Van den Veyver Ignatia B.

(Orcid ID: 0000-0002-0651-5924)

Prenatally diagnosed developmental abnormalities of the central nervous system and

genetic syndromes: a practical review.

Ignatia B. Van den Veyver, M.D.1

Affiliation: Department of Obstetrics and Gynecology, Baylor college of Medicine, Houston

TX

Running title: Prenatally diagnosed CNS defects and genetic syndromes.

1Corresponding author:
Ignatia B. Van den Veyver, M.D.
Department of Obstetrics and Gynecology
Baylor College of Medicine
Duncan Neurological Research Institute at Texas Children’s Hospital
1250 Moursund Street, Room DNRI 1025.14
Houston, TX 77030
Phone: (832) 824 8125
Fax: (832) 825 1271
email: iveyver@bcm.edu

Conflict of interest statement: No conflict of interest related to the contents of this

manuscript.

Funding: No grant support for this manuscript.

Word count: 6325

Table count: 2
Figure count: 3

What is already known about this topic?

 Developmental brain abnormalities are complex, difficult to diagnose prenatally and
etiologically diverse
 Genomic technologies such as chromosomal microarray (CMA) and genome
sequencing are rapidly expanding knowledge of genetic causes for brain abnormalities

What does this study add?

 Review of how ultrasound and MRI benefit prenatal diagnosis of brain abnormalities
 Review of detection rates by CMA and sequencing for different classes of brain
abnormalities
 Highlights classes of genes typically associated with different brain abnormalities

Data availability statement: Research data not shared

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/pd.5520

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Abstract

Developmental brain abnormalities are complex and can be difficult to diagnose by prenatal

imaging because of the ongoing growth and development of the brain throughout pregnancy

and the resolution limitations of ultrasound, often requiring fetal magnetic resonance imaging

as an additional tool. As for all major structural congenital anomalies, amniocentesis with

chromosomal microarray and a karyotype is the first-line recommended test for the genetic

work-up of prenatally diagnosed central nervous system (CNS) abnormalities. Many CNS

defects, especially neuronal migration defects affecting the cerebral and cerebellar cortex, are

caused by single gene mutations in a large number of different genes. Early data suggest that

prenatal diagnostic exome sequencing for fetal CNS defects will have a high diagnostic yield,

but interpretation of sequencing results can be complex. Yet, a genetic diagnosis is important

for prognosis prediction and recurrence risk counseling. The evaluation and management of

such patients is best done in a multidisciplinary team approach. Here we review general

principles of the genetic work-up for fetuses with CNS defects and review categories of

genetic causes of prenatally diagnosed CNS phenotypes.

Key-words: fetal brain – prenatal ultrasound – prenatal magnetic resonance imaging (MRI) –

genetic syndrome – chromosomal microarray – single gene disorder – neuronal migration –

macrocephaly – microcephaly – posterior fossa – corpus callosum.

Acknowledgements: The author acknowledges the contributions of Dr. Magdalena Sanz

Cortes, Dr. Christopher Cassady, and Dr. Wesley Lee for ultrasound and MRI images used in

figures.

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Introduction

Structural malformations of the brain have a prevalence at birth of 1-2:1,0001, or

potentially higher with newer imaging technologies2. Development and growth of the brain

and other parts of the central nervous system (CNS) is not completed until after birth. Major

brain structures are identifiable by the end of the first trimester3, allowing for early detection

of major abnormalities, such as acrania, exencephaly, anencephaly or holoprosencephaly3,4.

In contrast, defects of brain growth and differentiation are often not identified until later in

the pregnancy, mostly after 20-22 weeks, because the affected structures have not yet fully

developed before that stage5. These phenotypes also evolve throughout gestation and may not

be apparent until late second or early third trimester. Thus, prenatal diagnosis of CNS

abnormalities requires solid knowledge of CNS embryology and development and of the

limits of imaging at different gestational ages for accurate interpretation of findings on

screening prenatal ultrasound, dedicated fetal neurosonography, or fetal magnetic resonance

imaging (MRI). Although a detailed overview of CNS embryology, growth and

differentiation in perspective of prenatal imaging is beyond the scope of this review and can

be found elsewhere3,5,6, we will highlight relevant general concepts in the description of

specific categories of CNS abnormalities.

With the increasing use of new genomic technologies, including chromosomal

microarray (CMA)7-10 or next generation sequencing for gene-panel tests and whole genome

or exome sequencing (WES, WGS)11-16, new genes that cause syndromes and birth defects,

especially those involving the brain, are uncovered at an unprecedented pace17,18.

Thus, counseling about causes, associated syndromes, prediction of the prognosis for

neurodevelopment and function, seizures, and recurrence risk of structural brain

abnormalities is complex. Women with affected pregnancies are best cared for by a

multidisciplinary team (Table 1) with close communication between the primary prenatal

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care providers, maternal-fetal medicine specialists, sonographers and radiologists, prenatal

and pediatric genetics professionals, neonatologists, pediatric neurologists and neurosurgeons

with expertise in fetal medicine, other subspecialists as needed, and social workers that

support the families. For the most severe defects with anticipated life-threatening

complications in the newborn, consultation with an experienced palliative care team (at our

institution referred to as the perinatal pediatric advanced care team or PPACT) helps guide

patients with decision making about neonatal management. Clinical prenatal geneticists and

genetic counselors play a crucial role in this multidisciplinary team. They guide the genetic

evaluation, direct the choice of the most appropriate genetic testing strategy, and counsel

prospective parents about possible or confirmed presence of a genetic syndrome in their fetus.

Genetic counselors also help families cope with uncertainty, prepare for poor prognosis,

connect with support groups, and inform about resources for early interventions and

treatments that improve life quality or outcomes.

In this review, we first provide a general overview of genetic etiologies and testing

strategies for prenatally identified developmental brain malformations. We then review

specific classes of CNS defects with their currently known most common genetic etiologies.

