prenatal diagnosis and 47,xxy

American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 163C:64–70 (2013)

A R T I C L E

Prenatal Diagnosis and 47,XXYJOE LEIGH SIMPSON* AND CAROLE SAMANGO-SPROUSE

In this contribution, we consider detection of 47,XXY by a variety of available methods. These include traditionalinvasive procedures, screening with maternal serum analytes and fetal ultrasound, and most recently cell-freefetal DNA. Since its introduction in the late 1960s, prenatal genetic diagnosis has evolved greatly. Serendipitiousdetectionof 47,XXYwas not infrequentwhenprenatal genetic diagnosis routinely involved testingby the invasiveprocedures CVS and amniocentesis. In 2013 this is much less common and relatively few pregnancies in the U.S.and Europe are tested without prior screening protocols, traditionally maternal serum analyte and fetal ultra-sound (NT). These protocols are not designed to identify 47,XXY or other X-chromosome aneuploides and withscreening by analysis of cell-free DNA in maternal blood, this situation may or may not be altered. Increasednumbers of cases could be detected if intake increases and vendors offer information on 47,XXY. A furtherconsideration is that ability of array CGH to detect microdeletions or microduplications below resolution of akaryotype could make return to direct testing using an invasive procedure attractive.

� 2013 Wiley Periodicals, Inc.

KEYWORDS: prenatal screening; genetic diagnosis; 47,XXY; maternal serum analytes; fetal ultrasound; cell-free DNA

How to cite this article: Simpson JL, Samango-Sprouse C. 2013. Prenatal diagnosis and 47,XXY.Am J Med Genet Part C Semin Med Genet 163C:64–70.

INTRODUCTION

Since its introduction in the late 1960s

prenatal genetic diagnosis has evolved

greatly. Initially, only the invasive proce-

dure amniocentesis was available to di-

agnose chromosomal abnormalities by

amniotic fluid cell analysis. Second tri-

mester amniocentesiswas offered only to

women of ‘‘advanced’’ (35 years) mater-

nal age at time of delivery, or having a

previous trisomic offspring. Later, cho-

rionic villus sampling (CVS) allowed

diagnosis in first trimester. Although

the main indication for both CVS and

amniocentesis was detecting autosomal

aneuploidy, 47,XXYand 47,XXX were

serendipitously detected as well. The

prevalence of these sex chromosome

aneuploidies at first or second prenatal

genetic diagnosis was higher than in the

general population becausematernal age

was increased in both. Because it is

Although the main indication

for both CVS and

amniocentesis was detecting

autosomal aneuploidy,

47,XXY and 47,XXX were

serendipitously detected as

well. The prevalence of these

sex chromosome aneuploidies

at first or second prenatal

genetic diagnosis was higher

than in the general population

because maternal age was

increased in both.

now considered appropriate for women

of any maternal age to be offered

an invasive procedure for aneuploidy

detection, one might expect even

more polysomy X aneuploidies to be

identified serendipitously. However,

concurrent to introduction of CVS in

Advances in prenatal genetic diagnosis that impact on detection of 47,XXY.Joe Leigh Simpson, M.D. is senior vice president for Research and Global Programs at the March of Dimes where he oversees a multi-million dollar

research grant portfolio focused on the prevention of birth defects, premature birth, and infant mortality, including basic biological processesof development, genetics, clinical studies, studies of reproductive health, environmental toxicology, and studies in social and behavioral sciences.Dr. Simpson also serves at present as Professor and Chair, Department of Human and Molecular Genetics and Professor Obstetrics and Gynecology atFlorida International University, Miami. He has authored numerous books; articles, chapters, and reviews on topics ranging from diabetes and birthdefects, to safety and efficacy of prenatal genetic diagnosis, to recovery of fetal cells and cell-free DNA from maternal blood, to biosensors.

Carole Samango-Sprouse, Ed.D is an Associate Clinical Professor of Pediatrics at the George Washington University School of Medicine and HealthSciences. She is actively involved in the clinical and developmental care of children with rare neurogenetic disorders. She is the CEO of theNeurodevelopmental Diagnostic Center providing care for children with uncommon neurogenetic disorders from all over the world. She writesextensively about the relationship between brain function, neurodevelopmental profile and neurogenetic disorder. She has provided care for childrenwith 49,XXXXY for over 10 years.

