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