Because it is not possible to provide an all-encompassing overview that describes each

possible defect, we focus on broad categories and highlight the more commonly identified

conditions.

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Detection of aneuploidies, copy number gains and losses, and single gene defects with

prenatally diagnosed structural CNS abnormalities

Numerical and structural chromosomal abnormalities

Trisomy 13 and trisomy 18 are the most common aneuploidies found when there are

structural brain abnormalities, but other aneuploidies and structural chromosomal

abnormalities also commonly present with CNS defects19. CMA is now a well-established

genetic diagnostic tool and is the recommended first-line genetic test for fetal structural

abnormalities. CMA identifies clinically significant copy number losses (deletions) or gains

(duplications) in 6-7% of pregnancies with fetal anomalies and a normal karyotype8,20. A

meta-analysis published in 2014 on over 3300 reported cases found a pooled prevalence of

pathogenic or likely pathogenic copy number variants (CNVs) of 6.8% overall and 6.2%

(35/563 cases) for CNS defects7. Shaffer et al reported in 2012 that CMA revealed a

significant finding in 8.6% of cases with CNS defects and that within that group, the highest

yield was when there were abnormalities in other organ systems (11%), in particular if there

were abnormalities of the posterior fossa and cerebellum (13.8%-19.6%), holoprosencephaly

(10.6%) or agenesis of the corpus callosum, while spinal abnormalities had the lowest

detection rate of 3.8%)9.

Single gene defects

Based on the above detection rates, women are best offered a diagnostic procedure

(most commonly an amniocentesis) with as recommended first-line test a CMA, to which

some add a karyotype to detect rare instances of balanced rearrangements or clarify the

cytogenetic origin of trisomies or location of copy number gains, as a first step to find a

genetic cause for fetal brain abnormalities identified by prenatal imaging. For certain specific

CNS defects, there is a high likelihood that a single gene defects are the underlying cause,

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which may justify including a gene sequencing test early in the diagnostic work-up. This can

be a gene panel that contains from a few to hundreds of genes known to be associated with

specific categories of defects. These have the advantage of favorable cost and a lower

likelihood of detecting sequence variants of uncertain significance. The disadvantages are

that panel content may not be up to date with the newest disease gene discoveries because

they are typically developed based on information on disease genes for conditions that

present after birth and not optimized for prenatal presentations. A potentially more

successful strategy that we favor is genome-wide sequencing, such as diagnostic exome

sequencing, which has already shown benefit for prenatal cases with CNS abnormalities, but

full investigation of the clinical utility and their place in prenatal genetic testing is still

underway11,13,21-24.

Common prenatally diagnosed CNS defects

Choroid plexus cysts

Single or multiple choroid plexus cysts (CPCs) (Figure 1A) are present in 1-2% of all

pregnancies and among the most common findings on prenatal ultrasound of the brain in the

second trimester25. CPCs have been extensively studied because of their association with

aneuploidy, in particular trisomy 18. Although 40-50% of fetuses with trisomy 18 have

CPCs, they are not by themselves, without any other structural fetal anomalies, predictive of

trisomy 18 and can be present in many pregnancies with a normal outcome25-27. Other severe

syndromes with abnormal brain development can also present with CPCs, for example

Aicardi syndrome, but those typically have other identifiable CNS abnormalities28. Thus,

when isolated CPCs are found, counseling can be reassuring that the prognosis is favorable29.

It is helpful to include an explanation that CPCs form in non-neuronal tissues inside the

ventricles and are unlikely to impact future neurocognitive development.

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Neural tube defects

Depending on geographic location, approximately 0.2 – 10:1000 infants are born with

neural tube defects (NTDs)30,31 (Figure 1B-E). Of the NTDs detectable during pregnancy

40% are anencephaly, 50% myelomeningocele (MMC; spina bifida), 3% craniorachischis,

and 7% encephalocele31. The majority of NTDs are of multifactorial etiology, with evidence

for a gene-environment interaction and their incidence is lowered by increasing folic acid

intake6,32,33. A subset of NTDs have a specific genetic etiology and can be a features of a

known genetic syndrome. Ten percent of myelomeningocele cases overall and 25% when

other structural anomalies are present have chromosomal abnormalities. In addition, 3.6% of

patients with spina bifida have a significant CNV detected by CMA and an estimated 1%

with myelomeningocele have a genetic syndrome. Chromosomal abnormalities are found in

0.6-5% of anencephaly cases and in 6.5% of encephalocele cases. About 25% of fetuses with

encephaloceles have extra-CNS structural abnormalities, in particular polydactyly and renal

abnormalities. This combination of findings is highly suggestive of a genetic syndrome, in

particular Meckel-Gruber syndrome and related disorders, caused by predominantly

autosomal recessive mutations in a growing number of genes, including MKS1, TMEM67,

TMEM216, RPGRIP1L, CEP290, CC2D2A, TCTN2 31,32. Obtaining a genetic diagnosis for

these is important for counseling about recurrence risk and diagnostic options for future

pregnancies.

Ventriculomegaly and hydrocephalus

Ventriculomegaly and hydrocephalus, affecting 0.4-1:1000 liveborns, are common

abnormalities detected on prenatal ultrasound34 (Figure 1F-H). Ventriculomegaly can be

primary, due to an inherent developmental abnormality, or secondary, resulting from

environmental insults, such as prenatal infections (e.g. cytomegalovirus). The developmental

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prognosis depends on the size and symmetry of the ventricles and the presence of associated

abnormalities. Symmetrical ventriculomegaly has a higher incidence of associated

abnormalities (39% vs. 24%) than asymmetrical ventriculomegaly35. Ventriculomegaly can

be obstructive or non-obstructive and is divided into mild, moderate and severe, based on the

measurements of the lateral ventricle at the level of the antrum36,37. The risk for associated

CNS or non-CNS abnormalities is 10-40% with mild ventriculomegaly (10-12 mm) and 58-

78% with moderate (12.1 – 15 mm) and severe ventriculomegaly (>15 mm)37-39. Mild

ventriculomegaly in the second trimester is a well-known soft marker for Down syndrome

and an average of 2.7% of fetuses with isolated mild ventriculomegaly have chromosomal

abnormalities37. This increases to 14.2% with moderate and 17.4% with severe

ventriculomegaly38. Up to 7.6% of cases of ventriculomegaly with a normal karyotype have a

clinically significant CNV detected on CMA9.

