genetic biomarkers in dementias christos kroupis, …...hixson j.e. and d.t. ve rnier “restriction...
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Genetic biomarkers in Dementias
Christos Kroupis, MSc, PhD, EuSpLM
Asst. Prof. Clinical Biochemistry and Molecular Diagnostics
Attikon University General Hospital
Medical School, National and Kapodistrian University of Athens
Talk topics
Dementias
Genetic markers
in Peripheral blood:
Alzheimer’s disease (AD) genes
ApoE genotyping
Frontotemporal Dementia (FTD) genes
in CSF and plasma:
miRNAs
Dementias
Four most common types of dementia:
Alzheimer’s disease Lewy-body dementia
Frontotemporal dementia (FTD)
Vascular dementia
Other causes of cognitive impairment:
Parkinson’s disease (PD), Creutzfeldt-Jakob Disease, Wernicke-
Korsakoff syndrome, Normal-pressure Hydrocephalus (NPH) etc
Genetic markers for AD
Karch CM and AM Goate “Alzheimer's disease risk genes and mechanisms of disease
pathogenesis” Biol. Psychiatry (2015), 17, 43-51
Early-onset (5%) Late-onset (95%) Julianne Moore (“Still Alice”)
APOE GENE 19q13.2
E3 wt
Ε3/Ε3
Ε3/Ε4
Ε2/Ε3
Ε2/Ε4
Ε4/Ε4
Ε2/Ε2
Wu L. and L. Zhao “ApoE2 and AD: time to take a closer look” Neural Regen Res (2016) 11, 412–3
ApoE4 is present in about 20% of most populations; however, goes up to 40-50% of AD patients
4 helices
in N-term
(LDL-R, LPR, SR-BI)
APOE isoforms in AD
APOE4 confers greater AD risk in women
Altman A. et al., “Sex Modifies the APOE-Related Risk of Developing Alzheimer's Disease”
Ann Neurol (2014), 74, 563-74
ApoE is expressed in astrocytes and microglia cells in brain
FDA approved APOE testing
DTC (direct-to-consumer testing) from saliva DNA
Odds ratio for developing AD
APOE carriers site: https://www.apoe4.info/wp/
-dF1/dT (530 nm)
-dF2/dT
(640 nm)
dbSNP rs429358
E4 allele
C112R
dbSNP rs7412
E2 allele
R158C
APOE GENOTYPING: Real-time PCR MELTING CURVE
LightMix (Roche)
CE-IVD Kit
HhaI digest
APOE GENOTYPING: PCR – RFLP
Hixson J.E. and D.T. Ve rnier “Restriction isotyping of human APOE by gene amplification and cleavage with HhaI” J Lipid Res (1990), 31, 545-8
Confirmation method
EQAs important
ISO15189
accreditation
FrontoTemporal Dementia (FTD)
bvFTD SD
PPA (PNFA)
LPA
PSP
CBS
Primary
Progressive
Aphasia
behavioral (frontal) variant
FTD-MND (Motor Neuron Disease)
FTD/ALS
FTDP
-17
Josephs KA. Frontotemporal dementia and related disorders: deciphering the enigma. Ann Neurol 2008;64: 4-14
Kertesz A, et al. The evolution and pathology of frontotemporal dementia. Brain 2005;128: 1996-2005
Semantic
Logopenic
Progressive
Supernuclear
Palsy
Cortico-
Basal
Syndrome
Amyotrophic Lateral Sclerosis
either familial (30-50%) or sporadic, as common as Alzheimer's in
people<65 years old (majority of cases between 45 – 64 y)
FrontoTemporal lobar
Degeneration (FTLD) Anatomy
FTLD-TAU FTLD-U
[ubiquitin(+)]
FTLD-TDP
FTLD-FUS
FTLD-UPS
[ATYPICAL
TDP(- )/
FUS(-)]
Histology (IHC) +
Goedert M, et al. FTD: implications for understanding Alzheimer disease. Cold Spring Harb Perspect Med 2012;2: a006254
Rohrer JD. Structural brain imaging in frontotemporal dementia. Biochim Biophys Acta 2012;1822: 325-32
Rabinovici GD and Miller BL. FTD: epidemiology, pathophysiology, diagnosis and management. CNS Drugs 2010;24: 375-98
Genetic markers for FTD
MAPT FTLD-TAU FTLD-TAU
GRN
C9orf72 FTLD-TDP
TARDBP
VCP
FUS FTLD-FUS
CHMP2B FTLD-UPS
SQSTM1
TMEM106B
SQSTM1 ΣΠΑΝΙΕΣ ΠΕΡΙΠΤΩΣΕΙΣ
PSEN1, PSEN2
PRNP
FTLD-TAU
FTLD-TDP
FTLD-FUS
FTLD-UPS
GENES PROTEINOPATHY
rarely
Takada LT. “The Genetics of Monogenic Frontotemporal Dementia” Dementia & Neuropsychologia (2015), 9: 219-29
Goldman JS, et al., “Frontotemporal dementia: genetics and genetic counseling dilemmas” Neurologist (2004),10, 227-34
