Challenges in Genetic Counselling of ambiguous …bgcn.ir/MckUpload/file/اسلاید/Dr_...
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Challenges in Genetic
Counselling of ambiguous
Cytogenetics findings
including Mosaicism of
XX/XY, low grade mosaicism
for autosomes, marker
chromosome, tetraploidy,
and chromosomal
polymorphism
Farkhondeh Behjati , PhD
Professor of
Medical Genetics,
Head of Cytogennetic Lab,
Genetics Research Center,
University of Social Welfare and
Rehabilitation Sciences
&
Sarem Hospital & Sarem Cell
research Center
Tehran, Iran
Objective
• The Cytogenetics findings on most
occasions for prenatal diagnosis are
normal findings of male or female
karyotypes. However, in a few cases there
are karyotype findings which can create
uncertainty in the interpretation of findings
and its implication in the genetic
counselling and the patient’s care.
Ambiguous Findings??
• Patients with 46, XX/ 46, XY karyotype
and female or male gender sonography,
46, XX/92, XXXXX and normal female
sonography, mos 46, XY/47, XY,+mar (in
a minority of cells), inversion 9, large
heterochromatin of Y chromosome, and
long satellite stalk or double satellites in
acrocentric chromosome.
www.rcsiwomenshealth.com/images/amnio.jpg
Amniocentesis
Chorionic Villus Sampling (CVS)
Amniocentesis
• First 2-5 ml of fluid is discarded– To minimize risk of the contamination from maternal cells
• 20 to 30 ml, then, transferred to sterile tubes
• Real-time ultrasonographic guidance during amniocentesis– Reduce the frequency of multiple needle insertions
– Reduce fetal trauma
– Reduce fetal loss
• Extra observation by ultrasound in order to document fetal viability
• Reporting signs of infection, vaginal bleeding, leakage of amniotic fluid and regular uterine contractions
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• Culturing to grow in monolayers
– Adding small volume of tissue culture medium
(0.5 ml) and resuspending the cell pellet
– Highly supplemented tissue culture medium
• : Amniomax or Chang
– Possibility of culture failure:1 in 700 in
midtrimester
– Culturing at least two flasks
Interpretation of results
• Freshly blood-stained amniotic fluid :separately analyzed by a Kleihauer test and cell count to determine whether the new blood is maternal or fetal. Best to do Microsatellite analysis, QFPCR) in the fetus and mother
– If the blood is fetal,the AFAFP value may be elevated without a congenital anomalies
• Brown or green tinged amniotic fluid:
– Increased risk (5-9%)of perinatal mortality and pregnancy loss
– Discoloring pigment: hemoglobin
– Vaginal bleeding prior to amniocentesis
trc.ucdavis.edu/.../week4/amniocentisis.gif
AF cell Culture
Cytogenetics Methodology
Cytogenetics Methodology
Interpretation of results
• Balanced translocations:
– Inherited• Offspring phenotype will be normal
– De novo• Major fetal malformations and mental retardation
around 8-10%
Mosaicism in AF
• three levels of mosaicism
– Level I :single cell anomaly in an otherwise
normal colony
– Level II :abnormality observed in two or more
cells (flask method) or in two or more cells
from one colony (in situ) in the same culture
– Level III :abnormality observed in two or more
independent cultures.
Recommendations for Evaluation and Interpretation of Mosaicism(ACMG, ECA, ACGS)
• Level I mosaicism
– should not be reported
– No follow-up
• Level II mosaicism
– major differences in what cytogeneticists consider
– the number of additional independent metaphases to be
examined in addition to the original 10 cell analysis
– nature of the chromosome abnormality will influence the number
of additional metaphases to be analyzed
– usually not be reported
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• may report:
– Analysis of a minimum of 10 additional colonies/metaphases is
not possible
– If clinical findings are present:
• such as fetal anomalies, IUGR or suspected vanishing twin on
ultrasound
– chromosomes known to be associated with clinically significant
mosaic states
• Level III mosaicism
– All should be reported.
– Follow-up: Additional studies such as fetal ultrasound
E.C.A.
