Pre-examination

P
i ti Process
P
(f labs)
(for
l b )
Validation of test - II
Francesco Fiorentino
Lab Director
GENOMA - Molecular Genetics Laboratory
Rome Italy
fiorentino@laboratoriogenoma.it

MUTATION ANALYSIS
Mutation
Analysis
Indirect
Diagnosis

Direct
Diagnosis
Minisequencing
Sequencing
Restriction enzime
digestion
PCR products sizing
SSCP
SSCP--DGGE
ARMS / DD-ARMS
Molecular beacons

Direct
+
Indirect

Linkage Analysis
Exclusion testing
WGA + STR
haplotyping
Direct mutation testing
+ Linked STR markers

MUTATION ANALYSIS

Indirect
Diagnosis

Indications for indirect diagnosis

Direct mutation testing is not possible
The mutation is unknown
The mutation is a large deletion/insertion with unknown breakpoints

Direct mutation testing is not efficient

The gene region to be amplified is refractory to PCR (e.g. GC-rich)
Presence of a pseudogene

Genes with a wide spectrum of mutations

indirect diagnosis as a general protocol for different couples

Preimplantation HLA matching

flexible indirect HLA typing protocol applicable to a wide spectrum of
possible HLA genotypes

Exclusion testing
e.g. Huntington disease

Indirect diagnosis: Pros / Cons

Advantages:

No mutation analysis
same protocol useful for many couples

Useful for rare disorders with private mutations

Disadvantages:

Applicable to informative couples with family history

At least two affected family members needed

Not applicable in cases of de novo mutation and no previous

pregnancies

Principle of indirect diagnosis

Father
Mother

Child

PGD for 2121-OH deficiency by linkage analysis

Genetic Markers
Single nucleotide polymorphisms (SNPs)

single base change in genomic DNA

one per every 500 - 1000 base pairs
less informative than microsatellite markers
low mutation rate* ( 10-9)

Short Tandem Repeats (STRs)

or Microsatellites
1 every few kb, high degree of heterozygosity
mutation rate* 10-3
Types:
di- (CA)n,
tri- (CAG)n,
tetra- (GATA)n.
Penta (AAAAT)n nucleotide repeats

TGCATTGCGTAGGC
TGCATTG
TGCATTC
TGCATT
CCGTAGGC

TGCT
TGC
TCACACACACACA
ACACACACACAGC
GC
TGCTCACACA
TGCT
ACACA-----------GC
GC

100bp
100
bp

Minisatellites (VNTR - variable number

tandem repeats)
*per generation
1 every few kb
mutation rate* 10-1

Microsatellites Markers
microsatellite

the repeat region is variable between

samples while the flanking regions
where PCR primers bind are constant

(ca)n

Allele
gacctaatc ca ca taccgtta
gacctaatc ca ca ca ca ca taccgtta

197.2

223.4

2
Allele 5

Alleles distinguishable
by PCR product length

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another

Microsatellite Characteristics
Stutter Peaks: DiDi-nucleotide vs Tetra
Tetra--nucleotide Repeats

True Allele

Stutter 4bp

True Allele

Stutter 2bp

Stutter 4bp

Microsatellite Marker Types

Dinucleotide Repeat Markers
E.g.: (CA)(CA)(CA)(CA)n

Abundant coverage
Characteristic stutter patterns
Interpretation can be complex

Microsatellite Marker Types

Tetranucleotide Repeat Markers
E.g. (TCTA)(TCTA)(TCTA)n

Well characterised
Discrete allele peaks
Low, predictable and measurable stutter peaks
Easier interpretation

How to build the haplotypes?

haplotypes?

Selection of the STR markers linked to the disease causing

gene

The choice of linked STR markers

Telomere

Chromosome 11

1.50 Mb

D11S4146

0.70 Mb

D11S988

0.48 Mb

D11S4146

0.15 Mb

HBB
D11S1760

0.74 Mb

D11S1338

1.12 Mb

D11S1997

2.05 Mb

D11S1331
Centromere

How to build the haplotypes?

haplotypes?

