Download Postdoctoral Research Associate

Consanguinity and Genetics
PhD thesis title:
Population and family based studies of Consanguinity:
Genetic and Computational approaches
“Intermarriage has chiefly caused weakness of character leading to drink,
not lack of brains or a certain amount of physical strength, but a very inert
and lazy disposition”
Davenport C.B. (1911)
Quoting a correspondent in the Island of Bermuda
A. Mesut Erzurumluoglu
[email protected]
Contents




Intro to Consanguinity
Genetic effects of consanguinity per se
Theoretical section on using
Consanguineous populations
Rare diseases
◦ Primary ciliary dyskinesia
◦ Intellectual disability (Autosomal recessive)
◦ Papillon-Lefèvre syndrome
 Proxy molecular diagnosis


Guide: Identifying highly-penetrant variants
Discussion
Consanguinity

Union between second cousins or closer
◦ Mostly first cousins (F= 0.0625, ~70%)
e.g. Amish,
Mennonites,
Hutterites
Hamamy et al, 2011
Consanguinity
Example of difference
between union of (a)
unrelated (b) related
individuals

Everyone possesses LoF
variants
◦ Rare
◦ Unique to you/your
family
◦ Heterozygous

Erzurumluoglu et al, 2016
P(Hom|Unr) ≈ 0
Consanguinity and health

Can inflate risk of AR disorder

P(Hom|Cons) ≈
0.0625
Erzurumluoglu et al, 2016
Figure from PhD thesis
Why people marry their cousins?

Socio-Economic
◦ Keep money ‘within the family’
◦ Viewed as ‘less risky’
◦ Lower mahr* or dowry^


Ethno-religious
Tradition/culture
◦ Follow your parents’ example
◦ Do as you’re told



Power/politics
Family values/education
Geography
*mandatory payment (Islamic marriage), in the form of money/possessions paid by the groom
^transfer of parental property to groom at the marriage of a daughter (mostly Hindu tradition)
Contents
Intro to Consanguinity
 Genetic effects of consanguinity per se
 Theoretical section on using
Consanguineous populations
 Rare diseases

◦ Primary ciliary dyskinesia
◦ (Autosomal recessive) Intellectual disability
◦ Papillon-Lefèvre syndrome


Guide: Identifying highly-penetrant variants
Discussion
Consanguinity and health

Outbred offspring v First cousins
◦ Rarer the variant/disease, more distinct the
effect of the consanguinity
Hom FH
Erzurumluoglu et al, 2016
Consanguinity and health

Consanguineous v non-consanguineous
◦ Confounded by many environment and health
related factors
Erzurumluoglu et al, 2016
Genetics of Consanguinity

Rare diseases
◦ Rarer the disease, more apparent the effect

Common-complex disorders/traits
◦ Inconclusive evidence!
 Height, cognitive ability, educational attainment and FEV1?
(Joshi et al, 2015, Nature)
 Directional dominance?
◦ Genetic aetiology of disease (Gibson et al, 2012)
 Rare variant model – highest effect
 Broad sense heritability model – lower, variable
 Dependent on significance of GxE and GxG factors
 Infinitesimal model – lowest effect
 Offspring of 1st Cousins: Genome’s 15/16 ‘outbred’
Erzurumluoglu et al, 2016
Contents
Intro to Consanguinity
 Genetics effects of consanguinity per se
 Theoretical section on using
Consanguineous populations
 Rare diseases

◦ Primary ciliary dyskinesia
◦ (Autosomal recessive) Intellectual disability
◦ Papillon-Lefèvre syndrome


Guide: Identifying highly-penetrant variants
Discussion
Quasi-reverse genetic (QRG)
studies in human populations?

Model organisms
◦ Sometimes not applicable to humans
 Acquired different functions (or lost them)
 No homologs in model organisms
 ‘Orphan’ genes (~1100 human-specific)

Consanguineous populations
◦ Elevated levels of homozygosity
◦ Naturally occurring ‘complete knockouts’

Reverse genetics: Genotype -> Phenotype
◦ Clinical phenotype ascertainment bias
◦ Random sampling
 Whole-exome sequencing (or custom SNV array)
QRG studies (2)

McArthur DG et al., 2012
◦ Consequence: Missense, Stopgain, splice-site
acceptor/donor, frameshifting indels
◦ Arbitrary conservation score thresholds
◦ Most are in heterozygous form
◦ “The more interesting a result appears to be, the
more likely it is to be false” – McArthur DG

Icelandic study (Sulem et al, 2015)
◦ Largest (n>100k, 2k whole-genome sequenced)
◦ 1,171 genes ‘completely’ knocked-out (n=6,795)
QRG studies (3)

“LoF” mutations?
◦ Not much functional analysis to support their
claims
◦ Two different ‘LoF’ definitions
 Variants which reduce expression of a gene
 Variants which completely inactivate a gene when
homozygous
◦ ‘Predicted high impact’ (PHI, Φ) mutations
 Rare stopgains, frameshifting indels, missense, splice-site
acceptor/donor variants, start loss

