snp allele analysis: upd and then some - millennium science · snp allele analysis: upd and then...

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

Questions?

hutton.kearney@msj.org

Asheville, NC