Fetal and Pediatric Pathology

ISSN: 1551-3815 (Print) 1551-3823 (Online) Journal homepage: http://www.tandfonline.com/loi/ipdp20

A Dysmorphology Based Systematic Approach

Toward Perinatal Genetic Diagnosis in a Fetal
Autopsy Series

Shagun Aggarwal, Ashwani Tandon, Aneek Das Bhowmik, Jamal Mohamed

Nurul Jain Safarulla & Ashwin Dalal

To cite this article: Shagun Aggarwal, Ashwani Tandon, Aneek Das Bhowmik, Jamal Mohamed
Nurul Jain Safarulla & Ashwin Dalal (2018): A Dysmorphology Based Systematic Approach
Toward Perinatal Genetic Diagnosis in a Fetal Autopsy Series, Fetal and Pediatric Pathology, DOI:
10.1080/15513815.2017.1397070

To link to this article: https://doi.org/10.1080/15513815.2017.1397070

Published online: 16 Jan 2018.

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FETAL AND PEDIATRIC PATHOLOGY
, VOL. , NO. , –
https://doi.org/./..

A Dysmorphology Based Systematic Approach Toward Perinatal

Genetic Diagnosis in a Fetal Autopsy Series
Shagun Aggarwal a,b , Ashwani Tandonc , Aneek Das Bhowmikb , Jamal Mohamed Nurul
Jain Safarullab , and Ashwin Dalalb
a
Department of Medical Genetics, Nizam’s Institute of Medical Sciences, Punjagutta, Hyderabad, Telangana,
India; b Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, India;
c
Department of Pathology, Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India

ABSTRACT ARTICLE HISTORY

Background. This retrospective study assesses the contribution of Received  July 
genetic disorders in fetuses undergoing postmortem evaluation and the Revised  October 
performance of a clinical dysmorphology based systematic approach Accepted  October 
toward genetic diagnosis. Materials and Methods. Ninety fetuses, includ- KEYWORDS
ing spontaneous losses and terminated pregnancies, underwent a post- Fetal autopsy; fetal
mortem evaluation including dysmorphological examination, radiolog- dysmorphology; fetal
ical studies, and histopathological examination. Genetic testing includ- pathology; birth defects;
ing karyotyping, biochemical testing, Sanger sequencing, and exome exome sequencing
sequencing were performed selectively. Results. A genetic etiology was
concluded in 48 fetuses (55%). As a standalone test, dysmorphological
examination was able to ascertain a definite genetic diagnosis in sixteen
cases, histopathology in six; and karyotyping, biochemical testing and
exome sequencing in two cases each (Total 28). Additionally, dysmor-
phology findings indicated possible genetic disorder in 20 cases. Conclu-
sion. Genetic etiologies contribute significantly to fetuses undergoing
autopsy in this series. A systematic approach to postmortem fetal evalu-
ation guided by dysmorphological examination provides high diagnos-
tic yield toward perinatal genetic diagnosis.

Introduction
Fetal autopsy is one of the prime modalities for establishing underlying causative etiology in
cases of morphologically abnormal pregnancies or intrauterine fetal demise (1–3). Various
autopsy series in the past have demonstrated that a postmortem evaluation provides addi-
tional findings or modifies the antenatal diagnosis in 20%–50% cases (4–7). Antenatal series
have also shown that at least 50% of syndromic diagnoses are possible only after an autopsy
(8). Etiologically, chromosomal abnormalities are reported in a significant proportion of
pregnancy losses, the magnitude of contribution being inversely proportional to gestation
(9–11). In addition, chromosomal microarray studies in postmortem period report submicro-
scopic abnormalities in 2%–10% of stillbirths (11–13). Antenatal series with morphologically
abnormal fetuses also report 6%–10% of cases with submicroscopic abnormalities detected
by microarray (14,15) and some recent reports have found single gene defects in such fetuses

CONTACT Shagun Aggarwal shagun.genetics@gmail.com Department of Medical Genetics, th Floor, Specialty Block,
Nizam’s Institute of Medical Sciences, Punjagutta, Hyderabad, Telangana , India.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ipdp.
©  Taylor & Francis Group, LLC
2 S. AGGARWAL ET AL.

using exome sequencing, reporting yields as high as 20%–30% (16–20). Hence, an underlying
genetic etiology appears likely in a significant number of abnormal fetuses.
The establishment of etiological diagnosis in an abnormal fetus has obvious implications
for an ongoing pregnancy in the antenatal scenario, and has definite benefits in terms of emo-
tional closure, recurrence risk counselling, definitive prenatal diagnosis in subsequent preg-
nancies, in a postmortem scenario (1,2,7).
This study reports our attempt to assess the performance of a systematic approach involv-
ing dysmorphological, histopathological, radiological, and genetic evaluations in arriving at
a genetic diagnosis in case of fetuses undergoing autopsy.