FTD Diagnosis
MRI
PET /
SPECT
NEURO-
PSYCH
OLOGY
CSF
PLASMA
SERUM
FTLD-TAU PiD MAPT
Non-classified
cases
Type A GRN
FTLD-TDP Type D VCP
TARDBP
Types A/B C9orf72
Non-classified
cases
FTLD-FUS aFTLD-U FUS
FTLD-UPS CHMP2B
Histopathology Subtypes Related genes Clinical phenotypes
bvFTD, FTDP-17, PNFA, SD, CBS,
PSP, FTD/MND (ALS)
bvFTD, FTDP-17, PNFA, CBS
bvFTD, FTD/MND(ALS), SD, CBS
bvFTD, FTD/MND(ALS), SD, CBS
bvFTD, FTD/MND(ALS)
bvFTD, FTD/MND(ALS)
bvFTD, FTD/MND(ALS), PNFA
GENE
TESTING
Snowden J, et al. Frontotemporal lobar degeneration: clinical and pathological relationships. Acta Neuropathol 2007;114: 31-8
Warren JD, Rohrer JD, Rossor MN. Clinical review. Frontotemporal dementia. BMJ 2013;347: f4827
Benussi A, et al. Phenotypic Heterogeneity of Monogenic Frontotemporal Dementia. Front Aging Neurosci 2015;7: 171
Neary D, et al. Frontotemporal dementia. Lancet Neurol 2005;4: 771-80
C9orf72 gene (9p21.2)
Toxic RNA
(G-quadruplexes)
Dipeptide Repeat
Toxic peptides
(Gly-Ala, Gly-Pro, Gly-Arg)
Repeat
Associated
Non-ATG
translation
Unknown function
Hexanucleotide repeat
Taylor, J.P. et al., Nature (2016) e539, 197–206)
C9orf72 assay
DeJesus-Hernandez M. et al., “Expanded GGGGCC Hexanucleotide Repeat in Noncoding
Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS” Neuron (2011), 72, 245-56
Repeat-primed PCR
Fragment analysis
Long PCR
MICROTUBULE ASSOCIATED PROTEIN TAU
GENE (MAPT) 17q21.31
Wang JZ, Gao X, Wang ZH. “The physiology and pathology of microtubule-associated
protein tau” Essays Biochem (2014),56, 111-23
Tau hyperphosphorylation
Mazanetz MP and PM Fischer “Untangling tau hyperphosphorylation in drug design for
neurodegenerative diseases” Nature Reviews Drug Discovery (2007), 6, 464–79
Paired
Helical
Filaments Neurofilament Tangles
MAPT mutations
Iqbal K. et al., “Tau and neurodegenerative disease: the story so far” Nature Reviews
Neurology (2016), 12, 15–27
MAPT mutations in 3-14% FTD pts (9-38% / 50% in familial cases and 0-3% in
sporadic cases)
GRANULIN GENE (PGRN) 17q21.31
Nguyen AD, et al. “Progranulin: at the interface of neurodegenerative and metabolic diseases”
Trends Endocrinol Metab (2013), 24, 597-606
PGRN mutations
Gass J. et al., “Progranulin: An emerging target for FTLD therapies” Brain Res (2012),
1462, 118-28
PGRN Mutations in 1-16% of FTD pts (7-28% in familial cases and 1-4% in sporadic cases)
Mutations lead to haploinsufficiency (50% of PGRN activity)
Progranulin neurotrophic agent that acts along with its receptor
(SORT1) for protein lysosome transfer and degradation
Worst FTD prognosis
PGRN exon 3 screening
* Frameshift mutation: c.264delG, E88fs
• Female FTD patient, 63 years, with family PD history (father)
• Hasn’t been reported before (FTD MOLGEN mutation database)
http://www.molgen.ua.ac.be/ADMutations/
HRMA
in Rotor
Gene
DNA
Seq
PGRN exon 12 screening
* Missense variant: c.G1445A (p.C482Y)
• SD male
MAPT gene exon 13 screening
* Missense mutation: c.2092G>A, p.V698I
• CBD female 59 years, no family history
DNA Sequencing
Sanger: NGS (Next Generation Sequencing)
or Massive Parallel Sequencing:
Whole Genome Sequencing (WGS)
3 billion nucleotides
Whole Exome Sequencing (WES)
30 million nucleotides
(1-2% of whole genome)
Targeted sequencing
(100-200 genes)
Per genomic area:
4 runs per gel or per 1 capillary
NGS Steps Blood or tissue
Adding barcodes/adaptors
Potential for massive parallel sequencing of many different
samples altogether (e.g. 10-50 samples): each one with its
unique «barcode» nucleotide sequence (Tags 1 και 2) e.g.
Sample 1: ACTACCAGTG
Sample 2: TTGCAGTAAC
Sample 3: GGCATACTGA
……..