*Correspondence to: Dr. Joe Leigh Simpson, M.D., March of Dimes Foundation, 1275 Mamaroneck Avenue, White Plains, NY 10605.E-mail: [email protected]

DOI 10.1002/ajmc.31356Article first published online in Wiley Online Library (wileyonlinelibrary.com): 28 January 2013

� 2013 Wiley Periodicals, Inc.

the 1980s, non-invasive screeningmeth-

ods evolved using maternal serum ana-

lytes. Women of all ages began to be

screened to detect those having aneu-

ploidy risk approximating that of a

35-year-old. This permitted younger

women at increased risk for aneuploidy

to be identified, and offered an invasive

procedure when otherwise this would

not have occurred. The converse could

also occur in women older than age

35 years. Overall, however, the preva-

lence of Down syndrome has actually

not decreased. Nonetheless, multiple

non-invasive protocols are now offered

in all pregnancies and the number of

procedures has decreased. New non-

invasive technologies are being devel-

oped, most importantly autosomal

trisomy detection using cell-free fetal

DNA. As non-invasive protocols have

become increasingly utilized by pro-

viders and patients alike, fewer invasive

procedures are being performed. Thus,

largely serendipitous detection of

47,XXY has decreased.

In this contribution, we consider

detection of 47,XXX and 47,XXY by

the various available methods. These

include traditional invasive procedures,

screening with maternal serum analytes

and fetal ultrasound, and most recently

cell-free fetal DNA.

SAFETYOF INVASIVEDIAGNOSTIC PROCEDURES

For decades invasive procedures were

offered for aneuploidy detection only

to women at relatively high aneuploidy

risks was the rationale for this restriction

that these procedureswere considered to

have sustentative risk for procedure-

related pregnancy loss. The risk due to

amniocentesis at 15–22 weeks was stated

to be 1 in 200. However, the actual basis

was for years a NICHD collaborative

study [Anonymous, 1976], this study

actually showing a 0.5% arithmetic

increase in pregnancy loss in women

undergoing amniocentesis (N ¼ 1,040)

compared to a control group

(N ¼ 992). This was not a statistically

significant difference, but the familiar 1

in 200 procedure-related risk remained.

A single randomized trial by Tabor et al.

[1986] was later conducted in Denmark

in the 1980s, however, and showed a 1%

procedure-related loss. Since then pro-

cedure-related pregnancy losses have

logically decreased as quality ultrasound

was routinely applied [Simpson, 2005].

At present, the risk of procedure-related

losses following amniocentesis in single-

ton pregnancies is considered in experi-

enced hands to be approximately 1 in

400 [Simpson, 2005, 2012]. This low-

ered risk is of relevance because of

the recent development of array com-

parative genome hybridization (CGH),

which can detect not only all aneuploi-

dies but microdeletions and micro-

duplications below the resolution

of a karyotype. Array CGH requires

At present, the risk of

procedure-related losses

following amniocentesis in

singleton pregnancies is

considered in experienced

hands to be approximately 1

in 400. This lowered risk is of

relevance because of the recent

development of array

comparative genome

hybridization (CGH), which

can detect not only all

aneuploidies but

microdeletions and

microduplications below the

resolution of a karyotype.

CVS or amniocentesis, and if instituted

routinely would increase the number of

procedures.

In contrast to relative safety of tra-

ditional amniocentesis at 15–22 weeks,

the American College of Obstetricians

and Gynecologists (ACOG) states un-

equivocally that amniocentesis before

13–14 weeks’ gestation should not

be performed for genetic indications

[ACOG, 2001]. Untoward results

include higher rates of pregnancy loss,

talipes equinovarus (TE), and amniotic

fluid leakage [Nicolaides et al., 1996;

Anonymous, 1998; Philip et al.,

2004].