Selection of the STR markers linked to the disease causing

gene

Evaluation of the informativity of the markers:

Selection of the informative markers
Preferably fully informative (i.e., 4 different alleles, father a/b and
mother c/d)

Identification of the alleles associated with the

mutation/disease

Affected Child

Father

Mother

Informativity testing
Affected Child

Father

Mother

How to build the haplotypes?

haplotypes?

Selection of the STR markers linked to the disease causing

gene

Evaluation of the informativity of the markers:

Selection of the informative markers
Preferably fully informative (i.e., 4 different alleles, father a/b and
mother c/d)

Identification of the alleles associated with mutation/disease

Determination of the haplotypes

DETERMINING HAPLOTYPES FOR LINKAGE ANALYSYS

D11S4146
D11S988
D11S4181
HBB
D11S1760
D11S1338

162
156
120
126
109
105
N
107
134

156
162
126
120
105
109
IVSI--110 Father
IVSI
111
130

D11S4146
D11S988
D11S4181
HBB
D11S1760
D11S1338

Mother

162
120
109
IVSI--110
IVSI
111
130

160
132
116
IVSII--745
IVSII
103
136

Affected
Child

168
124
111
N
105
132

160
132
116
IVSII--745
IVSII
103
136

D11S4146
D11S988
D11S4181
HBB
D11S1760
D11S1338

DETERMINING HAPLOTYPES FOR LINKAGE ANALYSYS

D11S4146
D11S988
D11S4181
HBB
D11S1760
D11S1338

156
126
105
IVSI--110
IVSI
111 Grandfather
130

172
118
120
N
115
142

D11S4146
D11S988
D11S4181
HBB
D11S1760
D11S1338

162
120
109
N
107
134

Grandmother

156
126
105
IVSI--110 Father
IVSI
111
130

162
120
109
N
107
134

Mother

164
134
124
N
115
126

168
124
111
N
105
132

D11S4146
D11S988
D11S4181
HBB
D11S1760
D11S1338

160
132
116
IVSII--745
IVSII
103
136

D11S4146
D11S988
11S988
D11S4181
HBB
D11S1760
D11S1338

Affected
Child
D11S4146
D11S988
D11S4181
HBB
D11S1760
D11S1338

162
120
109
IVSI--110
IVSI
111
130

160
132
116
IVSII--745
IVSII
103
136

Linkage-based PGD protocols: general guidelines

Type of markers:
STRs, preferably tetra-nucleotide repeat (di-nucleotide repeat are
also acceptable)

Location of STR markers:

preferentially intragenic or extragenic, very closed to the gene (max 1
Mb of distance) to reduce the risk of recombination events

Heterozygosity of STR markers

High (>0.8) to improve informativity of the markers

No. of STR markers

Preferably 4, 2 upstream and 2 downstream

Size of the alleles

Small product size (preferably < 250 bp) to improve PCR efficiency

Number of family members

At least two generations or affected family members

Indirect Diagnosis

Exclusion
Testing

Exclusion of HD using linkage

1
2
3
4
5

D4S127
D4S1614
D4S3034
D4S412
D4S126

6
7
8
9
10

21
22
23
24
25

Mother

Father

21 26 D4S127
22 27 D4S1614
23 28 D4S3034

Female partner 24 29 D4S412

Male partner

25 30 D4S126

A/B - C/D
D
50% risk

D4S127
D4S1614
D4S3034
D4S412
D4S126

Embryo 2

?
1
2
3
4
5

6
7
8
9
10

D4S127
D4S1614
D4S3034
D4S412
D4S126

C D

A B

Embryo 1

26
27
28
29
30

Embryo 3
21
22
23
24
25

21
22
23
24
25

21
22
23
24
25

Embryo 4

1
2
3
4
5

C E

A or B E

EF

6
7
8
9
10

26
27
28
29
30

A or B F
50% risk

50% risk

21
22
23
24
25

26
27
28
29
30

C F

Indirect Diagnosis

WGA
+
Haplotyping

Whole Genome Amplification (WGA)