The developmental prognosis for isolated ventriculomegaly is usually good, with no

neurological deficit in 78-100% of described cases, but studies on long-term outcomes

assessing for milder neurobehavioral problems are limited34,37,40,41. In severe obstructive

hydrocephalus, brain damage due to pressure affects prognosis, which can be difficult to

predict prenatally. When ventriculomegaly is a secondary sign of syndromes with

developmental abnormalities of neuronal migration (described below), the prognosis depends

on the underlying condition37.

Five to ten percent of male fetuses with nonsyndromic congenital hydrocephalus have

X-linked aqueductal stenosis due to mutations in the L1CAM gene in Xq28, the most

common genetic form of hydrocephalus (1:30,000)42. One recent study on 138 male fetuses

with severe hydrocephalus showed that 41% have mutations in L1CAM43; conversely, 90% of

males with L1CAM mutations have aqueductal stenosis. Adducted thumbs are present in >50-

88% and agenesis of the corpus callosum in 68%42,43. A distinct feature is pyramidal tract

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hypoplasia42, which could be detectable on MRI tractography. Seven percent of L1CAM

mutations are de novo and the majority of affected males are simplex cases. In inherited

cases, >95% of carrier women are asymptomatic but they have a 50% chance of affected

male offspring.

Midline defects: Abnormalities of the corpus callosum and cavum septum pellucidum

Midline brain abnormalities are commonly diagnosed prenatally. Agenesis of the

corpus callosum (ACC) can be complete or partial and is found in up to 0.03-0.07% of

second trimester ultrasounds38,44 (Figure 1I-K). It affects 1.8/10,000 overall, but the

incidence of ACC is significantly higher in children with neurodevelopmental delay (2-3%)

and it is often part of a syndrome45-47. Prenatal diagnosis of ACC can be difficult and is often

delayed until after 20 weeks of gestation because the corpus callosum is not formed before

18-20 weeks46, but early signs, including absence of the cavum septum pellucidum, abnormal

course of the pericallosal artery and colpocephaly of the lateral ventricles can raise suspicion

for ACC44,46-48. When ACC is detected, it is important to search for other CNS and non-CNS

abnormalities. Isolated ACC has a favorable (although uncertain) prognosis with normal

intelligence but predisposition to learning and social deficits in 70-80% and

neurodevelopmental delay 20-30%, of which 12% is severe in some series49,50. Other CNS

abnormalities are present in ~50%-85% depending on the study, and extra-CNS abnormalities

are present in 65%47,49. When other abnormalities are present, there is a risk of 17% for

chromosomal abnormalities, but it is lower in isolated ACC. Furthermore, other CNS

abnormalities can be missed prenatally in up to 20% of cases44,49. The risk for significant

CNVs detected by CMA in a large postnatal series of patients with ACC is 7-9%, including

in “ACC-only” cases51. ACC is a component of one of >200 different genetic syndromes. It

has been associated with metabolic disorders such as pyruvate dehydrogenase or fumarase

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deficiency, Smith–Lemli–Opitz syndrome, and several others. Some of the more common

syndromes with ACC (with causative genes listed in parentheses), are Aicardi syndrome

(unknown genetic etiology), Mowat-Wilson syndrome (ZEB2), acrocallosal syndrome

(KIF7), ATRX-related disorders (ATRX), Opitz G syndrome (MID1), L1-related disorders

including Mental retardation Adducted thumbs, Shuffling gait, Ataxia (MASA) and X-linked

hydrocephalus with aqueductal stenosis (L1CAM; see also above)45,47,52. Other causes can be

environmental, such as alcohol, other teratogen exposure, and congenital infections 45,52.

The cavum septum pellucidum (CSP) is visible as early as 15 weeks and should be

seen between 18 and 37 weeks, after which it begins to narrow5,53. Absent CSP is rare and

usually an early sign of other midline forebrain anomalies, such as holoprosencephaly and

ACC, but it can be a normal variant with good prognosis48,53-55. If the CSP is not seen by 18-

20 weeks’ gestation, further evaluation, including MRI, is indicated to search for other brain

abnormalities53 that may indicate the presence of a genetic syndrome. When frontal horns are

small, partial or complete ACC with or without other migrational abnormalities can be

present. When frontal horns are normal and there is a corpus callosum, it could indicate

septo-optic dysplasia (SOD), better referred to as hypoplastic optic nerve syndrome54,55. This

constellation of abnormalities includes hypoplastic optic nerve, which may require postnatal

ophthalmologic exam for confirmation, abnormalities of the hypothalamic-pituitary axis, and

in some, agenesis of the corpus callosum and schizencephaly53,54. Genetic abnormalities are

found in only 1% of cases, but mutations in HESX1, SOX1, SOX2 (with severe eye defects

and other anomalies), SOX3, OTOX2, and FGF8 have all been implicated56. The significance

of an enlarged (>10 mm) CSP is controversial, but it can be a sign of a CSP cyst and

associated with other findings, including chromosomal abnormalities5,54,57.

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Midline defects: Holoprosencephaly

Holoprosencephaly (HPE) (Figure 1L,M) affects 1:250 conceptuses58,59, but is much

rarer (0.4-1.2/10,000) in live births because the majority of fetuses with HPE do not survive

the pregnancy52,60. The most severe form alobar HPE (40% of HPE) is associated with high

early lethality. The milder semi-lobar (43%) and lobar (17%) HPE as well as the rarer

interhemispheric and septo-preoptic variants are challenging to diagnose prenatally. Eighty

percent of fetuses with HPE have other anomalies, of which 80% are facial defects. Other

associated brain abnormalities include absent CSP, ACC, pituitary hypoplasia, neuronal

migration abnormalities and hydrocephalus56,59,61. The facial defects range from

hypotelorism, cleft lip and palate, single nostril, proboscis, to cyclopia and anophthalmia56,59.