• MOSAICISM IN PRENATAL STUDIES
– Two or three cultures should be set up for
each sample
– if the same abnormality is present in two
independent cultures, mosaicism is confirmed
– For in situ preparations
• analysing cells from one cell culture may be
sufficient or all from the same colony
Association for Clinical Cytogenetics
(ACGS)
POSTNATAL BEST PRACTICE
GUIDELINES (2007) v1.01
Reporting of variants
Well-documented polymorphic variant
chromosomes may not require
reporting or family follow up
Examples of variants that need not be
mentioned in the report are:
• pericentric inversions with the breakpoints in the heterochromatic
region, such as: inv(1)(p11q12), inv(1)(p12q12), inv(9)(p11q12),
inv(9)(p11q13), inv(16)(p11q11).
• heterochromatic size variants, including 1qh+, 9qh+, 16qh+, Yqh+,
Yqh-,
• acrocentric short arm variants resulting from the Yq
heterochromatin translocation and satellited Y chromosome
• Acrocentric short arm variants, with the exception of cases in which
further investigation of the possible involvement of euchromatin is
required.
Examples of variants that need not be
mentioned in the report are:
• fragile sites, excluding FRAXA and FRAXE.
• pericentric inversions that have been described as
presumed harmless variants
inv(2)(p11.2q13), inv(3)(p11q11) inv(3)(p11q12),
inv(3)(p13q12),
inv(5)(p13q13), inv(10)(p11.2q21.2)
• inv(Y)(p11q11).
• G-band euchromatic variants of 8p23.1, proximal 9p,
insertion of
euchromatin into 9qh, proximal 9q, 15q11.2 and
16p11.2.
Examples of variants that need not be mentioned
in the report are:
• It is recognised that specific clinical
situations may require further
laboratory investigations and/or family
studies to enable clarification
and certainty of coincidental status of
these findings; this is subject to
professional judgement.
Association for Clinical Cytogenetics
(ACGS)
PRENATAL DIAGNOSIS BEST
PRACTICE GUIDELINES (2009) V1.00
Reporting of mosaic findings
For situations where chromosomes are
known to be involved in Uniparental Disomy
(UPD), further studies should be considered.
A detailed ultrasound scan should be
advised.
UNIPARENTAL DISOMY STUDIES
• At present, adequate data is only available for CPM,
additional marker chromosomes and balanced
Robertsonian translocations
• UPD testing should be considered in cases of:
• Apparent CPM for chromosomes 7, 11, 14, and 15
• Homologous and non-homologous Robertsonian
translocations involving 14 and 15
• Marker chromosomes of chromosome origin 7, 11, 14
and 15.
• UPD studies for balanced reciprocal translocations are
not necessary.
UNIPARENTAL DISOMY STUDIES
• UPD testing in mosaic trisomy 16 pregnancies (a pregnancy with
both disomy and trisomy 16 cells lines in the placenta and/or fetus).
These cases present particular difficulties. In cases where UPD is
excluded, there is still a significant risk of adverse fetal outcome (as
judged by lower birth weight and/or fetal malformation).
• This is attributable to the presence of confined placental mosaicism
or cryptic trisomy 16 mosaicism in the fetus or both.
• For this reason it is particularly important that the implications of a
negative UPD result are considered and understood before such
testing is initiated.
Maternal Cell Contamination
• The possibility of maternal cell contamination
should be included in the text of an individual
report if there is considered to be a significant
risk of an incorrect result.
• Where maternal contamination is suspected
consideration should be given to confirming
fetal origin of cells using other techniques such
as QF-PCR.
69,XXX
46,XX,t(4;6)(q31.3;q25.1)mat
47,XX,+21
45,XX,rob(14;15)(q10;q10)matreferred for: raised maternal age
47,XX,+marreferred for: abnormal sonography
47,XX,+mar de novoreferred for: raised maternal age
47,XX,+mar pat referred for: previous child with MR
Follow
up
CommentsKaryotypeReferral
reason
No
18% Cells from only one
culture had an apparently
balanced reciprocal
translocation between
chromosomes 2 and 4.