Materials and methods

This study was conducted in a tertiary university hospital and associated federal laboratory
in a southern province of India over a period of four years. Approval from Institutional Ethics
committee was obtained. Inclusion criteria were all fetuses brought for an autopsy evaluation
including stillbirths and medically terminated pregnancies. Fetal autopsies involved a dys-
morphological examination and gross dissection by the clinical geneticist, full body antero-
posterior and lateral radiographs, gross and histopathological studies by the pathologist; and
laboratory genetic testing on fetal samples. Additionally, a detailed pedigree charting was
performed for all cases as part of clinical genetic evaluation. A single clinical geneticist and
pathologist recorded the dysmorphology and histopathology findings, respectively. In cases
where viable tissue was available, karyotyping was done, irrespective of the autopsy findings.
Targeted biochemical and/or molecular testing were performed selectively depending on the
clinical, radiological or histopathological findings, and availability of suitable sample. Molecu-
lar testing also included next generation sequencing (NGS) in four cases, which were selected
due to specific clinical features and/or family pedigree indicative of a monogenic disorder.
Different approaches of NGS (whole exome and clinical/targeted exome sequencing) meth-
ods were used; NGS methods and data analysis pipelines were as described earlier (21–23).
This approach simulates the actual clinical setting in most centers, where appropriate fetal
samples are often lacking and advanced technologies are limited by cost and access.
The findings of the dysmorphological evaluation and gross dissection were concluded
under three categories, indicative of a definite genetic disorder, suggestive of a possible genetic
disorder and miscellaneous category, which included acquired and unknown etiologies. A
definite genetic disorder was concluded if there were characteristic pattern of anomalies
and/or dysmorphic features. A possible genetic disorder was concluded in cases with sugges-
tive findings and/or positive family history, but with no specific diagnosis apparent. A compar-
ison was made with the ultrasound findings and the possibility of a specific genetic disorder
antenatally was also ascertained. The contribution of histopathological and radiological find-
ings toward making a genetic diagnosis was grouped under three headings, confirmatory of
clinical diagnosis, adding to clinical diagnosis, or independently providing a specific genetic
diagnosis. Although karyotype or molecular/biochemical test could not be performed in all
cases due to unavailability of suitable samples, the positive results were included in the final
analysis. The cases were finally reclassified as definite genetic, possible genetic and miscella-
neous after including results of histopathology, radiology and genetic testing. The data was
analyzed to look for the contribution of genetic etiologies and the performance of individual
evaluation steps toward establishing a genetic diagnosis. The assessment protocol followed in
this study has been depicted in Figure 1.
 FETAL AND PEDIATRIC PATHOLOGY 3

Figure . Diagrammatic representation of the assessment algorithm and the contribution of individual eval-
uation modalities toward the final classification as “definite genetic disorder,” “possible genetic disorder,” and
“miscellaneous.” Footnote: Genetic tests number in the figure represents the number of tests (not number of
subjects). Confirmatory findings in case of genetic tests and exome sequencing represent the cases where
the test result confirmed the dysmorphology diagnosis. Specific genetic diagnosis indicates the cases with
diagnosis not found on other evaluations, hence representing a stand-alone yield. A total of  cases were
reclassified on basis of histopathology, genetic testing or exome sequencing as “definite genetic disorders”.
These included  from “possible genetic disorder”group and  from “miscellaneous”group, this being a case
of hydrops fetalis found to be Monosomy X on karyotype.

Results
A total of ninety fetuses underwent autopsy. These ranged from 12 to 38 weeks 5 days gesta-
tion. The indications for autopsy included intrauterine fetal demise (20), stillbirths (8), and
pregnancy terminations due to abnormal ultrasonography [62]. Dysmorphologic examina-
tion, radiography, internal dissection and histopathology studies were performed in all cases.
Fetal karyotyping was possible in 26/90 cases, targeted biochemical and/or molecular genetic
testing in 12 cases and NGS in four fetuses.

Ultrasound findings
In 37 cases, the ultrasound findings indicated possibility of a genetic disorder, due to con-
stellation of multiple abnormalities and/or in conjunction with a positive family history. In
the rest of the 53 cases ultrasound findings were considered to be of miscellaneous etiology,
these including cases with isolated malformations or etiologically heterogeneous entities like
hydrops fetalis, ventriculomegaly, fetal growth restriction, or acquired conditions.
4 S. AGGARWAL ET AL.

Dysmorphology findings
Dysmorphological evaluation indicated a specific genetic disorder in 16 cases (Table 1).
These included the 13 cases with antenatal imaging findings indicating a possible genetic
disorder(Case1–13, Table 1) and three cases which were classified as miscellaneous(Case
14–16, Table 1). In 31 cases, a possible genetic etiology was considered likely, but no specific
diagnosis was apparent (Case 17–25, 27, 28, Table 1 and Case 1–20, Table 2). In the remaining
43 cases, dysmorphology and gross findings were concluded as miscellaneous.