Other extracranial abnormalities are present in 46% and depend on the etiology52. They

include urogenital anomalies (24%), postaxial polydactyly (8%), vertebral defects (5%), limb

reduction defects (4%), and transposition of great arteries (4%)59. HPE can be caused by

genetic or environmental factors. Environmental causes include teratogens, such as alcohol,

retinoic acids and maternal diabetes, which carries a 1% risk for HPE. Between 20-50% of

HPE are caused by chromosomal abnormalities56,60 of which 75% are trisomy 13 and others

include triploidy (20%) and trisomy 18 (1-2%)52. Trisomy 13 is the most likely differential

diagnosis when HPE is detected prenatally and other structural abnormalities are present52.

When the karyotype is normal, structural chromosomal abnormalities that can involve

multiple loci containing candidate genes for HPE are found in >10%9,62. In addition to

chromosomal abnormalities, genetic syndromes are the cause of 18-25% of HPE52,56, of

which 10% are related to abnormalities of cholesterol biosynthesis. The most common among

these is Smith-Lemli-Opitz syndrome (SLOS), a common autosomal recessive disease (birth

prevalence of 1:20,000-1:40,000) of variable severity, caused by mutations in DHCR7, the

gene that encodes 7-dehydrocholesterol reductase. Features of SLOS include fetal growth

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restriction, microcephaly, occasionally HPE, cleft palate, cardiac defects, hypospadias or

ambiguous genitalia in males, 2-3 syndactyly of the toes, postaxial polydactyly, distinctive

facial features, and the potential for severe neurodevelopmental disability 63. Recently,

expanded carrier screening has revealed a higher carrier frequency for DHCR7 mutations

than previously ascertained, suggesting that there may be high early prenatal lethality of

affected fetuses64. Other syndromes associated with holoprosencephaly include Pallister-Hall

syndrome, Meckel syndrome, Rubinstein-Taybi syndrome, pseudotrisomy 13, and severe

presentations of 22q11.2 deletion syndrome52,56,61. Some forms of isolated HPE have been

linked to dominant mutations with reduced penetrance in single genes52. The most common

one is SHH, which is mutated in 40% of familial and <5% of sporadic cases. Others include

ZIC2, SIX3, and TGIF1, in 5%, 1.3% and 1.3% of familial cases respectively, and more rarely

GLI2, PTCH1, DISP1, FGF8, FOXH1, NODAL, TDGF1, GAS1, DLL1 and CDON56. The

clinical findings in familial cases can be subtle, such as a single incisor in apparently

unaffected family members. Thus, examining parents is helpful for recurrence risk

counseling. This phenotypic variability has led to a “multiple genetic hit” proposal with

digenic inheritance in some cases65.

Malformations of cortical development

Development of the cerebral cortex and cortical malformations.

New neurons of the developing cortex proliferate in the ventricular zone, then divide

asymmetrically and migrate in an inward-out pattern to populate the cortex where they

differentiate, form axons and dendrites, and establish patterned connections with other

neurons66. Malformations of cortical development can thus be classified as defects in

proliferation, migration or connectivity, based on which of those processes is (most)

affected66-68. Defects in proliferation and migration result in brain abnormalities that can be

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visible prenatally, whereas connectivity defects primarily cause functional deficits resulting

in such conditions as intellectual disability, autism and epilepsies. Cortical malformations,

especially focal defects, such as heterotopia, polymicrogyria and focal cortical dysplasias are

often not visible until later in gestation, after the routine 20-week anatomy ultrasound, and

their diagnosis requires neurosonography and fetal MRI expertise. Typical imaging features

are abnormal (premature, delayed, absent, or wide) sulcation, thin or thickened irregular

hemispheres with abnormally developed and wide gyri, cortical clefts, or heterotopia which

present as nodular protrusions into the lateral ventricles or as intraparenchymal nodules68.

The genetics of cortical malformations is complex and mutations in multiple genes,

many of which are autosomal recessive, have been implicated. Although karyotype and CMA

are first-line diagnostic tests prenatally, a genetic diagnostic work-up by WES or WGS, may

be more successful in uncovering a genetic etiology. Genetic defects or insults that affect cell

proliferation will lead to reduced or excessive growth, causing microcephaly or

megalencephaly, respectively. Incomplete neuronal migration results in heterotopia and

classic (type 1) lissencephaly. Neuronal overmigration causes cobblestone cortical

malformation (cobblestone lissencephaly). Postmigrational defects that cause cortical

disorganization result in polymicrogyria and focal cortical dysplasias68. Malformations of

cortical development can also result from infectious, toxic or vascular insults, which are

beyond the scope of this review, but must be considered in the diagnostic work-up.

Disorders of proliferation – microcephaly

The developmental prognosis of congenital microcephaly (Figure 2A) is affected by

the presence of other associated intra- and extracranial abnormalities and negatively

correlates with the size of the brain. If the head circumference of -2SD, 10% have intellectual

disability and when it is -3SD, 50% have severe intellectual disability38. Microcephaly can be

secondary to environmental factors, including infections, such as cytomegalovirus and

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Zikavirus, or maternal genetic conditions, such as uncontrolled maternal phenylketonuria69. A

detailed discussion of secondary microcephaly is beyond the scope of this review, but must

be considered in the diagnostic work-up. Genetic causes of microcephaly include

chromosomal abnormalities and pathogenic CNVs (present in 5.4%)9. A large number of

genes have been associated with isolated primary microcephalies (MCPH: microcephaly

primary hereditary), many of which encode proteins with essential roles in chromosomal

segregation, mitotic cell division, centrosome and kinetochore function. Others have a role in

