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Finding the Fault in Nick's Genome
Nick 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 “Nick” to view video at Journal-Sentinel site
The case history
Nick was born October 2004, the third 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 Nick's DNA solve this mystery?
Nick's doctor and other specialists had already tested Nick for a
number of genetic mutations that could cause the observed
symptoms, and found nothing.
Now they wondered if they could sequence Nick'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
Nick's doctors decided to sequence Nick'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 Nick'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, Nick'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 Nick's parents have?
Nick has two older sisters. What stake do they have in Nick'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, what “incidental findings” in Nick's genome
should be notified to the family? For each mutation listed,
indicate “yes” or “no” to informing the family.
1) a mutation in the BRCA1 gene, known to be
associated with early breast cancer
a) yes
b) no
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
Nick's exome sequence was compared to the human reference
genome sequence, to identify differences, called "variants". If
variants are randomly distributed throughout the human genome,
how many variants would be expected in Nick's exome sequence?
Assume that the exome is 2% of the genome, and that humans are
99.9% identical in DNA sequence. Recall from the video or the
readings the size of the human genome.
A) 15,000
B) 60,000
C) 120,000
D) 600,000
E) 3,000,000
Slide 10
Liz Worthey identified 15,272 variants in Nick's exome sequence (Worthey et al.
2011, Genomics in Medicine 13, 255–262), a typical number of variants for exome
sequences from healthy people. Compare this number with the number expected
based on human DNA being 99.9% identical. Which of the statements below are
consistent with this information?
A) Mutations in protein coding sequences are more likely to be eliminated by natural
selection than mutations in the rest of the genome.
B) Mutations occur at a higher rate in non-coding sequences than in protein-coding
sequences.
C) Mutations in exons are more likely to be corrected by DNA repair enzymes than
mutations in introns or intergenic regions.
D) Mutations arise and persist in the human population at equal rates in non-coding
and protein-coding DNA.
Slide 11
Given over 15,000 variants in Nick's exome
sequence, how can we determine which variant is
causing his disease, if any?
Liz Worthey categorized each variant and devised
a scheme to filter them according to their likely
impact on the protein function.
Slide 12
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) an insertion or deletion of a single
nucleotide
4) an amino acid change in the
active site of an enzyme
5) a change of one nonpolar amino
acid to another, in a transmembrane
domain
Slide 13
Which of these criteria would be the least useful in
identifying the mutation (variant) responsible for Nick'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 14
Summary of protein-coding variants
Protein-coding variants
substitutions
Total
Novel
14,886
1,223
insertions
147
65
deletions
239
119
Nonsynonymous (changes
amino acid sequence)
7158
879
Homozygous for early stop
codons
13
2
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 15
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
mutations
1
*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 16
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 Nick, and the
bottom is from Nick's mother. Nick's mother is heterozygous for the
mutation; one copy of her X chromosome has a normal G, but the
other has an A. Nick inherited the A allele from his mother.
Slide 17
In Nick'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 18
What does the XIAP gene do?
It regulates programmed cell death (apoptosis) and the gut
immune system.
Just as Nick'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.
Nick's copy of gene results in a single amino acid change.
Why is this particular change so harmful?
Slide 19
Nick’s mutation changes a highly conserved
amino acid
Alignment of XIAP amino acid sequences from different species, from Worthey et al.
2011.
Nick'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 20
Nick's XIAP mutation is recessive. Nick inherited it from his
mother, who is a carrier with no symptoms. Nick's older
sisters now know they may also have inherited the same
mutation. What is the probability that Nick’s sister is a carrier
of the XIAP mutation?
A) 0
B) 1/4
C) ½
D) ¾
E) 1
Slide 21
With the diagnosis of Nick's XIAP mutation, Nick received
a bone marrow transplant. After surviving a few
harrowing months of recovery, Nick is now free of his
symptoms and enjoying eating steak and pizza and
everything else healthy kids like.
Nick 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 22
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 23
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 24