Universal first amplification step
WGA product analysis in conventional facilities
No requirement for development of special
single cell/mutation detection tests
MUTATION
ANALYSIS

ANEUPLOIDY

LINKED
MARKERS

SINGLE
CELL

DNA
FINGERPRINTING

HLA
HAPLOTYPING

Multiple Displacement Amplification

Isothermal, no cycling
involved (incubation at 30C)

Random priming using

exonuclease resistant
modified random hexamers

Polymerase
P l
makes
k strand
t d
and displaces other strand,
e.g. F29 polymerase

104-106-fold amplification
Obtaining gs of DNA

Spits et al., 2006, Nature Protocols, Vol 1(4): 1965-1970

MDA and PGD

Use for haplotyping in PGD for

monogenic disease (PGH)
High ADO rate, many markers have to be included
in the protocol

Use for array-CGH in PGS

A combination of both

MUTATION ANALYSIS

Direct + Indirect
Diagnosis

The use of STR markers in PGD procedure

Represents a diagnostic tool for indirect mutation analysis, providing
an additional confirmation of the results obtained with the direct
genotyping procedure
provides a control of misdiagnosis due to undetected ADO
provides an additional control for contamination with exogenous DNA
Provides information on embryos chromosomes copy number
PGD protocols for SGD are not appropriate for clinical practice
without including a set of linked STR markers

Allele drop-out
Allele drop-out (ADO) is defined as the non-amplification of one
allele when performing PCR at the single cell level.
This phenomenon can only be demonstrated in heterozygote
cells, which show a homozygous pattern when ADO has occurred
ADO occurs in all cell types
types, e
e.g.
g blastomeres
blastomeres, lymphocytes
lymphocytes,
buccal cells and fibroblasts.
An undetected ADO event leads to misdiagnosis

Avoidance of misdiagnosis due to ADO

Father

HBB gene
and markers
Telomere
D11S4146
D11S988
D11S4181
HBB
D11S1760
D11S1338
Centromere

HBB gene
and markers
D11S4146
D11S988
D11S4181
HBB
D11S1760
D11S1338

Embryo
5

162
120
109
-110
111
130

160
132
116
N
103
136

Affected
with ADO

Embryo
1

162
120
109
-110
111
130

168
124
111
N
105
132

Carrier

162
120
109
IVSI-110
111
130

160
132
116
IVSII-745
103
136

Carrier

HBB gene
and markers

Telomere
160
168
D11S4146
132
124
D11S988
116
111
D11S4181
IVSII-745 N
HBB
103
105
D11S1760
136
132
D11S1338
Centromere

156
126
105
N
107
134

Embryo
2

156
126
105
N
107
134

Mother

Embryo
3

156
126
105
N
107
134

168
124
111
N
105
132

Normal

Embryo
6

156
126
105
N
107
134

168
124
111
N
105
132

Normal

Embryo
7

156
126
105
N
107
134

160
132
116
IVSII-745
103
136

Carrier

Embryo
8

162
120
109
-110
111
130

160
132
116
-745
103
136

Affected

HBB gene
and markers
D11S4146
D11S988
D11S4181
HBB
D11S1760
D11S1338

STRmarkers:OtherapplicationinPGD

p
Preimplantation
HLA Matching

Preimplantation HLA Matching by STR haplotying

D6S439
HLA-DQ
DQCAR II
HLA-DRB
DRA-CA
TNF-a
HLA-B
HLA-BC
HLA-C
D6S265
D6S510
HLA-A
MOG-CA

190
3
155
5
148
128
7
130
9
148
270
11
135

198
4
162
6
154
133
8
139
10
160
288
12
155

Father

Mother

PGD

D6S439 190
HLA-DQ 3
DQCAR II 155
HLA-DRB 5
DRA-CA 148
128
TNF-a
7
HLA-B
HLA-BC 130
9
HLA-C
D6S265 148
D6S510 270
11
HLA-A
MOG-CA 135