First trimester chorionic villus sam-

pling (CVS) is considered comparable in

safety to traditional amniocentesis and an

advantage because pregnancy termina-

tion is safer in the first trimester [Lawson

et al., 1994]. In 1989 the first phase of

the U.S. Collaborative Clinical Com-

parison of Chorionic Villus Sampling

and Amniocentesis study [Rhoads

et al., 1989] reported that the pregnancy

loss rate after trans-cervical CVS was

no different than rates after second-

trimester amniocentesis. A random-

ized-controlled trial by the same group

later found no difference between trans-

cervical CVS and trans-abdominal CVS

[Jackson et al., 1992]. In this second

phase of the NICHD collaborative

CVS study, 1,194 patients were random-

ized to trans-cervical CVS and 1,929

to trans-abdominal CVS. Loss rates

in cytogenetically normal pregnancies

through 28 weeks were 2.5% and

2.3%, respectively. The overall loss

rate (i.e., background plus procedure-

related) during the randomized trial

was 0.8% lower than rates observed dur-

ing the mid-1980s. This likely reflected

increasing operator experience as well as

availability of both trans-cervical and

trans-abdominal approaches.

A 2003 Cochrane review [Alfirevic

et al., 2003] assessing comparative safety

of trans-abdominal CVS, trans-cervical

CVS, early amniocentesis, and second-

trimester amniocentesis concluded that

both second-trimester amniocentesis

and trans-abdominal CVS (TA-CVS)

were safer than either trans-cervical

CVS (TC-CVS) or early amniocentesis.

This conclusion was based in part on

reports in which some operators were

less experienced in this procedure than

TA-CVS. In theU.S. collaborative study

[Rhoads et al., 1989] all operators

were, by contrast, highly experienced.

Because trans-cervical CVS is more dif-

ficult to master than trans-abdominal

CVS, the difference in operator experi-

ence is likely significant.

ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 65

DETECTING 47,XXXAND 47,XXY USINGAMNIOCENTESIS ORCHORIONIC VILLUSSAMPLING

Geneticists are well aware that prenatal

cytogenetic studies (whether following

screening or a de novo invasive proce-

dure) are most commonly performed

because of advanced maternal age.

The incidence of trisomy 21 is 1 per

800 live births overall in the USA, orig-

inally justifying prenatal genetic diagno-

sis for women age 35 years and above

[Cross and Hook, 1982; Hook et al.,

1983]. The incidence of trisomy 21 in

the second trimester is about 1 in 270;

the risk of all aneuploidies is about twice

that. The incidence in the first trimester

is somewhat higher [Hook et al., 1988;

Hook and Cross, 1989]. Trisomy 13,

trisomy 18, 47,XXX, and 47,XXY

all increase with advanced age, but

not so marked as for trisomy 21. Thus,

in procedures performed because of

risk for trisomy 21, one will detect

a number of 47,XXY fetuses because

maternal age effect is in common with

both.

The American College ofObstetri-

cians and Gynecologists (ACOG)

endorsed in 2007 the right of any

woman to have an invasive procedure,

assuming near 100% detection of aneu-

ploidy [Anonymous, 2007]. ACOG

guidelines stated that neither age 35 years

nor any specific age should be used as a

threshold for invasive or non-invasive

screening: ‘‘All women, regardless of

age, should have the option of invasive

testing.’’ It was stated that ‘‘patients

informed of the risks, especially those

at increased risk of having an aneuploid

fetus, may elect to have diagnostic

testing without first having screening.’’

This allows women the ability

to achieve near 100% detection of

trisomy 21, possible only with an inva-

sive procedure. Indeed, the detection

rate by an invasive procedure is 10–

15% higher than that of traditional

non-invasive screening by maternal se-

rum analytes, which shall be discussed

below.

DETECTINGMICRODELETIONS ANDMICRODUPLICATIONS BYARRAY COMPARATIVEGENOME HYBRIDIZATION(CGH)

The traditional kayotype, as performed

either after a positive screening protocol

or de novo to detect all autosomal aneu-

ploidy, may soon become obsolete.

Its replacement will be chromosomal

microarrays, which have greater ability

than karyotypes to detect smaller dele-

tions or duplications. Conventional

karyotypes can detect alterations of 5–

10 million base pairs (Mb). Microarrays

(array CGH) detect alterations as small as

200 kilobases (200,000) or, if desired,

20–50 bases. These alterations occur

throughout the genome. The caveat is

that smaller changes are often benign.

