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!