Radiological findings
Specific radiographic findings were found in four cases, which provided additional infor-
mation in support of the clinical diagnosis (Case 2, 12–14, Table 1). In six cases the radio-
graphic findings were confirmatory of clinical findings (Case 11, 20, Table 1 and Case 8, 13–15,
Table 2). In others, the radiographs were noncontributory.

Histopathology findings
In six cases histopathology revealed specific findings, which indicated a genetic diagnosis
(Case 17–22, Table 1). Of these, in three cases the dysmorphology findings and family his-
tory provided important adjunct information for interpretation of histopathology findings.
Additional findings were found in five cases (Cases 8, 9, 12, Table 1 and Cases 2, 12, Table 2).
In seven cases, histopathology findings were confirmatory of the clinical diagnosis (Case 3–6,
13, 14, Table 1, and Case 9 Table 2). In other cases, histopathology indicated acquired etiol-
ogy (8) or was noncontributory [64] due to autolysis (12), nonspecific (10), or unremarkable
findings [42].

Cytogenetic testing
Karyotyping performed in 26 fetuses revealed two cases of Trisomy 13 and one case each of
Trisomy 18 and Monosomy X (Case 1, 15, 23, 26, Table 1, 4/26 positive). Two of these positive
cases were clinically suspected, while two were primarily cytogenetic diagnoses.

Biochemical and molecular testing

Phenotype driven targeted biochemical and/or molecular testing resulted in identification of
a genetic disease in six cases (Case 3, 6, 8, 9, 24, 25, Table 1); two of these being standalone bio-
chemical diagnoses and others merely confirmatory of clinical diagnoses.. In five cases with
hydrops fetalis, enzyme assays for five lysosomal storage disorders from cultured amniocytes
were unremarkable and one case with a cono-truncal anomaly was normal for 22q microdele-
tion (overall 6/12 positive).

Next generation sequencing

Targeted or Whole exome sequencing results were available in four cases (Case 14, 18, 27, 28,
Table 1). Case 14 concluded to be a connective tissue disorder, showed double heterozygous
putative pathogenic mutations in FBN1 and FBN2 on NGS, which cause Marfan syndrome
and Beals syndrome, respectively; supporting autopsy diagnosis. Case 18 was concluded to
Table . Cases classified as definite genetic disorders after complete evaluation.

Classification
based
on Classification
ultra- Dysmorphology based on Primary
Case sound evaluation dysmorphic Diagnostic
No. Ultrasound findings findings POG F/H findings findings Radiological Histopathology Genetic testing Diagnosis Basis

 Holo- Possible  wks No Alobar holopros- Definite Noncontributary Noncontributory Karyotype- Trisomy  Dysmorphology
prosencephaly, encephaly, Trisomy 
PAP,CLP,CHD, midline
multiple soft CLP,PAP,
markers complex CHD
 Short bones, limb Possible  wks Yes Cleft palate, facial Definite Additional Noncontributory Parental Oto-palato-digtal Dysmorphology
deformities, dysmorphism, findings Short karyotypes syndrome
vermis digital bones, mild normal DNA
hypoplasia anomalies, platy-spondyly, NA
vermis decrease skull
hypoplasia ossification
 Growth restriction, Possible  wks Yes Ichthyosis, Definite Noncontriutory Confirmatory Parents Gaucher Disease, Dysmorphology
cardiomegaly, hepato- Ichthyosis Heterozygous perinatal lethal
joint splenomegaly, carriers of phenotype
contractures cardiomegaly RecNil allele in
GBA
 CDH, unilateral CLP, Possible  wks No Facial Definite Nonconributory Confirmatory NA Fryns syndrome Dysmsorphology
cystic SOL dysmorphism, Abdominal phenotype
abdomen unilateral cleft cysts lined by
lip, digital intestinal
anomalies, epithelium
multiple
malformations
 Microcephaly, Possible  wks Yes Microcephaly, Definite Noncontributory Confirmatory Fetal karyotype Lissencephaly Dysmorphology
decreased lissencephaly, Lissencephaly normal RELN or TUB
gyri-sulci, vermis vermis Molecular mutation
hypoplasia hypoplasia testing NA
FETAL AND PEDIATRIC PATHOLOGY

(Continued on next page)

5
 6

Table . (Continued)

Classification
based
on Classification
ultra- Dysmorphology based on Primary
S. AGGARWAL ET AL.