DNA damage repair, DNA replication, and cilia function69-71. A distinct group of syndromic

disorders with microcephaly are the autosomal recessive microcephalic primordial

dwarfisms, which include Seckel syndrome caused by mutations in DNA damage response

genes ATR and ATRIP and centriole biogenesis genes. Another is Meier-Gorlin syndrome

caused by mutations in ORC1, ORC4, ORC6, and other genes that play a role in origin of

DNA replication. A third class of syndromes are the microcephalic osteodysplastic primordial

dwarfisms (MOPD)69-71. These conditions present prenatally with IUGR and microcephaly

out of proportion to fetal growth69-71. MOPD1 is caused by mutations in the non-coding RNA

gene RNU4ATAC, which codes for small nuclear RNA U4atac, a component of the minor

spliceosome. If exome sequencing is done for genetic work-up and this non-coding gene is

not included in the exome capture library, these mutations can be missed72. MOPD2 is caused

by mutations in PCNT, which encodes pericentrin, an essential component of the

pericentriolar material in the centrosome and essential for normal mitotic spindle formation.

The genetic etiology for MOPD3 is currently unknown.

Disorders of proliferation –megalencephaly

Megalencephaly (Figure 2B) or large brain can be primary or secondary73. Secondary

megalencephaly can be a sign of metabolic disorders that affect the brain, such as Canavan

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disease, glutaric aciduria type I and lysosomal storage disorders. These are usually gradually

progressive postnatally and usually not detected by prenatal imaging73. Primary

megalencephaly results from a cellular defect that causes a more general overgrowth

syndrome or can result in overgrowth of only the brain, which can be generalized, confined to

a single hemisphere (hemimegalencephaly), or focal (focal cortical dysplasia)73. Activating

dominant or somatic mutations in genes that control two main pathways with important roles

in cell growth regulation, RAS-MAPK (RASopathies) and PI3K-AKT-mTOR, have been

implicated in these conditions73.

A common prenatal-onset syndromic form is Sotos syndrome74, which affects

1:14,000 live births and is primarily caused by fully penetrant autosomal dominant mutations

in NSD1, 95% of which are de novo with low (<1%) recurrence risk. Newborn infants have a

length that is >2SD above expected. They have a large broad forehead and dolichocephalic

head shape, which is ascertainable on prenatal ultrasound. Additional prenatally detectable

findings include ventriculomegaly, hypoplasia or agenesis of the corpus callosum,

megacisterna magna and cardiac (in 20%) and renal (in 15%) anomalies. Mild to severe

intellectual disability is common and 3% of individuals with Sotos syndrome develop tumors,

including sacrococcygeal teratoma. Weaver Syndrome or EZH2-related overgrowth with

intellectual disability75 is caused by heterozygous mutations in EZH2, and is associated with

prenatal-onset somatic and brain overgrowth with a variable range of other findings on

postnatal MRI, including ventriculomegaly, periventricular leukomalacia, cerebellar

hypoplasia, infarcts, and polymicrogyria with and without pachygyria. Other anomalies, such

as cardiac defects are rare. There is also an increased risk for tumors. Simpson-Golabi-

Behmel Syndrome (SGBS)76 is an X-linked condition caused by hemizygous mutations in

males in GPC3, which encodes glypican 3. SGBS is characterized by macrosomia of prenatal

onset and craniofacial features that include macrocephaly, coarseness, macroglossia, cleft lip

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and palate, and bifid uvula. Affected males have mild to severe intellectual disability and can

have structural brain anomalies, a heart defect, diaphragmatic hernia, large kidneys with renal

dysplasia, cryptorchidism and hypospadias, gastrointestinal anomalies, skeletal anomalies

that include vertebral fusion, scoliosis, rib anomalies, and postaxial polydactyly. They are

also at increased risk for embryonal tumors. Other syndromes, caused by mutations in PTEN

include Bannayan-Riley-Ruvalcaba syndrome, Cowden, and PTEN-related Proteus (like)

syndrome. The more common form of Proteus syndrome is caused by heterozygous and

mosaic AKT1 mutations. Only Bannayan-Riley-Ruvalcaba syndrome is associated with

prenatal macrosomia and macrocephaly. All have a predisposition to tumors and hamartomas

and intellectual disability and there can be other anomalies

Segmental brain overgrowth phenotypes are associated with activating mutations in

genes that regulate the PI3K-AKT-mTOR signaling pathway73. Hemimegalencephaly and

megalencephaly with capillary malformation (MCAP) are caused by mosaic activating

PIK3CA mutations. Megalencephaly-Polymicrogyria- Polydactyly-Hydrocephalus (MPPH)

can be caused by mutations in PIK3R2, CCND2, or AKT3. Because these mutations are

mosaic, they can be challenging to detect prenatally as they may be present at only very low

levels in amniotic fluid. We have previous experience with a prenatal diagnosis of another

PIK3CA-related segmental overgrowth disorder (CLOVES syndrome), where a mosaic

mutation that was not detected on DNA prepared from amniocytes but became detectable

after cell culture. We hypothesized that this was the result of a proliferative advantage in

culture of the cells carrying the mutation77. Focal cortical dysplasias are another class of brain

malformations, with focal areas of dysplastic cortex abnormal gyri and sulci and caused by

somatic mutations that activate the mTOR pathway.

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Disorders of neuronal migration

Defective neuronal migration can cause malformations of development of the cerebral

and cerebellar cortex. Many of the known genes mutated in these conditions encode

cytoskeletal proteins, tubulin subunits, and kinesins. These mutations produce diverse

conditions that can present with a combination of heterotopias, gyration abnormalities, other

CNS abnormalities and extracranial structural defects66-68. Early prenatal detection is

challenging because neuronal migration and cortical folding occurs in the late second and

third trimester of fetal development and because of the limitations of ultrasound. Fetal MRI

may be needed for full evaluation.