All potential SNV stop-gains (~4.5m)
◦ Homozygous
◦ Nonsense mediated decay (NMD)
Erzurumluoglu et al, 2015 and 2016
No of PHI SNVs in the (reference)
genome
Next: Suitable population
Erzurumluoglu et al, 2016
Most suitable population

Riyadh, Saudi Arabia
◦ Highly consanguineous (~50%)
◦ Long tradition of consanguinity
 Higher F values
◦ Lots of recent migration
 Rich gene pool
◦ Migrants are also consanguineous
◦ Good infrastructure for collaboration
Source:
Wikimedia
All known disease causal variants

All known (highly penetrant) disease causal
variants
◦ Mendelian diseases/types

Highly penetrant protective variants?
◦ Known autosomal dominant mutations


Protein-protein interactions?
Molecular biology
◦ NMD mechanism
◦ Dosage compensation?
 Expression of het v homs (for stopgain and wild-type)
◦ Inbreeding de-repression in humans?
Potential findings
Disease
causal genes
Non-essential
genes
Non-essential
exons/parts
Protective
variants
Erzurumluoglu et al, 2016
Informative
Heterozygotes
Non-essential
regions
Also:
 Proxy molecular diagnoses (e.g. Erzurumluoglu et al, 2015)
 Nullizygous CNVs (e.g. Khalak et al, 2012)
Potential findings

BRCA2 and APC are well established
(dominant) disease genes for
breast/ovarian cancer and colon cancer
respectively
◦ Human knockouts for BRCA2 (primordial
dwarfism) and APC (severe limb
malformation) have very different phenotypes
from those of the established dominant
phenotype in haploinsufficient individuals
Potential findings

Single/few instances of highly penetrant mutations can inform
population based studies
◦ (Hom) Familial hypercholesterolemia (LDLR gene)
 Highly informative about role of LDL-C in cardiovascular disease
 Develop coronary heart disease by the time they’re 55
◦ PCSK9 knockouts protect individuals from cholesterol-driven
cardiovascular diseases
Harper et al, 2015, Nat Rev Genet
Potential findings

Analbuminaemia
◦ Metabolic defect characterised by an impaired
synthesis of serum albumin
◦ Albumin is the most common serum protein (ALB
gene)
◦ Benign condition

SNPs falling near Mendelian forms of certain
diseases
◦ Nephrotic syndrome
 Monogenic
 Multifactorial complex
Potential findings

Environmental factors can also be
important determinants
◦ FUT2 knockout may lead to (clinically
consequential) B12 deficiency only in nutrition
deficient states
Contents
Intro to Consanguinity
 Genetics effects of consanguinity per se
 Theoretical section on using
Consanguineous populations
 Rare diseases

◦ Primary ciliary dyskinesia
◦ (Autosomal recessive) Intellectual disability
◦ Papillon-Lefèvre syndrome


Guide: Identifying highly-penetrant variants
Discussion
Primary Ciliary Dyskinesia (PCD)
•
•
•
Autosomal recessive
Abnormal cilia function and/or structure
Chronic sino-pulmonary infections
Lee, 2010, Gene
CCDC151

Story
◦ Found homozygous p.E309* in CCDC151 in
one PCD patient
◦ Model organisms: Jerber et al, 2014
◦ Replicated by: Hjeij et al, 2014

Differences in respiratory cilia
◦ Outer dynein arms missing in Hjeij et al
◦ Inner and outer dynein missing in Jerber et al
(and our results)
DNALI1

Story
◦ Found homozygous p.R263* in DNALI1 in one
PCD patient
◦ Another PCD diagnosed sibling
 Heterozygous for the variant


Should not trigger NMD
Possible explanations
◦ Not contamination (repeated)
◦ Mosaicism?
◦ Other mutations?
 Monogenic cause
 Bigenic cause
DNALI1
(Predicted) Wild-type DNALI1p structure
(Predicted) Mutant DNALI1p structure
Images created using Robetta
Images created using STRING
Control
Patient 1b
Patient 1a
Patient 2a
Patient 2b
Figures from PhD thesis
Other genes

DNAAF3 - homozygous p.R136*
◦ Already reported – Mitchison et al, 2012

MNS1 - homozygous p.M263T
◦ Novel variant in novel gene – inconclusive

Same individual
◦ HEATR2 - homozygous p.G734Afs*2
 Novel variant in known gene – inconclusive
◦ LRRC48 - homozygous p.E328*
 Novel variant in novel gene – inconclusive
Intellectual disability

Widely used threshold: IQ < 70
◦ Affects ~2% of global population (all forms)


Autosomal recessive type
Dense SNP array data – 23andme
◦ 13 family members (6 affected)

