snp allele analysis: upd and then some - millennium science · snp allele analysis: upd and then...
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SNP allele analysis: UPD and
then some
Hutton M. Kearney, PhD , FACMG
Copy number + SNP microarrays
AGILENT AFFYMETRIX
OGT ILLUMINA
Affymetrix experience
• SNP 6.0 clinical use from 2008-2011
• CytoScan HD alpha testing site Spring, 2011
– Product evaluation, protocol and performance feedback provided
– “Proof of principle” exchanges with more than 100 laboratories
• CytoScan clinical service launch July, 2011
• Over 2,000 samples run on this platform
• Tissues types validated:
– peripheral blood
– buccal
– villi/POC, amniotic fluid, skin
– also by proxy: tumors, bone marrow, leukemic bloods
Allele detection (Affy simplified)
A
B
Probe designed specific to “A” genotype
Probe designed specific to “B” genotype
A
B
A
B
A
B
AA genotype BB genotype AB genotype
0.5 + 0.5
0.5 + 0.5
0.5
0.5
SNP allele calls
Each point represents a single SNP interrogated by “A” + “B” allele probes
Arbitrary fluorescence unit of haploid locus (single allele) = 0.5 Genotype “AA” = [0.5+0.5] – [0] = 1
Genotype “AB” = [0.5] – [0.5] = 0
Genotype “BB” = [0] – [0.5+0.5] = -1
AA
BB AB
We don’t typically genotype these samples, but instead derive general information regarding allele state
[A]-[B]=?
1 0
-1
Allele difference pattern supportive of deletion CNV
Deletions
A
B
Allele difference pattern supportive of duplication CNV
AAA
BBB
AAB
ABB
Allele difference pattern in large region of homozygosity
Which is it, LOH, AOH, ROH, LCSH?
• LOH: loss of heterozygosity – This term describes an event where heterozygosity (once
present) is now absent – Copy number neutral or deletion
• AOH: absence of heterozygosity – This term describes an observation where no heterozygosity is
present – Copy number neutral or deletion
• ROH: runs of homozygosity – This term is specific to copy number neutral homozygosity – Does not apply to hemizygous deletions
• LCSH: long contiguous stretch of homozygosity – This term is interchangeable with ROH
Homozygosity-significant? • Consanguinity/ Isolated ethnic population
– Relevant only to narrow focus on single-gene (recessive) disorders
– Rarely (or not so rarely…), uncovers cases of abuse/incest as an incidental finding
• Uniparental disomy – Inheritance of both homologues from the same parent
• Trisomy rescue, mitotic recombination (segmental)
– Implications for homozyosity, undetected mosaicism, as well as abnormal phenotypes due to differential methylation in maternal/paternal germline
Clinical utility of homozygosity detection
References:
Kearney, HM, Kearney, JB, and Conlin, LK. Diagnostic implications of excessive homozygosity detected by SNP-based microarrays: consanguinity, uniparental disomy and recessive single-gene mutations. Clinics in Laboratory Medicine 31(4):595-613, 2011
Papenhausen P, et al. UPD detection using homozygosity profiling with a SNP genotyping microarray. Am J Med Genet 155A:757-68, 2011
• Approximately 3% of our clinical cases have excessive homozygosity – <1% of our cases represent uniparental disomy
– Most LCSH is consanguinity (occasionally abuse suspected)
– ~10% of these cases (UPD or consanguinity) have successful autozygosity mapping to confirm homozygous mutation responsible for recessive disease.
Homozygosity reporting criteria
• LCSH is filtered at ≥3 Mb to unclutter the analysis
– Most individuals have numerous LCSH < 3 Mb totaling up to 2-5% of genome
• Any LCSH greater than 3-15 Mb is scrutinized
– Common/recurrent LCSH disregarded (maintain BED file) – Single chromosome involved?
• Consider UPD and associated mechanisms • Many (or most!) are not associated with UPD
– Throughout genome? • We report cases with > 2% autosomal LCSH (>3 Mb)
– Consider autozygosity mapping for recessive conditions in all cases
Isolated LCSH leading to UPD detection
UPD: Trisomy rescue mechanism
Gametes
Somatic Tissue
Zygote
disomy trisomy
Normal Trisomy “rescue”
UPD
OR
Non-disjunction to restore disomy
Meitoic recombination will generate regions of iso- and heterodisomy
Meiosis I nondisjunction (heterozygous at centromere)
Figure: Kearney et al (2011) Clinics in Laboratory Medicine 31(4):595-613
M I
M II
Meiosis II nondisjunction (homozygous at centromere)
Figure: Kearney et al (2011) Clinics in Laboratory Medicine 31(4):595-613
M I
M II
UPD: Trisomy/monosomy rescue
Figure: Kearney et al (2011) Clinics in Laboratory Medicine 31(4):595-613
Not all UPD events generate LCSH
Heterodisomic vs Isodisomic UPD
Heterodisomic segments are still UNIPARENTALLY derived (and methylated as such)…the placement of LCSH relative to imprinting loci is irrelevant when whole-chromosome UPD is involved.