Disorders of incomplete migration: Heterotopias

The most common forms of heterotopias are periventricular heterotopias (PVH)

(Figure 2C) wherein neurons do not migrate out of the periventricular zone, and subcortical

band heterotopia (SBH) wherein migration is incomplete along the trajectory to the cerebral

cortex creating a bandlike layer of neurons within the white matter, referred to as “double

cortex”. A common form of PVH are Bilateral Periventricular Nodular Heterotopia (BPNH)

caused by mutations in FLNA in 49% of all cases, more specifically 100% of familial and

25% of sporadic cases. In this X-linked dominant condition, females are variably affected due

to mosaicism or X-inactivation patterns. Loss of function mutations in FLNA are prenatally

lethal in males, but there are surviving males with hypomorph and mosaic mutations. BPHN

is typically detected by MRI which reveals bilateral nearly contiguous PVH at the lateral

ventricular walls with an otherwise normal appearing cortex. The corpus callosum can be thin

and there can be posterior fossa and cerebellar abnormalities. It clinically manifests primarily

as a seizure disorder in affected girls and women, who have normal intelligence to borderline

intellectual disability and may have higher risk for cardiovascular disease78. A rarer recessive

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form of BPNH is caused by mutations in ARFGEF2, which presents with associated

microcephaly and severe developmental delay and seizures66,68,78-80.

Subcortical band heterotopia is caused by heterozygous mutations in females in the X-

linked DCX gene, encoding doublecortin. Affected girls and women have focal or generalized

seizures and either normal intelligence, mild learning disability, or intellectual disability of

variable severity, depending on the severity of the mutation, X-inactivation, and mosaicism66-
68,81,82
. When women transmit the DCX mutation to males, it causes classic lissencephaly

(see below)82. Not all heterotopia are caused by these two genes and other genetic defects,

external exposures (such as ischemia or infections) must be considered.

Tuberous sclerosis

Tuberous sclerosis (TSC) should be considered if periventricular nodules are

identified on prenatal ultrasound 83. Prenatal intracranial features include cortical tubers in

70% and subependymal nodules in 90%, but the most prominent prenatal findings are single

or multiple cardiac rhabdomyomas. In contrast to CNS findings, these typically resolve after

birth, but their discovery should prompt detailed evaluation of the brain. TSC is highly

clinically variable: 80% of affected individual have seizures and 50% have

neurodevelopmental and behavioral impairment. Of individuals with clinical criteria for TS,

85% have autosomal dominant heterozygous mutations in either TSC1 (31%) or TSC2 (69%),

which is the more severe form. Two-thirds have de novo mutations, and one third are

familial. Germline (in 5%) and somatic mosaicism (in 1%) have both been reported in TSC.

Knowing whether germline mosaicism is present affects recurrence risk counseling.

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Disorders of incomplete migration: Classic or type I lissencephaly

The term lissencephaly is derived from the Greek words “lissos” (smooth)

“enkephalos” (brain). It is divided in classic or type I lissencephaly and cobblestone or type II

lissencephaly (see below). Classical lissencephaly affects 1.2:100,000 births and is

characterized by a smooth thick disorganized 4-layer cortex and absence of deep sulci and

gyri, the result of neuronal undermigration, with absence of other major brain anomalies. It

can be divided in 6 grades from mild (grade 6) to complete agyria (grade 1)52,66,68,80,81. There

are three main genes for classic lissencephaly: the first is LIS1 (PAFAH1B1) in 17p13.3. Loss

of function of LIS1 causes Miller-Dieker syndrome (Figure 2D,E). This is often due to a

microdeletion, which is de novo in 80% and inherited from a parent with a balanced

translocation in 20%, but other LIS1 mutations have been found in isolated lissencephaly. A

second common cause of classic lissencephaly are mutations in DCX in Xq22.3 in males, of

whom 25% inherit the mutation from their mother and the remainder are caused by de novo

mutations66,80. Mutations in these two genes cause 76% of all lissencephaly52 and mutations

in TUBA1A cause an additional 4%52. Other findings associated with lissencephaly can

provide clues to the molecular etiology. Lissencephaly associated with microcephaly should

raise suspicion for TUBA1A mutations or other tubulinopathies (TUBB2B, TUBG1, etc).

Lissencephaly with cerebellar hypoplasia (LCH) presents with a spectrum of cerebellar

abnormalities from vermian hypoplasia, to underdevelopment or cerebellar agenesis and can

be caused by autosomal recessive TUBA1A (30% of cases) or RELN mutations. Other

variants include X-linked lissencephaly with ACC and ambiguous genitalia in males

(XLAG), caused by mutations in the ARX gene. Microlissencephaly is associated with a head

circumference of <3SD for developmental stage and can suggest a primordial dwarfism

(discussed above). Other more rarely implicated genes include VLDLR, CDK5, ACTB,

ACTG1, NDE1, DYNC1H1, KIF2A66,68,80,81.

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Disorders of overmigration: Cobblestone cortical malformation or type II lissencephaly

Cobblestone cortical malformation (Figure 2F-H), now considered a better name than

type II lissencephaly, affects 1:100,000 births. These present with a more nodular cerebral

cortical surface caused by overmigration of neurons and glia. This phenotype is associated

with a very disorganized cortex with multiple coarse gyri and areas with no gyri, together

with other CNS abnormalities, including abnormal pons and kinked brain stem, cerebellar

abnormalities and callosal abnormalities. It most often results from mutations (the majority

autosomal recessive) in genes that are important for dystroglycan production and function,

resulting in a collection of congenital muscular dystrophies or “dystroglycanopathies”. The

most common ones are Walker-Warburg syndrome (WWS), muscle-eye-brain disease

(MEB), and Fukuyama congenital muscular dystrophies (FCMD). They have a uniformly

guarded prognosis for neurodevelopment and are differentiated by additional features that are

not all ascertainable on prenatal imaging. WWS is often associated with mortality in the first

year of life, while children with MEB may survive longer. Patients with WWS often have eye

abnormalities, cleft lip, renal dysplasia and sometimes hypoplastic genitalia. Congenital

contractures and akinesia or severe hypotonia can also be present66,68,81.