The smaller the copy number variants

(CNV), the less likely a pathogenic ef-

fect. However, even small CNVs can be

significant if involving a deleted gene.

CNVs (duplications or deletions) are

more likely to be polymorphisms if

inherited from a clinically normal par-

ent. However, incomplete penetrance

can exist within a family. Confidence

and precision in determining signifi-

cance of a given CNV will increase as

data accumulate. Still, the clinical value

of microarrays is clear, as first shown in

evaluating children with developmental

delay. Of these, 5–7% have clinically

significant CNV variants, a far greater

yield than a karyotype. Array CGH

is now considered a standard part of

postnatal evaluation in those children

[Kearney et al., 2011].

The most relevant prenatal data

came from the recently reported

NICHD prenatal cytogenetic array

study, which involved 4,401 women

having these indications: 46% maternal

age; 26% fetal ultrasound anomaly;

18% increased risk by maternal serum

analyte screening; and 9% others

[Wapner et al., 2012]. All 316 autosomal

trisomies and all 57 sex chromosome

aneuploidies detected by karyotypes

were detected by array CGH. The array

CGH platforms used interrogated 84

chromosomal regions too small to be

detected by conventional karyotypes.

All microdeletions or microduplications

predicted in advance to be detected

(e.g., 22q11 deletion syndrome) were

detected. In addition, theNICHD study

found other clinically significant CNVs

in 5.8% of fetuses with a normal karyo-

type, and in 1.7% in which the indica-

tion was only maternal age or only

increased risk due to serum analyte

screening. Half a dozen other salutary

studies exist as well, the most being the

report of Shaffer et al. [2012] of over

5,000 cases. Of those having an abnor-

mal fetal ultrasound but still viable,

Shaffer et al. found 6.5% to have a clini-

cally significant CNV.

A strong case can thus be made for

offering invasive testing in every preg-

nancy, rather than initially performing

non-invasive screening and then if

‘‘screen positive’’ perform an invasive

procedure to detect autosomal aneuploi-

dies. If array CGH were to become

routine in the general obstetrical popu-

lation, it follows that sex chromosomal

abnormalities (e.g., 47,XXY) would be

detected in increased numbers.

SCREENING FORANEUPLOIDY BYMATERNAL SERUMANALYTES ANDULTRASOUND

If an invasive procedure to detect all

chromosomal abnormalities by kayotype

If an invasive procedure to

detect all chromosomal

abnormalities by kayotype or

array CGH were not chosen,

traditional screening involves

maternal serum analyte and

ultrasound screening. As of

December 2012, ACOG states

that this approach is still

applicable to ‘‘low risk’’

66 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

or array CGH were not chosen, tradi-

tional screening involvesmaternal serum

analyte and ultrasound screening. As of

December 2012, ACOG states that this

approach is still applicable to ‘‘low risk’’

pregnancies. More specifically, in high

risk pregnancies cell-free fetal DNA tests

is considered an option for fetal aneu-

ploidy screening [ACOG Committee,

2012]. However, several studies on rou-

tine populations had not been published

at the time ACOG deliberations were

underway and publication prepared.

First trimester screening for fetal

aneuploidy is best performed between

11 and 14 weeks gestation. The most

effective first trimester maternal markers

are plasma protein A (PAPP-A) and free

beta human chorionic gonadotropin (b-hCG) along with measurement of fetal

nuchal translucency (NT), the sonolu-

cent space behind the fetal neck. PAPP-

A levels are reduced, hCG increased and

NT measurement increased in trisomy

21. No marker alone or in combination

can achieve the near 100% possible by an

invasive procedure. NT measurement

alone has only about a 70% detection

rate with a false-positive (procedure re-

quired) rate by definition of 5% [Nico-

laides, 2004]. Even this detection rate

requires a robust quality NT assurance

program. When NT is combined with

biochemical markers, the detection rate

is 85–87%with a false-positive rate of 5%

[Wald et al., 2003; Wapner et al., 2003;

Malone et al., 2005].

In the second trimester, aneuploidy

screening preferably utilizes four bio-

chemical analytes—maternal serum

alphafetoprotein (MSAFP), hCG, un-

conjugated estriol (uE3), and dimeric

inhibin-A. Detection rate for trisomy

21 is approximately 75% in women

less than 35 years of age, but over 80%

in women 35 years of older. This

approach also necessitates a 5%

amniocentesis rate (false positive rate)

[Wald et al., 2003; Malone et al., 2005].

