Insights from a Genetic Isolate

Miles Benton

Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia

AGTA 2016 - Pullman Hotel, Auckland, New Zealand

9-12th October 2016


  1. Norfolk Island

  2. 3 2 'Case studies':
    • Phenomescan
    • WGS, founder effects and glaucoma
    • Allele-specific methylation

Norfolk Island

...and the Norfolk Island Health Study...

~6000 member pedigree

40% of current population haplogroup B

Benton MC et al.,: “Mutiny on the Bounty”: the genetic history of Norfolk Island reveals extreme gender-biased admixture. Investigative Genetics 2015, 6:11.

Norfolk Island Health Study

Increased risk of metabolic disorders

WGS, founder effects and glaucoma

Other contributors: Rod Lea, David Eccles, Donia Macartney-Coxson, Heidi Sutherland, Larisa Haupt, Lyn Griffiths

Whole Genome Sequencing

© 2016 Illumina, Inc.

108 core-pedigree members

Illumina HiSeq X10 at the Garvan Institute

Decent amount of data (David Eccles)

QUT HPC facility

Founder Effect Variants

Filtering strategy outline

Total variants:

Functional* variants:

*Functional = predicted to be damaging in 5 in-silico tests (SIFT, POLYPHEN2, MUTATIONTASTER, PROVEAN, MUTATION ASSESSOR)

ACOT4 (Acyl-CoA Thioesterase 4)

  • Regulation of lipid metabolism
  • Amino acid = p.TYR190ASN
  • Freq A = 0.0099 (EUR), 0 (AFR) and 0 (EAS)
  • Freq A in NI = 0.26
  • Rare allele most likely came from England via Bounty Mutineers and then increased in frequency in NI due to founder effect

Founder Effect Variants


There is a higher than usual prevalence of Glaucoma on NI, we explored the WGS data for associated variants

Variant Filtering Strategy
  1. filtered vcf files at 14% MAF
  2. filtered based on genotypic models, both dominant (DOM) and allelic (ALL)
  3. this resulted in 1083 for the DOM model and 1437 for the ALL model
  4. the final filter was aimed to assess functional impact, we chose to go with SIFT and PROVEAN
  5. this returned 1 variant

Variant information

  • Esterase-D (ESD)
  • reference allele = C
  • alternate allele = T
  • MAF CASES = 0.43 vs MAF CONTROLS = 0.05
  • ALL p-value: CC_ALL=2.849-07

Predicted as deleterious/damaging

Gene has been previously associated with Retinoblastoma and Wilson's disease (copper deposits around the cornea)

Allelic-specific methylation

(very recent data)

Other contributors: Rod Lea, Donia Macartney-Coxson, Nicole White, Daniel Kennedy, Heidi Sutherland, Larisa Haupt, Kerrie Mengersen, Lyn Griffiths

Identification of allele-specific methylation profiles across generations
Proof of principle pilot study

  • measuring genome-wide allele-specific methylation (ASM)
    • NGS bisulphite sequencing
    • SeqCap Epi CpGiant (Illumina HiSeq)

  • collected data for 24 NI individuals
    • comprising a close 3 generation pedigree

  • currently generating data for another ~90 samples

  • Fully customised QC and analysis pipeline:
    • fastqc, trimgalore
    • bismark, sambamba, picard tools
    • PileOMeth, R and methylkit

  • parallel processing enabled for local and remote machines

  • filtered at minimum 10 counts
Initial metrics
  • coverage ~40x ave
  • lowest number of CpGs for a given sample is 2.67M
  • highest number of CpGs for a given sample is 7.52M
  • the average across all samples is 3.48M
  • on-target mapping rate across the 24 samples >95% average

~1.12M CpG sites in common across the 24 samples with at least 10 times coverage and on-target



Good concordance between Illumina arrays and BS-seq

Allelic-specific methylation

  • for an initial look-see used methpipe suite
    • estimates ASM at each CpG site based on strand ratios of methylation

  • moving forward we want to use read data:
    • assign parent of origin and then infer ASM
    • gives the actual info rather than just knowing where an ASM event is

  • promising method recently published for mice ASM - SNPsplit (Krueger 2016)

ASM regions

  • Using a custom clustering method we identified ~1800 ASM regions (AMRs) conserved within the pedigree

  • Many of these AMRs map to known and predicted imprinted genes

Visualising ASM

Documented and predicted human imprinted loci are displayed in darkred.

ASM plot of chromosome 20 for a nuclear family (father, mother, son, daugther)

Presence of SNPs

  • 1,127,867 total methylation sites
  • 444,330 have a SNP present on C/G (39.4%)


Considering MAF:

  • 231,452 SNPs have recorded MAF info
  • 12,670 SNPs >= 0.05 MAF
  • 26,935 SNPs >= 0.01 MAF

Epigenetic inheritance

Next thing is to move onto modelling the inheritance...
...with the help of our brilliant Bayesian statisticians!


  • expanding upon our multi-layered NI data

  • identification of founder effect variant associations

  • initial ASM region and imprinting loci identification


NZ collaborators
Lyn Griffiths
John Blangero
Donia Macartney-Coxson (ESR)
Rod Lea
Joanne Curran
David Eccles (Gringene Bioinformatics)
Larisa Haupt
Harald Goring
Geoff Chambers (VUW)
Heidi Sutherland

Michelle Hanna

...the rest of the GRC IHBI lab group
Nicole White
Daniel Kennedy
Kerrie Mengersen

Garvan Institute of Medical Research / Kinghorn Centre for Clinical Genomics Melanie Carless (Texas Biomedical Research Institute) NHMRC

Claire Bellis (Genome Institute of Singapore)

Greg Gibson (Georgia Institute of Technology, USA)

The people of Norfolk Island who who volunteered for this study.

Thank you


PhD scholarship and opportunity
Donia Macartney-Coxson, Mik Black, & Miles Benton