194
6
158
4
152
130
2
132
8
155
268
10
145

Affected
child

190
3
155
5
148
128
7
130
9
148
270
11
135

188
7
150
5
144
120
3
128
1
150
260
9
130

194
6
158
4
152
130
2
132
8
155
268
10
145

194
6
158
4
152
130
2
132
8
155
268
10
145
HLA
identical
embryo

HLA STR haplotyping

Indirect typing of the HLA region by
segregation analysis of the STR alleles
The HLA identity of the embryos with
the affected sibling is ascertained
evaluating the inheritance of the
matching haplotypes.
A panel of 50 different STRs is studied
during the pre-clinical work-up
At least 8 informative markers, evenly
spaced throughout the HLA region are
selected to be used in clinical PGD
Achievement of an accurate mapping of
the whole HLA region

Informativity testing for preimplantation HLA matching

MARKER

MOTHER

FATHER

CHILD

TNF1

107

114

105

114

105

114

D6S510

155

140

151

148

151

140

D6S426

120

128

120

124

120

128

MIC A

168

171

171

168

171

171

D6S273

142

144

136

142

136

144

D6S276

218

220

216

211

216

220

LH1

144

146

141

144

141

146

DQ CAR II

131

118

130

123

130

DRA CA

130

137

130

139

130

137

MOG CA

215

206

225

206

225

206

117

129

120

129

HLA BC CA

118

132

120

D6S265

110

114

120

116

120

114

D6S291

156

160

158

158

158

160

TNF2

111

111

113

111

113

111

82--1
82

111

111

104

111

104

111

G51152

145

145

147

147

147

145

D3A

200

202

202

202

202

202

RING3 CA

126

124

124

126

124

124

62

156

163

156

163

156

163

D6S439

120

122

120

122

120

MIB

177

177

172

177

172

177

D6S105

141

141

139

153

139

141

122

The use of STR markers in HLA matching procedure

The same strategy can be used for different cases (and allele
combinations)
STRs provide an additional control for contamination with
exogenous DNA
The whole HLA complex can be covered, allowing the detection
of recombination events between HLA genes.

Avoidance of misdiagnosis due to recombination

D6S439
DQCAR II
DRA-CA
TNF-a
HLA-BC
D6S265
D6S510
MOG-CA

198
162
154
133
139
160
288
155

190
155
148
128
130
148
270
135

Father

Mother

188
150
144
120
128
150
260
130

194
158
152
130
132
155
268
145

PGD
D6S439
DQCAR II
DRA-CA
TNF-a
HLA-BC
D6S265
D6S510
MOG-CA

190
155
148
128
130
148
270
135

194
158
152
130
132
155
268
145
Affected
child

190
155
148
128
130
148
270
135

190
155
148
128
130
148
270
135

194
158
152
130
132
155
268
145

194
158
152
130
128
150
260
130
Recombinant
embryo

HLA
identical
embryo

STRmarkers:OtherapplicationinPGD

Detection of
chromosomal
Aneuploidies

Aneuploidy Detection by using STR markers:

microsatellite

AMA
RIF
RM

(ca)n
gacctaatc ca ca taccgtta
gacctaatc ca ca ca ca ca taccgtta
gacctaatc ca ca ca ca ca ca ca taccgtta