46,XX,t(2;4)(
q31; p14)[17]/
46,XX[90]
AMSSTCase 1
In two cultures46,XY,t(3;10)(p26
;p11.2)[8]/
46,XY[111]
AMSSTCase2
In one culture46,XX
t(1;13)(q10;10)[5]
/46,XX[65]
AMSSTCase 3
In one culture46,XX
t(9;14)(q34;11)[3]
/46,XX[73]
AMSSTCase 4
In all three
cultures
46,XY,add(17)(q2
5)[28]/46,XY[32]Abnormal
Sonography+
AMA
Case 5
NormalIn one culture46,XX,t(7;13)(p2
2;q11)[49]/46,XX[
121]
AMACase6
46,XX t(9;14)(q34;11)3 cells in one culture only
46,XY,add(17)(q25)
In all three cultures
46,XY,t(3;10)(p26;p11.2)
In two cultures
46,XY,t(3;10)(p26;p11.2)
46,XX[25]/46,XY[25]
Report: 46,XX[25]/46,XY[25]
• CELL ANALYSIS
• Number of Cultures: 3
• Number of Cells analyzed: 6 Number of Cells Counted: 14
• Number of Cells Screened: 30
• Total Number of Cells Examined: 50
• Banding Technique/s: GTG
• Banding Resolution: 430-480 bph
• RESULT
• Karyotype:46,XX[25]/46,XY[25]
• Comments: A normal female karyotype was present in two different cultures. However, the examined cells in the third culture were normal male karyotype. Maternal cell contamination can not be excluded.
• Recommendations:
• 1-Sonography monitoring of the fetus is recommended.
• 2- Genetics counseling is strongly recommended.
• Maternal cell contamination, single gene, small chromosomal abnormalities, low level mosaicism /chimerism and cell/s from other twin/s can not be excluded.
46, XY, Inv(9)(p11.2q13)
Report
• CELL ANALYSIS
• Number of Cultures: 3
• Number of Cells analyzed: 6Number of Cells Counted: 14
• Number of Cells Screened: 30
• Total Number of Cells Examined: 50
• Banding Techniques: GTG
• Banding Resolution: 420-460 bph
• RESULT
• Karyotype: 46,XX,inv(9)(p11.2q13)
• Comments: An apparently normal female karyotype. However one chromosome 9 had a pericentric inversion around centromere. This is usually regarded as a normal population variant.
•
• .
•
• Recommendation: Sonography monitoring of the fetus is recommended.
•
• Maternal cell contamination, single gene, small chromosomal abnormalities, low level mosaicism /chimerism and cell/s from other twin/s can not be excluded.
46,X,inv(Y)(p11.2q11.23)
Report
• Report:
• CELL ANALYSIS
• Number of Cultures: 2
• Number of Cells analyzed: 5Number of Cells Counted: 15
• Number of Cells Screened:
• Total Number of Cells Examined: 20
• Banding Techniques: GTG
• Banding Resolution: 500-520bph
• RESULT
• Karyotype: 46,X,inv(Y)(p11.2q11.23)
• Comments: A male karyotype with a pericentric inversion around the centromere of
• chromosome Y. This is usually regarded a normal population variant.
•
•
• Recommendation: Genetic counseling is strongly recommended.
•
• Low level mosaicism, single gene and small structural chromosomal abnormalities can not be excluded.
46,X,Yqs
Report
• Report:
• CELL ANALYSIS
• Number of Cultures: 2
• Number of Cells analyzed: 5Number of Cells Counted: 10
• Number of Cells Screened:
• Total Number of Cells Examined: 15
• Banding Techniques: GTG
• Banding Resolution: 450-470bph
• RESULT
• Karyotype: 46,X,Yqs
• Comments: A male karyotype was observed. However chromosome Y was satellitedat terminal part of its long arm. This is usually regarded a normal population variant.
•
• Recommendation: Genetic counseling is strongly recommended.
•
• Single gene, small chromosomal abnormalities and low level mosaicism can not be excluded.
•
•
46,XYqh+
Constitutive heterochromatin
banding (C-banding)
Report
• Report:
• CELL ANALYSIS
• Number of Cultures: 2
• Number of Cells analyzed: 5
Number of Cells Counted: 10
• Number of Cells Screened: 15
• Total Number of Cells Examined: 30
• Banding Techniques: GTG
• Banding Resolution: 440-500bph
• RESULT
Karyotype: 46,XY
Comments:
Referral for: Fetus with 4qs
Karyotype: 46,XX,4qs mat
Nuclear organizer region staining
Nuclear organizer region staining
• Report:
• CELL ANALYSIS
• Number of Cultures: 2
• Number of Cells analyzed: 5Number of Cells Counted: 10
• Number of Cells Screened: 15
• Total Number of Cells Examined: 30
• Banding Techniques: GTG
• Banding Resolution: 440-500bph
• RESULT
• Karyotype: 46,XX,4qs
• Comments: A female karyotype was observed. One chromosome 4 has a satellite at the end of the long arm This is usually regarded of no clinical significance.