Case sound evaluation dysmorphic Diagnostic

No. Ultrasound findings findings POG F/H findings findings Radiological Histopathology Genetic testing Diagnosis Basis

 Severe Possible  wks Yes Bilateral enlarged Definite Noncontributory Confirmatory PKHD mutation Autosomal Dysmorphology
oligohydramnios, kidneys, with ARPKD recessive
bilateral multiple small polycystic
enlarged cysts kidney disease
echogenic
kidneys with
small cysts
 Bilateral Possible  wks Yes Postaxial Definite Noncontributory Noncontributory Molecular testing Ciliopathy Dysmorphology
ventriculomegaly hexadacytyly, NA
bilateral lateral
ventriu-
lomegaly
 Clenched hands, Possible  wks Yes Hydrops, hepato- Definite Noncontributory Additional Homozygous bp Gaucher disease, Dysmorphology
club feet, absent (Twin ) splenomegaly, findings deletion GBA perinatal lethal
stomach bubble arthrogryposis Lamellar phenotype
multiplex, calcification
severe cerebral with
atrophy vasculopathy
brain
 Clenched hands and Possible  wks Yes Hydrops, hepato- Definite Noncontributory Additional Homozygous bp Gaucher Disease, Dysmorphology
club feet, dandy (Twin ) splenomegaly, findings Same deletion GBA perinatal lethal
walker arthrogryposis as case  phenotype
malformation, multiplex,
absent stomach severe cerebral
bubble atrophy
 Hydrops fetalis Possible  wks Yes- Hydrops, Definite Noncontributory Noncontributory DNA NA Heterotaxy Dysmorphology
Hydrops overgrowth,
situs
ambiguous
 Short bones, Possible  wks Yes  Facial Definite Confirmatory- Noncontributory Molecular testing Skeletal dysplasia Dysmorphology
arthrogryposis dysmorphism, Short long NA with joint
multiplex short stout bones, multiple dislocations
bones, multiple joint likely Omani
joint dislocations type
deformities
 ICEF, short bones, Possible  wks No Binder facies, Definite Additional Additional Molecular testing Chondrodysplasia Dysmorphology
absent stomach brachytelepha- findings findings NA Karyotype punctata,
bubble, lyngy Epiphyseal Ectopic normal brachytelepha-
polyhydramnios stippling calcification langic type ?
Keutel
syndrome
 Binder facies Possible  wks Yes Binder facies, Definite Additional Confirmatory Molecular testing Chondrodysplasia Dysmorphology
brachytelepha- findings NA punctata,
lyngy Epiphyeal brachytelepha-
stippling langic
type
 Arthrogryposis Miscell-  wks No Facial Definite Additional Confirmatory Double Connective tissue Dysmorphology
multiplex, aneous dysmorphism, findings Thin Loose heterozygote disorder
polyhydramnios multiple joint ribs, cortical connective for FBN and
contractures irregularity in tissue, myxoid FBN
and long bones, changes in mutation∗
dislocations of multiple joint deep dermis,
knee joints dislocations and articular
cartilage
 CHD- transposition / Miscell-  wks No Facial Definite Noncontributory Noncontributory Karyotype- Trisomy  Dysmorphology
DORV, aneous dysmorphism, Trisomy 
polyhydramnios overlapping
fingers,
multiple
malformations
FETAL AND PEDIATRIC PATHOLOGY

(Continued on next page)

7
 8

Table . (Continued)

Classification
based
on Classification
S. AGGARWAL ET AL.

ultra- Dysmorphology based on Primary

Case sound evaluation dysmorphic Diagnostic
No. Ultrasound findings findings POG F/H findings findings Radiological Histopathology Genetic testing Diagnosis Basis