NGS Steps #2
Obtaining info in NGS
Image capture
per nucleotide
addition cycle
Sequence alignment
Validated bioinformatics software necessary for analysis
Barcodes
hg19
(Human Genome
version 19)
Base calling in NGS: Mutation or
polymorphism detection
Coverage 23x (reads):
11 G’s (48%) and 12 A’s (52%) → Heterozygosity G/A
NGS collaboration for FTD in Greece
Frontotemporal dementia spectrum: first genetic screen in a Greek cohort (submitted)
familial (n=46) and
sporadic (n=57) cases
TARDBP p.Ile383Val in 3 patients (1 bvFTD, 2 svPPA)
2nd Neurology
Dept.
Attikon
General
University
Hospital
(Assoc. Prof.
S. Papageorgiou)
Biogenesis of miRNAs Small, non-coding RNA, 22 nt (mature miRNAs)
DGCR8: Di
George
Critical
Region 8
TRBP: Transactivator
RNA
Binding
Protein
RISC: RNA-
Induced
Silencing
Complex
Passenger miRNA
(leading)
Majority of
miRNAs in regions
between genes
(60%), the rest in
gene introns
6% of human miRNAs
are processed by
RNA editing
(IsomiRs)
Kuzhandai Velu V. et al., Journal of Clin and Diagn Res (2012), 6, 1791-95
Action of miRNAs in mRNA
mRNA target cleavage Translational repression mRNA deadenylation
miRNA ↑ mRNA ↓
miRNA ↓ mRNA ↑
miRNAome
1881 miRNA sequences in database:
http://www.mirbase.org/
When the expression of a miRNA is deregulated (through deletion,
polymorphism, epigenetic modifications, induction etc),
It can have as a consequence the expression change of many gene targets!
One mRNA could be a target of many miRNAs
Up to 30% of genes are miRNAs’ targets
[Lim L.P., et al., Nature (2005), 433, 769–73]
Correlations with diseases: http://www.mir2disease.org
miRNAs expression
in AD brain tissues
Hill J.M. et al., Front. Neur. (2015), 6, 232
Isolation of total RNA → cDNA
CFH
mRNA
Microarray evaluation
AD
Reduced expression of hsa-miR-27a-3p in CSF of
patients with Alzheimer disease
Collection of CSF samples → RNA→ cDNA
Frigerio C.S. et al., Neurology (2013), 81, 2103–6
Test cohort Validation cohort
Negative
controls
Real-time qPCR Higher concentrations → lower
Ct
Threshold
Baseline
Relative quantification with reference gene 2-ΔΔCt Method
Ct =
Cycle
threshold
miRNA screen
752 miRNAs were evaluated and those with >75% positivity in
one of the two groups were recorded
86 miRNAs were positive in controls (11%) and 65 miRNAs in AD (9%)
Normalize results with miR-30 και miR-101-3p as a reference
In CSF cDNA samples from 8 AD and 8 CT (controls)
V2.0 miRCURY LNA Universal RT microRNA PCR Human panel I+II and SYBR Green
Universal Master Mix (Exiqon) in LightCycler 480 real-time PCR instrument (Roche)
miRNA screen
results
Lower expression in AD (< -1):
hsa-miR-451a, hsa-miR-27a-3p,
hsa-miR-16-5p, hsa-miR-20a-5p,
hsa-miR-143-3p, hsa-miR-23a-3p,
hsa-miR-486-5p, hsa-miR-150-5p
Higher expression in AD (>1):
hsa-miR-216a-5p, hsa-miR-2110
Validation of 10 selected miRNAs
with separate real-time qPCR
assays in cohort Α
miR-27a-3p qPCR results
n=20 n=19 n=15 n=19
AD controls AD controls
Cohort A Cohort B
Validation in B cohort
Relative
expression
Correlation with established CSF biomarkers
r = -0.6025
p < 0.0001
r =-0.5304
p =0.0005
r = -0.3829,
p = 0.0279
r= -0.4030
p =0.0201
r= 0.4361
p= 0.0055
r= 0.4692
p= 0.0059
Cohort A
Cohort B
Effect of hsa-miR-27a-3p in other genes
BACE1: 60% reduction, p=0.0054; GSK3B: 44% reduction, p=0.0010;
MAPT: 35% reduction, p=0.0038; PSEN1: 44% reduction, p=0.0046
TargetScan (target prediction
algorithm)
Limitations – future goals
Limited number of CSF samples for miRNA screen due to cost
(less statistical value)
Post-mortem CSF samples were evaluated as well: 445 were
detected in 4 AD and 4 CT samples possibly due to different
sampling area in post-mortem, compromised integrity of the blood-
brain barrier and/or of brain cells after death
miRNAs more stable molecules and therefore,
ideal biomarkers in CSF or in blood in the future
And the quest continues…
Molasy M. et al., Journal of Human Genetics (2017), 62, 105–12
Investigation of miRNAs in CSF in other type dementias (FTD etc)
Thank for your attention
Keep your brains healthy!