Referral for: Father of Fetus with 15pss
Karyotype: 46,XY,15 pss
Report
• Report:
• CELL ANALYSIS
• Number of Cultures: 2
• Number of Cells analyzed: 5Number of Cells Counted: 15
• Number of Cells Screened:
• Total Number of Cells Examined: 20
• Banding Techniques: GTG
• Banding Resolution: 490-510bph
• RESULT
• Karyotype: 46,XY,15 pss
• Comments: An apparently normal male karyotype. One Chromosome 15 appears to have double satellite on it`s short arm .This is usually regarded as a normal population variant.
•
•
• Recommendation:
•
• Low level mosaicism, single gene and small structural chromosomal abnormalities can not be excluded.
The finding of a mixture of both 46,XX and
46,XY cells in amniotic fluid culture
• A 29 Years-old woman was referred for genetic
counseling.
• Gestational age : 16 weeks
• Maternal biochemical serum screening test : high risk
for Down Syndrome
• She underwent amniocentesis for chromosome study
using standard high resolution GTG banding
technique
Result:
• Karyotype result was 46,XY and sonography revealed a female fetus with normal internal genital organs.
• A normal baby girl was borned with 46,XX karyotype. Upon reviewing of amnio GTG slides (200 cells) both 46,XY and 46,XX cell lines (4 cells) were detected.
• The patient admitted that she had experienced a miscarriage and heavy bleeding early in pregnancy.
• The sonography at the time of amniocentesis had shown a shrinking cyst next to the fetus.
• These findings strengthened the vanished or resorbed twin as the reason for such a finding.
46,XY prenatal result with 46,XX outcome:
• vanishing twin phenomenon or hermaphroditism?
• An accurate obstetric history and thorough sonographycan be of great value in correct assessment of such situations.
• A vanishing twin, also known as fetal resorption, is a fetus in a muliti-gestation pregnancy which dies in utero and is then partially or completely reabsorbed by the twin.
• The occcurrence of this phenomenon is sometimes referred to as twin embolisation syndrome or vanishing twin syndrome(VTS).
Interpretation of the Results
• The fetus was most likely a non-identical twin.
• Most of the grown cells were from the vanished male
fetus which led to misdiagnosis.
• Thorough genetic counseling with the view of
obstetric history is of great value in such situations.
FISH
Follow upCommentsFISH ResultsReferral
reason
No
Cultural
Artifact
NormalTetraploidy in
cultures
Case 1
?Hermaphrodi
t
46,XY, [90]/
46,XX[10]
46,XY[14]/
46,XX[22]
and in
repeated
amnion:
46,XY, [18]/
46,XX[2]
Case 2
FISH & QF-
PCR Results:
Normal
Positive NIPT
Results
Case 3
46,XY, [90]/ 46,XX[10]
Tetraploidy
Tetraploidy in cultures
FISH Results: Normal
NIPT Results :positive
Karyotype, FISH & QF-PCR Results: Normal
Referral Reason: Abnormal maternal serum screening test
Nuc ish 13q14(D13S1195×2)
Nuc ish 18p11.1 q11.1(D18Z1×2)
Nuc ish 21q22(d21S65×2)
Rapid FISH
Referral Reason: Abnormal maternal serum screening test
Nuc ish 18p11.1 q11.1(D18Z1×3)
Rapid FISH
Referral Reason: Patau syndrome screen positive
Nuc ish 13q14(D13S1195×3)
Rapid FISH
Referral Reason: Mosaicism for trisomy 21
Nuc ish 21q22(d21S65×3)
Nuc ish Xp11.1-q11.1(DXZ1×1)[42]/ Xp11.1-
q11.1(DXZ1×3)[7]/ Xp11.1-q11.1(DXZ1×2)[221]Referral Reason: Abnormal maternal serum screening test
Acknowledgements
• Mr. Iman Bagherizadeh
• Mrs. Fahime Mousavi
• Dr. Zahra Hadipour
• Dr. Fatemeh Hadipour
• Dr. Yousef Shafaghati
• Dr. Abootaleb Saremi
• The Referring Clinicians
• Sarem Cytogenetics laboratory Staff
Thanks for your attention