 Hydrops Miscell-  wks No Hydrops, Cystic Definite Noncontributory Noncontributory Common Multiple pterygia Dysmorphology
aneous hygroma, aneuploidies syndrome
Multiple excluded
pterygia and
joint
contractures
 Severe Possible  wks Yes Increase skin Possible Noncontributory Specific diagnosis DNA NA Neonatal Histopathology
oligohydramnios wrinkling, facial Liver with hemochromatosis-
dysmorphism, pigment laden possible
limb macrophages monogenic
deformities showing iron
deposits on
Perls stain
 Corpus callosum Possible  wks Yes Corpus callosum Possible Noncontributory Specific diagnosis EPG mutation∗ Syndromic cortical Histopathology
agensis agenesis Cortical dysgenesis
microdysgene- with corpus
sis callosum
agenesis
 IUD,unilateral Miscell-  wks No Features of Possible Noncontributory Specific diagnosis Parental Lissencephaly Histopathology
ventricu- aneous coagulopathy, Lissencephaly karyotypes with
lomegaly,obliteration facial normal Mother coagulopathy
of umbilical dysmorphism, with low AT
arteries SUA levels
 Encephalocele, Miscell-  wks No Posterior Possible Confirmatory Specific diagnosis DNA NA Dystro-glycano- Histopathology
ventriculo- aneous encephalo- Occipital Cobblestone pathy
megaly, cele,ventriculomegaly, defect, lissencephaly
SUA corpus overlapping
callosum skull bones
agenesis,
dysmorphism
 Hydranencephaly, Miscell-  wks No Hydranencephaly, Possible Noncontributory Specific diagnosis DNA NA Fowler syndrome Histopathology
cerebellar aneous prominent Cerebellar
hypoplasia meningeal heterotopia,
vessels, dys- prominent
morphism,VSD, perforating
cerebellar menigocortical
hypoplasia vessels
 Inferior vermis Possible  wks Yes Inferior vermis Possible Noncontributory Specific diagnosis Molecular testing Lissencephaly- Histopathology
hypoplasia hypoplasia, Lissencephaly NA RELN or TUBA
Normal Sylvian mutations
fissure
 Complex CHD, left Possible  wks No Facial Possible Noncontributory Noncontributory Karyotype- Trisomy  Targeted Genetic
renal ectopia, dysmorphism, Trisomy  test
bilateral postaxial
enlarged polydactyly,
kidneys, CDH, multiple
Dandy Walker malformations
malformation
 Hydrops fetalis Possible  wks Yes Hydrops, Hepato- Possible Noncontributory Noncontributory Low beta- MPS VII Targeted Genetic
splenomegaly d/t autolysis glucuronidase test
activity
(Continued on next page)
FETAL AND PEDIATRIC PATHOLOGY
9
 10

Table . (Continued)
Case Ultrasound findings POG F/H Dysmorphology Classification Radiological Histopathology Genetic testing Diagnosis Primary
No. Classification evaluation based on Diagnostic
based findings dysmorphic Basis
on findings
S. AGGARWAL ET AL.

ultra-
sound
findings

 Hydrops fetalis Possible  wks Yes Hydrops fetalis, Possible Noncontributory Noncontributory Low beta- MPS VII Targeted Genetic
bilateral talipes d/t autolysis glucuronidase test
equinvarus, activity
hepatomegaly
 Hydrops fetalis Miscell-  wks No Hydrops fetalis Miscellaneous Noncontributory Noncontributory Karyotype- Monosomy X Targeted Genetic
aneous Monosomy X test
 IUGR, Anydramnios Possible  wks Yes Small kidneys, Possible Noncontributory Noncontributory ACE mutations∗ Possible renal Exome
hypoplastic d/t autolysis pathology sequencing
bladder
 Cystic hygroma, Possible  wks Yes Left Possible Noncontributory Additional ZFPM mutation∗ Multiple Exome
Diaphragmatic diaphragmatic findings malformation Sequencing
hernia hernia, Cystic Multicystic syndrome
hygroma, dysplastic
digital kidneys
anomalies

: One fetus with heterotaxy, hemivertebrae, ventriculomegaly, echogenic kidneys.

: sib with short trunk short stature and joint dislocations.
: mother and maternal grandfather with similar facies, normal stature.
: Sib with club feet, digital anomalies, posterior neck swelling.
∗ Mutations identified on Exome Sequencing.
ARPKD: autosomal recessive polycystic kidney disease; AT: antithrombin III; CDH: congenital diaphragmatic hernia; CHD: congenital heart disease; CLP: cleft lip/palate; CTEV: congenital talipes equino varus;
DORV: double outlet right ventricle; F/H: family history; ICEF: intracardiac echogenic focus; IUD: intrauterine demise; IUGR; intrauterine growth restriction; NA: not available; PAP: postaxial polydactyly;
POG: period of gestation; SOL: space occupying lesion; SUA: single umbilical artery; VSD: ventricular septum defect.
Table . Cases classified as possible genetic disorders after complete evaluation.

Classification
based
on Classification
ultra- Dysmorphology based on Primary
Case sound evaluation dysmorphic Diagnostic
No. Ultrasound findings findings POG F/H findings findings Radiological Histopathology Genetic testing Diagnosis Basis

 Ventriculomegaly Possible  wks Yes Facial Possible Noncontributory Noncontributory Molecular testing Syndromic ven- Dysmorphology
dysmorphism, NA triculomegaly
gut
malrotation,
ventricu-
lomegaly
 Tricuspid atresia, Possible  wks No Tricuspid atresia, Possible Noncontributory Noncontributory NA Multiple Dysmorphology
multiple soft Postaxial malformation
markers polydactyly, syndrome
facial
dysmorphim
 Complex CHD, Possible  wks No TGA with single Possible Noncontributory Noncontributory Karyotype NA Multiple Dysmorphology
thymic atrium, facial q deletion malformation
hypoplasia dysmorphism, negative syndrome
digital
anomalies,
Thymus
present
 Arthrogryposis Possible  wks Yes Arthrogryposis Possible Noncontributory Noncontributory NA Arthrogryposis Dysmorphology
multiplex, multiplex, multiplex with
micrognathia, facial dysmorphism
polyhydramnios dysmorphism,
narrow chest,
digital
anomalies
(Continued on next page)
FETAL AND PEDIATRIC PATHOLOGY
11
 12