Postmigrational defects: disorders of cortical disorganization

Gyration abnormalities

Gyration abnormalities are postmigrational defects, which are not visible until later in

pregnancy and can be isolated or associated with other intra- and extracranial abnormalities.

They include polymicrogyria (PMG) (Figure 2I,J), agyria and pachygyria and constitute

20% of all malformations of cortical development. They can be difficult to see on prenatal

ultrasound and are best evaluated by MRI, performed in the late second or early third

trimester (after 20-22 weeks)52. Recent studies have suggested that an abnormal shape and

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size of the sylvian fissure in early pregnancy can be an early sign of migration abnormalities

and indication for further imaging by ultrasound and MRI at a later gestational age 84,85. The

most common location is in the bilateral perisylvian region, but they can be unilateral, and

have an anterior-posterior or posterior-anterior gradient. The cortex in the abnormal gyri can

be unlayered with only 2 layers or layered with 4-6 layers. Schizencephaly is a subtype of

PMG that presents as a cleft in the cerebral cortex that connects the subarachnoid space to the

ventricles. It can have an open or closed lip and be unilateral or bilateral and is typically lined

by unlayered PMG66,68.

PMG can be sequelae of a non-genetic insult to the developing CNS by infections

(such as congenital CMV) or ischemia. Genetic causes include chromosomal abnormalities,

among which the 22q11.2 deletion, which is the most common prenatally detected

microdeletion and is well known for its association with cardiac anomalies but can

occasionally present with perisylvian PMG. Another common deletion syndrome with PMG

is the 1p36 deletion syndrome. The remainder of cases are caused by a variety of autosomal

dominant de novo, autosomal recessive and X-linked single gene mutations. Among these are

metabolic and lysosomal disorders, including Zellweger syndrome, neonatal

adrenoleukodystrophy, Pelizaeus-Merzbacher, glutaric aciduria type 2, maple syrup urine

disease and many others. Another class are collectively referred to as tubulinopathies (many

of which are de novo dominant)

For some conditions, such as Aicardi syndrome, the genetic cause is unknown, This condition

affects females only and is suspected when there are PMG, heterotopias and cysts, but it can

only be confirmed after birth by demonstration of the typical chorioretinal lacunae on

fundoscopic eye exam66,68,80,86.

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Cerebellar and posterior fossa abnormalities

Prevalence and classification of cerebellar defects

Cerebellar abnormalities affect 1:5000 live births and are often associated with other

CNS and non-CNS anomalies. Overall, 20-100% of children with cerebellar abnormalities

have impaired neurological outcome, but the severity depends on the type of defect and the

degree by which the vermis and cerebellar hemispheres are affected. Types of posterior fossa

abnormalities include megacisterna magna, Dandy-Walker malformation, vermian agenesis,

rhombencephalosynapsis, pontocerebellar hypoplasia, cerebellar atrophy, posterior fossa

arachnoid cyst, and Blake’s pouch cyst (which indicates delayed closure of the vermis)87,88.

Although terms like Dandy-Walker Variant, Dandy-Walker Spectrum or Dandy-Walker

complex were previously used, it is now considered better to abandon these designations for

this more descriptive terminology87.

Mega-cisterna magna

Megacisterna magna (MCM) (Figure 3A,B), or a transverse measurement of the

posterior fossa >10 mm with normal appearing cerebellar vermis and hemispheres is the most

common prenatally diagnosed posterior fossa abnormality, comprising 40% of all findings in

the posterior fossa88,89. The neurodevelopmental outcome is normal in 86-100% of isolated

MCM cases88-90 with no increased risk for aneuploidy. Additional CNS abnormalities are

found on 12.6%, most commonly ventriculomegaly, and other anomalies are found in

16.6%90. Small series suggest that when MCM is associated with other CNS and non-CNS

defects, only 29% to two-thirds develop normally and one-third have cognitive, language and

motor delay and have an increased risk for chromosomal or other genetic abnormalities91.

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Cerebellar hypoplasias

Vermian hypoplasia (Figure 3E) with intact cerebellar hemispheres is associated with

other anomalies in 70% of cases. When it is isolated, 77% are predicted to have normal

development, but 23% can have neurological abnormalities that include, gross and fine motor

disability, social and communication defects, behavioral abnormalities (15%) and hypotonia

(23%)87,88,92. Primary cerebellar hypoplasia is associated with chromosomal abnormality in

16.3% and 13.7% of those with a normal karyotype have a pathogenic copy number variant

by CMA87,88. Metabolic conditions including Smith-Lemli-Opitz syndrome, Molybdeen

deficiency, and adenoylsuccinase deficiency can be associated with cerebellar hypoplasia87.

Specific single gene disorders with cerebellar hypoplasia include Ritscher-Schinzel

Syndrome, caused by mutations in RTSC1/SPG8 (KIAA0196), RTSC2 (CCDC22), Joubert

spectrum disorders, which can be caused by mostly autosomal recessive mutations in

multiple genes and loci (see below). Other syndromes with cerebellar hypoplasia include

CHARGE Syndrome, associated with multiple other anomalies and caused by mutations in

CHD7, and acrocallosal syndrome caused by KIF7 mutations. When cerebellar hypoplasia is

associated with other brain malformations such as Dandy-Walker malformation,

rhombencephalosynapsis, pontocerebellar hypoplasia (often with cortical malformations), the

suspicion that it is part of a genetic syndrome should be high. Unilateral abnormalities are

more suggestive of cerebellar damage, for example by infection or ischemia, but can also be

caused by COL4A1 mutations or found in genetic syndromes such as PHACE syndrome, a

neurocutaneous syndrome characterized by posterior fossa brain malformations,

hemangiomas of the face (large or complex), arterial anomalies, cardiac anomalies, and eye

abnormalities. 87,88.