Various approaches have been pro-

posed for utilizing both first and second

trimester screening to increase the de-

tection rate over that achieved by screen-

ing in either trimester alone. Detection

rates model a 93% detection rate for

trisomy 21 given a false positive rate of

5%. Several different approaches com-

bine first and second trimester results. In

the U.S. the most popular protocol is

contingency screening. In this method,

women do not proceed to second tri-

mester screening if their risk is low on

the first trimester screen. Further testing

is indicated if not. With this approach,

65% of trisomies would be detected in

the first trimester with only 1.5% of

patients having a CVS procedure

[Malone et al., 2005]. Second-trimester

screening and amniocentesis increases

the overall detection rate to 93% for

trisomy 21 cases at a 4.3% false-positive

rate.

Maternal serum analyte and ultra-

sound screening are not designed to de-

tect autosomal or sex chromosomal

aneuploidies other than 18 or 21 karyo-

type [Simpson et al., 2003]. The

multiple of themedian in 47,XXYpreg-

nancies is slightly increased for second

trimester HCG and nearly equal to the

general population forMSAFP and uE3.

First trimester NT shows a 2.07 MOM

but PAPPA and HCG do not differ from

the general population [Spencer et al.,

2000]. It is for these reasons that fewer

47,XXY cases are being detected in pre-

natal genetic diagnosis programs.

SCREENING FORANEUPLOIDY BY INTACTFETALCELLSORCELL-FREEFETAL DNA IN MATERNALBLOOD

The long-term goal of prenatal genetic

diagnosis is definitive or near definitive

non-invasive prenatal diagnosis. One

hopes to use a maternal blood sample

to detect fetal aneuploidy without need

for an invasive procedure. CVS or am-

niocentesis might be performed only to

exclude rare, confounding circumstan-

ces that would result in false positive

results. When performed for a positive

screen, CVS or amniocentesis would

expect to almost always confirm aneu-

ploidy. There are, however, biologic rea-

sons for false positive results. An example

is a ‘‘vanishing twin,’’ a deceased aneu-

ploid embryo from which persistent

placental tissue continues to release

chromosome 21 transcripts.

Definitive non-invasive fetal diag-

nosis first involved recovery and analysis

of fetal cells in maternal blood. In 1991

detection of fetal trisomy 18 was made,

using nucleated fetal red blood cells

recovered from maternal blood [Price

et al., 1991]. Subsequent detection and

confirmation was made for trisomy 21

[Elias et al., 1992; Simpson and Elias,

1993; Bianchi et al., 1999]. Recovery

of intact fetal cells is still being pursued

with notable success by Paterlini-

Brechot and her colleagues at Rare Cells

Diagnostics (Paris) [Mouawia et al.,

2012].

Cell-free fetal DNA in maternal

blood is increasingly the topic of greater

interest. Cell-free DNA has been appre-

ciated in peripheral blood since at least

the 1970s, known initially from individ-

uals with cancer. During pregnancy ma-

ternal blood contains both cell-free

maternal DNA and cell-free fetal

DNA [Lo et al., 1997], now estimated

to be 5–10% fetal. Confirmation that

cell-free fetal DNA exists in maternal

blood was initially made by finding

DNA that cannot be of maternal origin.

If aY sequence is present, for example,Y

DNAmust be fetal in origin because the

mother has no Y-sequences.

Cell-free DNA inmaternal blood is

being aggressively pursued for detection

of fetal aneuploidy, specifically trisomy

21. Aneuploidy detection is more diffi-

cult than single gene detection because

detecting fetal trisomy must reflect

quantitative differences between affect-

ed and unaffected pregnancies. Current

strategies are based on counting the total

numberof chromosome 21 transcripts in

maternal blood. The current method is

massive parallel genomic sequencing of

both maternal and fetal sequences. A

pregnancy carrying a trisomy 21 fetus

has more chromosome 21 transcripts

than one carrying a normal fetus because

pregnancies. More specifically,

in high risk pregnancies

cell-free fetal DNA tests is

considered an option for fetal

aneuploidy screening.


the trisomic fetus has three chromo-

somes 21, whereas the euploid fetus

has only two. The mother’s 21 tran-

scripts remain the same in both situa-

tions. If 10% of cell-free DNA in

maternal blood is of fetal origin, a triso-

my 21 pregnancy contributing 50%

more fetal chromosome 21 transcripts

than disomic fetal chromosome 21 tran-

scripts would yield an overall difference

of 5%, a finite difference yet detectable.