A sampling of UPD15 and associated LCSH patterns (courtesy of Sarah South/ARUP)
15 Mb
6 Mb
19 Mb
All three cases were confirmed UPD 15 by methylation – Prader Willi
More UPD15 (Proof of principle study courtesy of Stuart Scott/ Mount Sinai)
Homozygosity around the centromere suggests MII error
UPD15 (trio analysis and mechanism)
mother
proband
LCSH block seen in mother (also in common LCSH track maintained by lab)
Tracking AOH experience
AOH block seen in >5% of all cases
Trio analysis (heteroUPD segment)
HeteroUPD: One parent will have
apparent violations of paternity; all
alleles will agree with UPD parent (both parental
alleles inherited)
Trio analysis (isoUPD segment)
IsoUPD: One parent will have
apparent violations of paternity: all
proband’s alleles will be homozygous
and dervied from UPD parent
Small regions of LCSH associated with UPD (Proof of principle study courtesy of Erik Thorland/Mayo Clinic)
~5 Mb terminal homozygosity on chromosome 15; confirmed PraderWilli syndrome
Segmental UPD
When UPD is created through somatic structural events (mitotic crossover, break-induced replication), it DOES matter whether the region of homozygosity maps to imprinted loci. e.g.: segmental UPD 11p (usually mosaic) is common in Beckwith Wiedemann syndrome.
Figure: Kearney et al (2011) Clinics in Laboratory Medicine 31(4):595-613
Single segment LCSH; consanguinity?
26 Mb LOH stretch on chr 10 Parents very distantly related (3rd cousins once removed)
27 Mb LOH stretch on chr 17 Parents very distantly related (2rd cousins once removed)
Carriers of balanced rearrangements that suppress recombination ???
11 meiotic exchange opportunities
9 meiotic exchange opportunities
Genome-wide LCSH seen in consanguinity
Figure: Kearney et al (2011) Clinics in Laboratory Medicine 31(4):595-613
% Autosomal LCSH
751,053 ÷ 2,881,033 = ~26% autosomal LCSH (>3 Mb)
% Autosomal LCSH
Relationship Degree of Relationship
Coefficient of Inbreeding for offspring
% Expected homozygosity
Full siblings First 0.25 25%
Parent/child First 0.25 25%
Half siblings Second 0.125 12.5%
Uncle/niece or Aunt/nephew
Second 0.125 12.5%
Double first cousins Second 0.125 12.5%
Grandparent/ grandchild Second 0.125 12.5%
First cousins Third 0.0625 6%
First cousins once removed
Fourth 0.03125 3%
Second cousins Fifth 0.015625 1.5%
(assuming outbred population…)
Chromosome ~start ~stop ~ size (Mb)
1 52 76.5 24.5
2 0.6 11.2 10.6
2 169.5 175.3 5.8
2 220.3 238.7 18.4
3 67.7 77.8 10.1
5 135.5 146 10.5
5 159 173.5 14.5
6 12.3 20.7 8.4
8 26.3 53.5 27.2
8 128 146.2 18.2
10 62 82.3 20.3
12 5.2 10.2 5
12 52.8 60.7 7.9
15 18.5 30.7 12.2
15 55.4 72.6 17.2
TOTAL: 210.8
1-22 TOTAL 2,868
% LCSH
(conservative
estimate)
7.4%
Estimation of degree of parental relationship
Presumed first cousins, confirmed by family history
1/2
1/8
1/16=6.25%
Our method: ∑ autosomal LCSH >3Mb in size/total autosomal length
Previous language for consanguinity cases >2% IBD
“…Notably, numerous independent regions of
apparent homozygosity were detected in this
specimen, encompassing greater than 18% of
this individual's genome. This result is not
diagnostic of, but raises the suspicion for the
possibility of a recessive single gene disorder…”
American College of Medical Genetics and Genomics standards and guidelines for reporting suspected
consanguinity as an incidental finding of genomic testing
Genetics in Medicine (in press) C. Rehder, K. David, B. Hirsch, H. Toriello, C. Wilson, and H. Kearney
“The guidelines presented here are designed to assist clinical laboratories in the management and reporting of microarray and exome/genome sequencing findings that suggest parental consanguinity, with a primary focus on detection and reporting language.”