Allele 1
Allele 2
Allele 3

Embryo with trisomy 21

Alleles are
distinguishable
by PCR product
length
197.2

Chromosomes:
13, 14, 15, 16, 18,
21, 22, X ,Y

223.4

Aneuploidy Detection by using STR markers

13
18

21

Y
21
21

18

13

18

Trisomy
21

18

13
21
13
13
21

Trisomy
13

STRmarkers:OtherapplicationinPGD

Detection of
unbalanced
chromosomal
translocations

Segregation of Robertsonian Translocations

Adjacent 1

Chr 13

23
13

Chr 14
22
12

21
11

22
12

Gamete 1
(Unbalanced)

23
13

24
14
Gamete 2
(Unbalanced)

24
14

21
11
Gamete 3
(NORMAL)

23
13
41
31

21
11

22
12

22
12

22
12

24
14
41
31

21
11

43
33

41
31

21
11

Gamete 5
(Unbalanced)

43
33

Balanced

24
14

Gamete 6
(Unbalanced)

Gamete 4
(BALANCED)

24
14
22
12

23
13

Normal
24
14

23
13

23
13

43
33

Trisomy 13
Monosomy 13

Gametes

Alternate

23
13
21
11

Adjacent 2

24
14

41
31

43
33
22
12

43
33

Embryos

41
31

Trisomy 14
43
33
21
Monosomy 11

14

41
31

14

23
13

13
13

43
33

D13S634

D13S631 D13S217

14

41
31

13

22
12

21
11

Trisomy 13
13
14
14

41
31

43
33

24
14

Monosomy 13

14

24
14

43
33

21
11

41
31

13
14
13

Normal

13
13

14

D14S553

D14S549

23
13

14

24
14

D14S617

43
33

ADO
14

22
12

41
31

Trisomy 14

43
33

13

41
31

21
11

13
14

Monosomy 14

14

24
14

43
33

21
11

41
31

13
14
13

Normal

Segregation of Reciprocal Translocations (in gametes

gametes))
Adjacent 1

7
8
4

C
D
1

Alternate

Gamete 1
(Unbalanced)

Gamete 2
(Unbalanced)

3
6

6
4

1
2

Gamete 9
Gamete 7
Gamete 8
(Unbalanced) (Unbalanced) (Unbalanced)

7
8

7
8

5
3

4
Gamete 11
(Unbalanced)
Gamete 10
(Unbalanced)

7
8

Gamete 6
(Unbalanced)

3:1
3:
1

Gamete 4
Gamete 3 (BALANCED)
(NORMAL)

3
6

3
6

Gamete 5
(Unbalanced)

35

7
8

7
8
4

3:1
3:
1

3
6

7
8

Adjacent 2

5
4

Gamete 12
(Unbalanced)

1
4

Gamete 13
(Unbalanced)

3
6

7
8

Gamete 14
(Unbalanced)

Segregation of Reciprocal Translocations (in embryos

embryos))

3 11

11

15

12

11

16
4

12

12

1
2

15

16
3

11
12

11

12

15

16

16

7
8

11

11

12

12

15

Embryo 4 (BALANCED)

5
11

11

12

15
16

12

Embryo 9 (Unbalanced)

15
16

11
12

12

Embryo 10 (Unbalanced) Embryo 11 (Unbalanced) Embryo 12 (Unbalanced) Embryo 13 (Unbalanced)

3
8

1
6
2

4 11

16

12

3 15
16

Embryo 8 (Unbalanced)

7
8

16

16

16

15

15

Embryo 3 (NORMAL)

Embryo 6 (Unbalanced) Embryo 7 (Unbalanced)

15

5
7
8

15

Embryo 2 (Unbalanced)

15

11

12

Embryo 5 (Unbalanced)

16

12

Embryo 1 (Unbalanced)

11

15

11

16

15
16

Embryo 14
(Unbalanced)