Autozygosity mapping
◦ D. Pike’s algorithm for determining LRoHs
◦ LRoHs viewed in IGV

Replicated by Alazami et al, 2013
◦ Found ADAT3 to be the causal gene (p.V128M)
ADAT3
Figures from PhD thesis
Papillon-Lefèvre syndrome



Autosomal recessive
Premature loss of the primary and secondary teeth,
palmoplantar hyperkeratosis
All caused by mutations in CTSC (>70 reported)
Source URL: www.rxdentistry.net
Proxy molecular diagnosis
Het for
p.G300D in
CTSC
8/9 chance that at
least one PCD
affected sibling is
heterozygous for PLS
causal variant
PCD
(+WES)
Erzurumluoglu et al, 2015
PLS
(-WES)
Proxy molecular diagnosis (2)
Validation of NM_001814.4:c.899G>A:p.(G300D) in all family members using *ARMS-PCR
L1-L6: using allele-specific (AS) primer for wild type. L7-L12: using AS primer for mutant.
L1&7: Mother. L2&8: Father. L3&9: PLS Proband. L4&10: PLS Affected brother. L5&11: Unaffected sibling – homozygous for
CTSC wild type allele. L6&12: PCD affected sibling who is a carrier for PLS. Ladder’s three bands are 100bp (bottom),
200bp and 300bp (top)
*Amplification refractory mutation system-polymerase chain reaction
Erzurumluoglu et al, 2015
Contents
Intro to Consanguinity
 Genetics effects of consanguinity per se
 Theoretical section on using
Consanguineous populations
 Rare diseases

◦ Primary ciliary dyskinesia
◦ (Autosomal recessive) Intellectual disability
◦ Papillon-Lefèvre syndrome


Guide: Identifying highly-penetrant variants
Discussion
Guide to analysing NGS data

Story
◦ BioRxiv – preprint service

Identifying highly-penetrant disease causal
variants
◦ Mendelian v Common complex
◦ Outbred v Consanguineous offspring




Systematically filtering/ranking variants
Tools/databases to use
Data formats to expect at each step
AutoZplotter – visual autozygosity mapper
Erzurumluoglu et al, 2015
Guide to analysing NGS data
Mendelian
Erzurumluoglu et al, 2015
Common-complex
AutoZplotter
p.E309* in CCDC151 was located within a LRoH region (~17Mb) on chr 19
Erzurumluoglu et al, 2015
Contents
Intro to Consanguinity
 Genetics effects of consanguinity per se
 Theoretical section on using
Consanguineous populations
 Rare diseases

◦ Primary ciliary dyskinesia
◦ (Autosomal recessive) Intellectual disability
◦ Papillon-Lefèvre syndrome


Guide: Identifying highly-penetrant variants
Discussion
Discussion Points
Consensus on ‘loss of function’ variants?
 Family based studies can be applicable to
population as a whole

◦ Familial hypercholesterolaemia
◦ Gene function: Mendelian v GWAS

Stopgain SNV array feasible?
◦ Pilot study – 100 individuals?
◦ Grant proposal
 “Screening for all possible single-nucleotide
nonsense variants in consanguineous populations”
Discussion Points (2)

Gene to phenotype
◦ ‘Black box’ in between
◦ Rare diseases

Genetic counsellors v Consanguineous
couples
◦ Lack of understanding on both sides
◦ Grant proposal - submitted
 “Taking an interdisciplinary approach to reducing
the burden of consanguineous marriages in Eastern
Turkey”
Conclusions
Consanguineous populations ‘as a whole’
can potentially be a great source for
identifying (real) LoF variants in a
homozygous state
 Educating not just the public, but genetic
counsellors (about sociological aspects)
and sociologists (about genetics of
consanguinity) can help reduce the
burden of diseases due to consanguinity

Brain teaser
F=?
Email: ame26
Appendices
Comparison of techniques
Consanguinity and Religions
Consanguinity and Law
Methods (PCD)
- 25 known human PCD genes (from literature)
- ~2000 ‘suspected’ Ciliome genes (from ciliome.com)
- Pipeline makes use of VCF files (both SNPs & Indels)
and VEP annotations
- Autozygous regions
- Predictions from FATHMM, SIFT, Polyphen-2
- Made use of dbSNP, EVS, ExAC and1000GP data
- GERP scores, STRING and Robetta predictions
- Search literature for biological plausibility
- Confirm variant status in parents and other siblings
- PCR & Sanger Sequencing
- PCR-RFLP or ARMS-PCR
Methods (PLS)

Screen the CTSC gene
◦ Heterozygous
◦ Rare or absent in public databases
◦ High impact mutation (e.g. stop gain, splicesite acceptor/donor, deleterious missense)
◦ Conservation
◦ Literature
◦ Confirm variant status in parents and PLS
affected siblings
PCR
ARMS-PCR
Amplification refractory mutation system-polymerase chain reaction
Answer