ACMG recommended reporting for consanguinity >10%
“Several large regions of homozygosity (_ Mb or larger) were detected, encompassing >_% of the genome. Although this result is not diagnostic of a specific condition, it raises the possibility of a recessive disorder with a causative gene located within one of these regions. Additionally, these results could indicate a familial relationship (first or second degree) between this individual’s parents. A genetics consultation is recommended.”
Case of consanguinity for discussion
• Newborn born to a 15 yo
• Child diagnosed with pseudohypoaldosteronism – Defect in sodium transport with
salt wasting, FTT, cardiac arrythmia (with hyperkalemia)
• Rare recessive seen in mainly consanguineous families
• CMA revealed 32% genomic homozygosity, SCNN1A gene included
• Molecular testing for SCNN1A revealed homozygous mutation, predicted deleterious
SCNN1A: chr12:6,326,272-6,356,784
Ethical considerations
• Results discussed with mother, she disclosed the FOB was her paternal half brother (reportedly consensual, also a minor)
– Half brother would expect to yield 12.5% LOH…32% LOH seen in this child
– How certain is this prediction for 1st degree?
• Case was discussed with legal team at Mission, and no further follow up was recommended.
A second case for discussion…
• 17yo with cleft lip, juvenile arthritis, hypermobility
• CMA revealed >18% homozygosity
– 1st degree (25%) vs. 2nd degree (12.5%)
• Very unusual situation:
– mother was adopted with no family history available
– father was raised in foster care, but he reports large and detailed biological family history
Ethical considerations
• Possible siblings/ half-sibs?
• Happily married with no knowledge of relationship
• No future children (mom with hysterectomy)
• Not diagnostic of any specific condition
• Family was not informed of homozygosity
Considerations for consanguinity detection
• Serious ELSI implications
• Homozyosity measurement is an indirect measure of consangunity and does not substitute for a paternity test
– Caution when implying degree of relationship!!
• Parents should be counseled clearly BEFORE test is administered, preferably with formal consent form upfront
Identification of homozygous mutations in cases with excessive LCSH
(IBD or UPD)
Pop quiz
Is it helpful to include consider regions of homozygosity on the X in a female
when performing autozygosity mapping???
Autozygosity mapping: Case 1
• 35 yo female
• Retinitis pigmentosa
• Hearing loss
• Suspected Usher syndrome type unknown (9+ targets)
• CMA revealed no copy number imbalances, but several large stretches of LSCH seen (below reporting criteria of 2%)
• LCSH regions checked for Usher-related genes
USH2A homozygous mutation within region of LCSH
Using .bed files to aid in autozygosity mapping-Joubert syndrome example
Autozygosity mapping: Case 2
• 22 yo male referred for dev delay
• 10% of genome homozygous
• Follow-up with family revealed hx of abuse
(uncle-niece)
– Predicted level at 12.5%
• Several cells showed abnormalities involving the pericentromeric region of chr 1
• These abnormalities are hallmarks of ICF syndrome – Immunodeficiency-Centromeric
Instability-Facial Anomalies Syndrome
– less than 30 cases reported world-wide
• Follow-up with physician revealed that patient suffered lifetime of immunodeficiency
Radial formation
Isochromosome 1p
Information from chromosome screen pointed a new finger…
DNMT3B in region of homozygosity
DNMT3B
• G587: Highly conserved residue (human, mouse, zebrafish) – Located in catalytic domain (methyltransferase)
• Predicted deleterious mutation in protein prediction models for G587D substitution
DNMT3B ICF mutation spectrum
Modified from: http://bioinf.uta.fi/DNMT3Bbase
G587D
patient
control
DNMT3B sequencing revealed a novel homozygous mutation
www.ccs.miami.edu/cgi-bin/ROH/ROH_analysis_tool.cgi
Genet Med advance online publication 25 October 2012
Acknowledgements
Fullerton Genetics Center
• Joseph B. Kearney, PhD
• Microarray technologists:
– Pablo Sagaribay
– Renee Casey
– Connie Monroe
– Melissa Shook
• Bill Allen, MD
• Ellen Boyd, MD
• Fullerton genetic counselors
Fullerton array team
Colleagues (data presented)
• Laura Conlin, PhD (CHOP)
• Stuart Scott, PhD (Mt. Sinai)
• Sarah South, PhD (ARUP)
• Erik Thorland, PhD (Mayo)
Affymetrix
• Richard Shippy