Table . (Continued)

Classification
S. AGGARWAL ET AL.

based
on Classification
ultra- Dysmorphology based on Primary
Case sound evaluation dysmorphic Diagnostic
No. Ultrasound findings findings POG F/H findings findings Radiological Histopathology Genetic testing Diagnosis Basis

 Nuchal edema, Possible  wks No Facial Possible Noncontributory Noncontributory Karyotype normal Dysmorphic Dysmorphology
unilateral talipes dysmorphism, syndrome
Bilateral simian
crease, digital
anomalies,
talipes
equinovarus
Excessive neck
skin folds
 Ventriculomegaly, Possible  wks No Bilateral complete Possible Noncontributory Noncontributory Karyotype Normal Multiple Dysmorphology
unilateral cleft lip cleft lip & malformation
& palate and palate, facial syndrome
short bones dysmorphism,
bilateral ven-
triculomegaly
 Hydrops fetalis Possible  wks Yes Hydrops fetalis Possible Noncontributory Noncontributory Enzyme assay for Recurrent Hydrops Dysmorphology
Increase and cystic LSDS normal, fetalis
NT hygroma, mild Microarray
facial normal
coarsening
 Absent Possible  wks No Facial Possible Confirmatory Noncontributory NA Syndromic radial Dysmorphology
radii,vertebral dysmorphism, Vertebral ray defect
defect, CHD,SUA radial ray segmentation
defect, anal defect in
atresia, thoracic region
vertebral and spina
splaying, bifida in
tracheo- lumbar region,
esophageal bilateral absent
fistula, radii
pulmonary
artery stenosis
 IUGR,Bilateral Possible  wks No Tricuspid Possible Noncontributory Confirmatory NA Multiple Dysmorphology
MCDK,cardiomegaly stenosis,MCDK Multicystic malformation
dysplastic syndrome
kidneys
 Ventriculomegaly, Possible  wks Yes Hydrops, facial Possible Noncontributory Additional Karyotype Normal Dysmorphic Dysmorphology
posterior fossa dysmorphism, findings syndrome with
cyst, fetal limb Proliferating meningeal
hydrops anomalies, menigeal vasculopathy
hep- vessels with
atosplenomegaly, perforation
ventricu- into cortex &
lomegaly, areas of
hemorrage in meningeal
posterior fossa hemorrage
 Hydrops, Increased Possible  wks Yes Facial Possible Noncontributory Noncontributory Parental Hydrops fetalis Dysmorphology
HC dysmorphism, karyotypes and with
hydrops, enzyme assays macrocephaly
unilateral for LSDs
upper limb normal
deformity,
macrocephaly
(Continued on next page)
FETAL AND PEDIATRIC PATHOLOGY
13
 14

Table . (Continued)

Classification
based
on Classification
ultra- Dysmorphology based on Primary
Case sound evaluation dysmorphic Diagnostic
No. Ultrasound findings findings POG F/H findings findings Radiological Histopathology Genetic testing Diagnosis Basis
S. AGGARWAL ET AL.

 Unilateral ventricu- Possible  wks No Facial Possible Noncontributory Noncontributory Karyotype normal Dysmorphic Dysmorphology
lomegaly, dysmorphism, syndrome
PUV PUV, Bilateral
mild ventricu-
lomegaly
 Bilateral radial ray Possible  wks No Facial Possible Confirmatory  Noncontributory Karyotype normal Syndromic radial Dysmorphology
defect and single dysmorphism, ribs, absent ray defect
umbilical artery Bilateral club radii, bent ulna
hands, Radial
ray defect, SUA,
horseshoe
kidney
 Bilateral absent Possible  wks No Facial Possible Confirmatory Noncontributory Karyotype Normal Syndromic radial Dysmorphology
radius, clubfeet dysmorphism, Bilateral absent ray defect/
radial ray radii, right tibia Mesomelic
defect, bilateral bowing, dysplasia
CTEV Bilateral CTEV
 Cardiomegaly, short Possible  wks No Facial Possible Confirmatory Claw Noncontributory NA Dysmorphic Dysmorphology
bones, dysmorphism, hands, long syndrome
hypoplastic kyphoscoliosis, digits
cerebellum, small joint
Hydronephrosis contractures,
excessive skin
folds, arachn-
odactyly,
hydrouretronephroses
with PUV,
cerebellum
small
  IUD Miscell-  wks No Facial Possible Noncontributory Noncontributory NA Dysmorphic Dysmorphology
aneous dysmorphism, syndrome
nuchal edema,
digital
anomalies
 IUD,hydrops, cystic Miscell-  wks No Facial Possible Noncontributory Noncontributory NA Dysmorphic Dysmorphology
hygroma aneous dysmorphism, syndrome
cystic hygroma,
digital
anomalies
 Ebstein anomaly Miscell-  wks No Facial Possible Noncontributory Noncontributory NA Dysmorphic Dysmorphology
aneous dysmorphism, syndrome
Ebstein
anomaly
 Hydrops Miscell-  wks No Hydrops, Facial Possible Noncontributory Noncontributory Karyotype normal Dysmorphic Dysmorphology
aneous dysmorphism, Enzyme assay syndrome
short neck, for LSDs
clinodactyly normal
 IUD Miscell-  wks No Facial Possible Noncontributory Noncontributory NA Dysmorphic Dysmorphology
aneous dysmorphism syndrome

