAP); the purple arrow
denotes the location of the amino acid change. That species from fruit flies to people all
have a cysteine (“C”) at this position indicates that this amino acid is critical. Nick has a
tyrosine (“Y”) instead of cysteine, making his protein non-functional. As a result, his
intestinal immune system overreacts and causes cell death in his intestinal epithelial cells.
Slide 18
Nic's XIAP mutation is recessive. Nic's mother is a carrier with
no symptoms. For one of Nic's older sisters, what is the
probability that she is also a carrier of the XIAP mutation?
A) 0
B) 1/4
C) ½
D) ¾
E) 1
Slide 19
With the diagnosis of Nic's XIAP mutation, Nic received a
bone marrow transplant. After surviving harrowing
months of recovery, Nic is now free of his symptoms and
enjoying eating steak and pizza and everything else
healthy kids like.
Nic was the first to have a mystery disease diagnosed by
genome sequencing. Since 2009, other genome
sequencing centers have started genome sequencing to
diagnose mystery illnesses. They are able to identify
causal mutations in about 50% of their patients.
Slide 20
Human genetic variation: what to expect if you have
your own genome sequenced
Results from whole genome and whole exome sequencing of healthy
people from all over the world (MacArthur et al. 2012) tell us:





Healthy people have millions of differences in their DNA
sequence.
The vast majority of these variants have no phenotypic effect.
Most (but not all) variants with phenotypic effects will be in the
exome (protein-coding DNA).
Each person has about 100 variants of unknown significance, that
may damage or alter the function of the gene.
Further study is required to determine the effects of these rare
variants of unknown significance.
Slide 21
Bibliography and sources:
Mark Johnson and Kathleen Gallagher. One in a Billion: A boy's life, a medical mystery.
Milwaukee-Wisconsin Journal Sentinel, series published starting Dec 18, 2010. Intro to
series and video introduction here:
http://www.jsonline.com/news/health/111224104.html
MacArthur, DG et al. 2012. A systematic survey of loss-of-function variants in human
protein-coding genes. Science 335: 823-828 DOI: 10.1126/science.1215040
http://www.sciencemag.org/content/335/6070/823.full
Worthey et al. 2011. Making a definitive diagnosis: Successful clinical application of
whole exome sequencing in a child with intractable inflammatory bowel disease.
Genetics in Medicine 13, 255–262.
http://www.nature.com/gim/journal/v13/n3/full/gim9201146a.html
Slide 22