Segregation of Robertsonian Translocations

(1PB testing
testing))
4

3
2

3
2

Trivalent forms of
Chromosomes in synapsis
phase during Meiosis I

3
4
2

3
2

Chr 14

Chr 13

1
4

Unbalanced Unbalanced
CHR13: 1/2
CHR14: 3

CHR13: null
CHR14: 4

Adjacent 1

14

3
2

3
2

3
21

3
2

NORMAL

BALANCED

Unbalanced Unbalanced

CHR13: 1
CHR14: 4

CHR13: 2
CHR14: 3

CHR13: 2
CHR14: 3/4

Alternate

CHR13: 1
CHR14: null

Adjacent 2

PCGD for Robertsonian Translocations by 1PB testing

D13S240
3
2

3
21

Unbalanced
CHR13: 1/2
CHR14: 3

14

NORMAL
CHR13: 1
CHR14: 4

D13S243

D13S252

PCGD for Robertsonian Translocations by 1PB testing

D14S121
4

3
2

D14S122

D14S551

3
2

Unbalanced
CHR13: 2
CHR14: 3/4

14

NORMAL
CHR13: 1
CHR14: 4

STR--based PGD for translocations: advantages

STR
Easy procedure and data interpretation
Amenable to automation
Rapid procedure (<12 h)(4-6 h for 1PB testing)
Cell fixation (PBs or blastomeres) is not necessary
Solve suboptimal fixation problems, easier procedure for transport PGD
Overcome to several technical limitation of FISH procedure:
Overlapping signals, split signals, lack of signals, cross-hybridization,
pol morphisms limited availability
polymorphisms,
a ailabilit of the probes
probes, combination of colo
colours
rs
Possibility to perform combined testing
e.g. Translocation + PGS; Translocation + SGD with or w/o PGS
Post-hybridization wash and re-probing are not necessary for combined testing
UPD can be detected
Lower error rate
Low expensive
A DNA fingerprint is achievable from each embryos
Identification of embryos that have implanted

STR--based PGD for translocations: disadvantages

STR

Affected by contamination

Affected by ADO Preferential Amplification

Recombination risk in cases of 1PB testing

Future vision: unique protocol for PGD

PGD
WGA
Translocation

Aneuploidy

SGD

Combined

Reciprocal

RIF

SGD + Aneuploidy

Robertsonian

RM

Translocation +
Aneuploidy

Inversion

AMA

All Chr

Pre--examination Process (for labs)

Pre

Validation of the
PGD Protocol

Developing a PGD protocol

Confirmatory testing of the mutation(s)
Choice of the PGD strategy (direct mutation diagnosis, linkage)
Informativity testing for linked/un
linked/un--linked STR markers
Test multiplex
p
PCR: combination of primers,
p
etc.
Application to the single cell level (i.e. single lymphocytes,
fibroblast, buccal cells, etc.)
Amplification efficiency, contamination and ADO rates in
heterozygous samples.
samples.
Test on spare blastomeres (depending on availability )
Ready for PGD

Preliminary PGD workwork-up: parameters to be evaluated

Pre-clinical tests on single cells (lymphocytes, fibroblasts,
amniocytes, buccal cells, etc.)
At least 50 cells in total

Amplification efficiency > 90 % should be aimed

Lower efficiency coincides with higher ADO
ADO rates should be as low as possible (preferably <10%)
ADO is mostly influenced by PCR method (conventional vs.
fluorescent)

The contamination rate should be less than 5% (preferably zero)

At least 50 cell wash blanks

ESHRE PGD Consortium guidelines, Hum Reprod. 2005 Jan;20(1):35-48

One--cell versus two

One
two--cells biopsy
Doubts and discussion about one vs two-cell biopsy
Risk of misdiagnosis (e.g. autosomal dominant disorders)
Claims that higher implantation rate is achieved after one-cell biopsy

Randomized control trial (Goossens V. et al., Hum Reprod 2007)

Removal of two blastomeres significantly decreases the likelihood of
blastocyst formation
f

one-cell biopsy results in a significantly lower diagnositic efficiency

live birth delivery rates were not statistically different for one- and
two-cell biopsy

RECOMMANDATION: diagnosis of only 1 cell

requires a robust PGD protocol