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Dandy-Walker malformation

In Dandy walker malformation (DWM) (Figure 3C,D) there is complete or partial

agenesis of the cerebellar vermis, cystic dilation of 4th ventricle, and enlarged posterior fossa

with upward displacement of the tentorium87,88. Up to 86% of fetuses with DWM have other

abnormalities, of which 49% have other CNS anomalies. Development can be normal in up to

a third of prenatally detected DWM; in particular when the vermis is normally lobulated,

82%-90% have normal intelligence. However, with associated CNS or extra-CNS

malformations or absent or abnormal vermis lobulation, 50% will have neurological

abnormalities, 50% will have hypotonia, 42% will have cerebellar dysfunction and 5% will

show hemiparesis. Genetic abnormalities associated with Dandy-Walker include

chromosomal abnormalities in 16.3%50, as well as a variety of single gene mutations.

Joubert syndrome and related disorders

In this group of overlapping conditions, the cerebellar/posterior fossa abnormality is

characterized by an abnormally deep interpeduncular fossa, more horizontally oriented

enlarged superior cerebellar peduncles, and hypoplastic cerebellar vermis. This combination

of findings results in the diagnostic molar tooth sign which is easily recognizable with fetal

MRI, but can also be observed by high-resolution prenatal ultrasound imaging87,88 (Figure F-

H). Joubert syndrome-related disorders can have associated renal, hepatic, and eye

abnormalities and polydactyly. There is significant overlap with other conditions such as

Meckel-Gruber syndrome, hydrolethalus syndrome, nephronophthisis syndrome, acrocallosal

syndrome, Bardet-Biedl syndrome and orofacial digital (OFD) syndrome88. Joubert syndrome

and these overlapping conditions are associated with developmental delays, hypotonia,

breathing anomalies, ataxia, abnormal eye movement, and facial dysmorphia. Some, such as

Meckel-Gruber syndrome, are lethal due to the severity of the kidney abnormalities or other

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defects. Collectively these conditions are caused by recessive mutations in more than 30

autosomal genes, as well as mutations in a few X-linked genes. Many of these have a role in

cilia function, leading to the term “ciliopathies”. Gene panel or exome testing yields a

molecular diagnosis in 62-94% of cases87,88,92-94.

Summary and conclusions

Structural abnormalities of the brain are complex with multiple genetic and non-

genetic etiologies and evaluation and management of women with affected pregnancies is

best done in a multidisciplinary team approach. Identifying the cause is essential for accurate

prognosis and recurrence risk counseling. In most cases, this requires a combination of high-

resolution neurosonography and prenatal MRI with expert interpretation of findings, and a

recognition that some features will not be apparent until later in gestation. Genetic testing

should be offered and include as first-line approach a chromosomal microarray analysis and

karyotype. Many structural brain abnormalities are caused by single-gene mutations.

Considering the large number of possible causative genes, exome sequencing is the strategy

most likely to yield a molecular diagnosis but is not yet widely available prenatally. Multi-

gene panels relevant to distinct phenotypes can also be considered in the diagnostic work-up

of these patients.

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Table 1: Multidisciplinary team in the clinical work-up for pregnancies with prenatally

identified central nervous system abnormalities

Prenatal care provider

Sonographer
Maternal-fetal medicine specialist
Radiologist with expertise in prenatal ultrasound and fetal MRI
Prenatal and pediatric geneticist and/or genetic counselor
Neonatologist
Pediatric neurologist
Neurosurgeon
Other subspecialties (to address associated defects)
Perinatal Hospice team (PPACT: Perinatal Pediatric Advanced Care Team)

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Table 2: Categories of central nervous system defects detectable on prenatal imaging

Common prenatally diagnosed central nervous system defects

Choroid plexus cysts
Neural tube defects
Ventriculomegaly and hydrocephalus
Midline defects
Abnormalities of the corpus callosum and cavum septum pellucidum
Holoprosencephaly
Malformations of cortical development
Disorders of proliferation – microcephaly
Disorders of proliferation – megalencephaly and hemimegalencephaly
Disorders of proliferation – focal cortical dysplasia
Disorders of neuronal migration
Incomplete migration: Heterotopias
Incomplete migration: Classic or type I lissencephaly
Overmigration: Cobblestone cortical malformation or type II lissencephaly
Postmigrational defects: disorders of cortical disorganization
Gyration abnormalities
Cerebellar and posterior fossa abnormalities
Mega-cisterna magna
Cerebellar hypoplasias
Dandy-Walker malformation
Joubert syndrome and related disorders

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 Figure 1: Commonly identified central nervous system abnormalities. (A) Second-
trimester ultrasound image of bilateral choroid plexus cysts (white arrows) in the lateral
ventricles. (B-E) Ultrasound images of neural tube defects: (B,C) Intracranial anomalies with
spina bifida at 17 weeks 1 day. (D) Myelomeningocele at 21 weeks 5 days. (E) Posterior
encephalocele. (F-H) Ventriculomegaly with aqueductal stenosis on ultrasound (F) and MRI
(G,H). (I-K) Agenesis of corpus callosum on ultrasound (I) and MRI (J,K). (L,M) Alobar
holoprosencephaly at 28 weeks 3 days on ultrasound (I) and MRI (J,K).

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 Figure 2: Malformations of cortical development. (A) Microcephaly at 25 weeks and 3
days. (B) Megalencephaly at 21 weeks. (C) Periventricular heterotopia at 21 weeks 5 days.
(D,E) Miller-Dieker syndrome lissencephaly at 32 weeks 1 day. (F-H) Cobblestone (type II)
lissencephaly at 25 weeks 5 days. (I-J) Polymicrogyria at 35 weeks 5 days. [All are MRI
images, except D and F].

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Figure 3: Posterior fossa and cerebellar abnormalities. (A,B) Megacisterna magna at 32
weeks 4 days on ultrasound and MRI. (C,D) Dandy-Walker malformation at 34 weeks and 3
days (ultrasound and MRI) . (E) Vermian hypoplasia at 20 weeks 0 days (MRI). (F-H)
Joubert syndrome at 32 weeks and 1 day on ultrasound (F) and MRI (G,H). Red circle
indicates “molar tooth” sign.

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