CHD: congenital heart disease; CTEV: congenital talipes equinous varus; F/H: family history; HC: head circumference; IUD: intrauterine demise; LSD: lysosomal storage disorder; MCDK: multicystic dysplastic
kidneys; NA: not available; POG: period of gestation; PUV: posterior urethral valves; SUA: single umbilical artery; TGA: transposition of great arteries.
FETAL AND PEDIATRIC PATHOLOGY
15
16 S. AGGARWAL ET AL.

be a syndromic corpus callosum agenesis with cortical microdysgenesis on basis of gross and
histopathological findings, and the history of a previously affected sibling. NGS results showed
presence of biallelic EPG5 mutations, indicating a diagnosis of Vici syndrome, which was con-
cordant with clinical features of elder sib. Case 27 was a fetus with early onset unexplained
anhydramnios with a similarly affected previous sibling. No definite diagnosis was possible
on autopsy, but NGS revealed biallelic ACE mutations confirming autosomal recessive renal
tubular dysgenesis as causative. Case 28 had autopsy findings suggestive of a multiple malfor-
mation syndrome. NGS results reported a heterozygous ZFPM2 mutation, which was inher-
ited from the father. ZFPM2 variants are reported to be associated with congenital diaphrag-
matic hernia as seen in this fetus, but this mutation could not explain the additional finding
of cystic kidney disease. No other pathogenic variant causative of renal abnormalities could
be identified.
A definite genetic diagnosis was concluded in 28/90 cases after systematic assessment based
on dysmorphology, histopathology, radiology and genetic testing with relative contributions
as depicted in Table 1. Twenty fetuses were concluded to be of possible genetic etiology
(depicted in Table 2) and 42 of miscellaneous origin, 11 of these being of acquired etiolo-
gies. Figure 1 depicts the diagnostic performance of the various evaluation modalities toward
the final genetic diagnosis.

Discussion
This study investigated the overall genetic contribution toward fetal pathological states in
a mixed cohort of spontaneously lost as well as terminated pregnancies due to abnormal
sonographic findings. The study also reports the diagnostic performance of a comprehensive
workup inclusive of clinical genetic, histopathological, radiological, and genetic evaluations
toward synthesis of a genetic diagnosis.
Out of ninety cases, a genetic diagnosis (single or a group) could be inferred on basis of
dysmorphological evaluation alone in 16, on histopathology alone in 6; and on biochemical
testing, cytogenetic testing and exome sequencing alone in 2 cases each. Overall, 28/90
(31%) cases were of a definitive genetic etiology. In addition, at least 20 cases had significant
abnormalities on dysmorphological evaluation indicating a possible genetic etiology. Taken
together, definite and possible genetic etiologies contributed to fetal pathology in more than
half (48/90) of autopsied fetuses. Interestingly, at least 28/48 of these were of monogenic
etiology, as apparent from the evaluation findings or positive family history. Available liter-
ature also implicates a genetic etiology in 25%–35% of congenital malformations in neonates
(2,24,25), 15%–30% intrauterine deaths (11,12,25,26) and at least 10%–30% of prenatally
detected malformations (14,15,27,28,29). The diagnostic bases in most studies are clinical
findings, karyotyping and chromosomal microarray. Recent exome sequencing studies in
morphologically abnormal fetuses have shown a diagnostic yield of 20%–30%, indicating a
large contribution of single gene disorders (16–20). The findings of this study also corrobo-
rate the magnitude of overall and relative genetic contributions, with a significant proportion
of monogenic disorders. Also, excluding the 11/42 cases with definite acquired etiologies,
of the remaining 31 cases concluded as miscellaneous, there were at least 19 fetuses with
hydrops fetalis, central nervous system involvement, isolated structural malformation or
developmental field defects, all of which are known to have a significant genetic contribution
(11,15,30–32).
Dysmorphological evaluation by a clinical geneticist, where soft dysmorphic features and
characteristic constellation of abnormalities act as important diagnostic handles, plays an
important role in the ascertainment of a genetic diagnosis in the outpatient clinic setting.
 FETAL AND PEDIATRIC PATHOLOGY 17