MASSIVE PARALLELGENOMIC SEQUENCING(MPGS)

In the U.S. four companies are offering

cell-free DNA assessment for trisomy

21. In two of these, information on

X-aneuploidy is available. In MPGS all

cell-freeDNApresent inmaternal blood

is sequenced. Those sequences for the

chromosome of interest (e.g., 21) are

then quantitatively enumerated and

compared to those expected in a normal

pregnancy. Sequenom, San Diego, CA

In MPGS all cell-free DNA

present in maternal blood is

sequenced. Those sequences for

the chromosome of interest

(e.g., 21) are then

quantitatively enumerated and

compared to those expected

in a normal pregnancy.

uses the approach patented by Lo

et al. [1997]. Palomaki et al. [2011] per-

formed a blinded, nested case-central

studyof archived samples for Sequenom,

matching 7 controls to each trisomy 21

case. A Z-score of three or above is

considered indicative of a trisomy 21

pregnancy. The detection rate was

98.6% (209/212), the false positive

rate 0.8% (3 of 1,471). Ehrich et al.

[2011] then reported results of 480 ma-

ternal plasma samples, some obtained

prospectively whereas others archived

material; all were analyzed in batch

and results not acted upon clinically.

Of the 480, 13 had ‘‘insufficient vol-

ume’’ and 18 failed quality control

parameters. In the remaining 449, all

39 trisomy 21 samples were correctly

identified; one false positive sample

was recorded. Trisomies 13 and 18

were also less efficiently tested in initial

studies, but the technology later was

better able to detect trisomies 13 and

18 [Chen et al., 2011]. No information

was provided on X-aneuploides. Chiu

et al. [2011] used the same technology

to study plasma samples prospectively

collected from pregnant women and

archived. Of 576, 1.7% ‘‘did not meet

recruitment criteria’’ and 5.6% samples

‘‘failed to pass the specimen quality

requirements’’. All 86 trisomy 21 cases

were detected, with 97.9% trisomy

18 and trisomy 13 were also detected

[Fan et al., 2008].

A second company, Verinata (San

Carlos, CA) uses the MPGS technology

developed by Quake and colleagues

[Chiu et al., 2011]. Sehnert et al.

[2011] performed a validation study

using archived samples. In 2012, Bianchi

et al. [2012] then used this technique in

samples prospectively collected and pre-

sumably analyzed in batch. The mean

gestational age in this study was

16 weeks. The non-informative rate

due to insufficient fetal DNA was 3%.

All 89 trisomy 21 cases were detected.

Detection rate for trisomy 18 was 35 of

36 and for trisomy 13 it was 11 of 14.

There were no false positives for chro-

mosomes 21, 18, 13 in this dataset.

Although not the stated intent of the

study, 45,X and 47,XXX cases were

also detected.

The largest published experience

using this method of MPGS comes

from BGI-Shenzhen [2012] [Dan et

al., 2012]. A total of 11,263 pregnancies

were samples, 1,387 without a ‘‘specific

risk factor.’’ All 143 trisomy 21 cases and

all 47 trisomy 18 cases were detected,

with no false negatives. There was only

one false positive trisomy 21 and one

false positive trisomy 18.

The largest published

experience using this method

of MPGS comes from

BGI-Shenzhen [2012]. A

total of 11,263 pregnancies

were samples, 1,387 without a

‘‘specific risk factor.’’ All 143

trisomy 21 cases and all 47

trisomy 18 cases were detected,

with no false negatives. There

was only one false positive

trisomy 21 and one false

positive trisomy 18.