The use of a similar approach in this study showed a high diagnostic yield of dysmorphology
as a standalone evaluation in a postmortem fetal cohort. A definitive genetic diagnosis could
be made in 16 and a possible diagnosis in 31, of which 11 could be confirmed by subsequent
evaluations. Dysmorphology findings also guided further targeted genetic testing as well as
interpretation of histopathology findings in at least 7 of these. The utility of fetal dysmorphol-
ogy has been indicated by other authors in literature (20,33,34).
Radiological evaluation provided additional or confirmatory findings in this study, and
remained primarily a supportive modality. Literature reports indicate its role in diagnosis of
perinatal lethal skeletal dysplasias (35). The two cases of chondrodysplasia punctata reported
here were primarily clinical diagnoses, and no other lethal dysplasias were present.
Histopathological studies also contributed significantly to the post mortem genetic diag-
nosis in this study, providing a specific diagnosis as standalone test in six cases, and additional
findings in another four. This reiterates the superior diagnostic performance of conventional
autopsy and histopathological studies as compared to virtual autopsies. Previous studies
also indicate utility of histology in diagnosis of genetic diseases, especially conditions with
central nervous system and renal parenchymal abnormalities, storage disorders and skeletal
dysplasias (35–37).
Genetic testing yields in this study were 4/26 for karyotyping (15%), and 6/12 (50%) for
targeted biochemical/molecular testing. Literature reports indicate a similar yield for chro-
mosomal abnormalities in stillbirths. Diagnostic utility as a standalone test after excluding
dysmorphologic and histopathologic diagnoses was in 4 cases, three with hydrops fetalis and
one with clinically nonspecific multiple malformation syndrome. In all cases where a specific
genetic disorder was concluded clinically, and relevant genetic test performed, the clinical
diagnosis was confirmed indicating a high accuracy of the systematic approach used. The
negative test results were seen in 4 cases with hydrops fetalis and 1 fetus with cono-truncal
defect, both being etiologically heterogenous phenotypes.
Chromosomal microarray studies have shown high diagnostic yield of 2%–10% in
stillbirths, while recent exome sequencing studies have revealed monogenic etiologies in
20%–30% of anomalous fetuses (11–13,16–20). In this study, their application in the cases
classified as possible genetic disorders (20) and the miscellaneous group [42] would have lead
to a further refined estimate of the genetic contribution toward abnormal fetal phenotypes.
However, these experiments were not utilized as a routine as they are not readily available
in the clinic in our setting. In all four cases where whole/targeted exome could be per-
formed, causative pathogenic variants could be ascertained, indicating the high diagnostic
performance of this technology. Exome sequencing confirmed the autopsy diagnosis in
two cases, and led to a specific genetic diagnosis as a standalone test in two cases where
no definite diagnosis could be concluded on autopsy. In one of the cases, only part of the
complex fetal phenotype could be explained by the exome results, reflecting the limitation
of this technology and need for further testing in the form of whole genome sequenc-
ing. Our systematic approach helped in careful selection of cases with strong suspicion
of monogenic diseases, thereby contributing to the higher diagnostic yield in this study
compared to literature series (16–20). Further, besides aiding in case selection, careful phe-
notyping played an important role in interpretation of the raw data and identification of the
pathogenic variant/s, as has been demonstrated in prior studies pertaining to pediatric cohorts
(20,38,39).
A limitation of this study was incomplete histopatholgical evaluation in 12 cases due to
tissue autolysis and inability to complete genetic evaluation in all cases. Though genetic test-
ing remains the gold standard for the final diagnosis, this may not be possible in all cases in
18 S. AGGARWAL ET AL.

the postmortem setting due to limitations of sample unavailability or cost and accessibility
concerns in resource poor settings.

Conclusion
A genetic etiology is causative in at least half of fetuses undergoing a postmortem examination
in this series. A systematic approach including a dysmorphological evaluation, histopatho-
logical and radiological studies and genetic testing as indicated has a high diagnostic perfor-
mance and is integral to establishment of the final diagnosis. Dysmorphological evaluation is
the modality which is crucial to recognition of a genetic disorder and should be included
as part of fetal autopsy protocols to maximize diagnostic yield and guide genetic testing.
Histopatholgical examination of fetal tissues is an important diagnostic as well as support-
ive modality, and remains relevant in the era of virtual autopsy. Exome sequencing is a new
technology, which can provide additional diagnoses in carefully selected cases guided by the
postmortem findings and pedigree information.

Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing
of the article.

ORCID
Shagun Aggarwal http://orcid.org/0000-0001-8738-9888

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