Two other groups in the U.S. are

seeking to detect fetal aneuploidy by

a modified MPGS-approach targeted

cell-free fetal DNA sequencing. A tar-

geted approach requires fewer sequences

(‘‘reads’’) are necessary to diagnose au-

tosomal trisomies of interest, perhaps

not 25 million but 1 million. One group

is Ariosa (San Jose, CA), which began

with salutary validation reports based on

setting of a 99% likelihood (threshold)

for trisomies 21, 18, or 13 (positive); less

than 1% likelihood was defined as not

aneuploid. A test set of trisomy 21 cases

and controls showed 100% detection of

trisomy 21 and 18 [Ashoor et al., 2012;

Sparks et al., 2012a,b]. This group pro-

vides a pregnancy-specific risk figure,

based not only on quantitative values

for number of 21 sequences, but also

maternal age and percent fetal DNA in

maternal blood. Norton et al. [2012]

used this approach in reporting a cohort

study involvingwomen of advancedma-

ternal age (mean 34.3-year) from which

samples were collected at mean gesta-

tional age 16 weeks. The non-informa-

tive rate was 4.6% (1.8% inadequate fetal

DNA; 2.8% assay failure).Detection rate

was 81 of 81 trisomy 21, and 37 of 38

trisomy 18 cases. One (1) of 2,228 sam-

ples was a false positive. A final report

using this method is different in design,

a ‘‘routinely screened’’ first trimester

series, with maternal age 31.8 years

[Nicolaides et al., 2012]. The non-

informative rate was 4.8% (2.2% < 4%

fetal DNA; 2.6% assay failure). Eight (8)


of 8 trisomy 21 cases and 2 of 2 trisomy

18 caseswere detected. The false positive

rate was 0.1%. In updated experience

(Ken Song, Personal Communication),

all 241 trisomy 21 cases, 103 of 105

trisomy 18 cases, and 8 of 10 trisomy

13 have been detected. False positives

were <0.1%. The accumulated sample

size at Ariosa involves ‘‘over 6,000

patients including over 2,000 low risk

patients.’’

A second group using targeted

sequencing is Natera (San Carlos,

CA). Their method requires knowledge

of parental single nucleotide polymor-

phisms (SNPs) [Demko et al., 2012].

Knowing parental SNPs allows one

to enumerate all possible trisomic, diso-

mic, and monosomic fetal genotypes

expected in euploid versus aneuploid

fetuses. Predictive values for individual

cases can then be compared to expect-

ations. Zimmerman et al. [2012] studied

166 samples, targeting 11,000 SNPs on

chromosomes 13, 18, 21, X andY. Their

sample included 2 trisomy 13 cases, 3

trisomy 18, 11 trisomy 21, 2 45,X and 2

47,XXY. The correct chromosome

number was reported in all 20 aneuploid

cases and 146 euploid cases. The

reported non-informative rate was

12.6% but this rate was defined as inabil-

ity to obtain information on any of

the five chromosomes. Non-informa-

tive rates in other studies were based

on fewer chromosomes interrogated.

Suppose cell-free DNA approach

replaces maternal serum analytes and

NT as the primary screening test.

What will be the impact on frequency

of 47,XXY fetuses? Fewer cases could be

detected given that less than 1% false

positive rate will result in fewer invasive

procedures than with maternal serum

analytes and NT screening with its 5%

false positive rate. On the other hand,

more women may opt for a less compli-

cated and less burdensome approach.

Whether vendors will offer results on

47,XXY remains unclear. If so, more

47,XXY cases should be detectable

than at present.

CONCLUSIONS

Serendipitous detection of 47,XXY was

not infrequent when prenatal genetic

diagnosis routinely involved testing by

the invasive proceduresCVS and amnio-

centesis. In 2013 this is much less com-

mon. Relatively few pregnancies in

the U.S. and Europe are tested without

prior screening protocols, traditionally

maternal serum analyte and fetal ultra-

sound (NT). These protocols are not

designed to identify 47,XXY or other

X-chromosome aneuploides. With

screening by analysis of cell-free DNA

in maternal blood, this situation may or

may not be altered. Increased numbers of

cases could be detected if intake increases

and vendors offer information on

47,XXY. A further consideration is

that ability of array CGH to detect

microdeletions or microduplications be-

low resolution of a kayotype could make

return to direct testing using an invasive

procedure attractive.

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