Relationship between prenatal ultrasound signs and genetic abnormalities for fetal malformations of cortical development

Abstract

Purpose

To explore the relationship between ultrasound signs of suspected fetal malformation of cortical development (MCD) and genetic MCD.

Materials and Methods

The retrospective study involved fetuses with one of the following 10 neurosonography (NSG) signs: (A) abnormal development of the Sylvian fissure; (B) delayed achievement of cortical milestones; (C) premature or aberrant appearance of sulcation; (D) irregular border of the ventricular wall or irregular shape of the ventricle; (E) abnormal shape or orientation of the sulci; (F) hemispheric asymmetry; (G) non-continuous cerebral cortex; (H) intraparenchymal echogenic nodules; (I) persistent ganglionic eminence (GE) or GE cavitation; (J) abnormal cortical lamination.

Results

95 fetuses were included in the study. Chromosomal microarray (CMA) combined with exome sequencing (ES) was available in 40 fetuses, CMA was abnormal in nine and ES in 22. Sign C (7/7, 100%), sign H (2/2, 100%), sign A (18/19, 94.7%), and sign B (12/13, 92.3%) were the signs leading to the highest probability of genetic MCD. The incidence of genetic MCD for sign E, sign I, and sign D was 66.7–73.7%. Only one or none of the fetuses with sign J, sign F, or sign G underwent CMA+ES. The signs in the fetuses with FGFR3, CCND2, FLNA, or TSC2 mutations had the expected features. The other fetuses with different gene mutations showed several non-specific NSG signs.

Conclusion

Several reliable signs for genetic MCD can be detected by NSG, and the probability varies with different signs. Most signs are not associated with a specific gene. Therefore, CMA combined with ES is preferred.

Zusammenfassung

Ziel

Untersuchung des Zusammenhangs zwischen den Ultraschallzeichen bei Verdacht auf eine fetale Fehlbildung der kortikalen Entwicklung (MCD) und einer genetischen MCD.

Material und Methode

Die retrospektive Studie umfasste Föten mit einem der folgenden 10 neurosonografischen Zeichen und Merkmalen (NSG): (A) abnorme Entwicklung der Sylvischen Fissur; (B) verzögertes Erreichen der kortikalen Entwicklungsstadien; (C) verfrühtes oder abweichendes Auftreten der Sulkation; (D) unregelmäßiger Rand der Ventrikelwand oder unregelmäßige Form des Ventrikels; (E) abnorme Form oder Ausrichtung der Sulci; (F) hemisphärische Asymmetrie; (G) nicht kontinuierliche Großhirnrinde; (H) intraparenchymale echogene Knötchen; (I) persistierende ganglionäre Eminenz (GE) oder GE-Kavitation; (J) abnorme kortikale Laminierung.

Ergebnisse

95 Föten wurden in die Studie inkludiert. Chromosomen-Mikroarray (CMA), kombiniert mit Exom-Sequenzierung (ES), war bei 40 Föten verfügbar, CMA war bei 9 und ES bei 22 abnormal. Merkmal C (7/7, 100%), H (2/2, 100%), A (18/19, 94,7%) und B (12/13, 92,3%) waren die Veränderungen mit der höchsten Wahrscheinlichkeit für eine genetische MCD. Die Inzidenz der genetischen MCD für die Zeichen E, I und D lag bei 66,7–73,7%. Nur bei einem oder keinem der Föten mit Merkmal J, F oder G wurde eine CMA+ES durchgeführt. Die meisten Föten mit verschiedenen Genmutationen zeigten verschiedene unspezifische NSG-Zeichen.

Schlussfolgerung

Mit dem NSG können mehrere zuverlässige Zeichen für eine genetische MCD festgestellt werden. Die meisten Zeichen sind nicht mit einem bestimmten Gen verbunden. Daher ist die CMA in Kombination mit ES vorzuziehen.

Keywords

malformations of cortical development - neurosonography - chromosomal microarray - exome sequencing - prenatal ultrasound

Introduction

Malformations of cortical development (MCDs) include a wide range of developmental disorders that occur during the formation of the cerebral cortex and may lead to neurodevelopmental deficits, mental retardation, cerebral palsy, and epilepsy [1] [2]. The precise incidence of MCDs remains unknown, although they appear to be more common, particularly in patients with epilepsy, than their incidence in the pre-magnetic resonance imaging (MRI) era [3]. The definitive diagnosis of MCD is based on neuropathological findings. However, pathological tissues are rarely available. Therefore, for most practical purposes, diagnosis begins with neuroimaging, associated clinical phenotyping, and genetic findings [1]. Due to the lack of clinical symptoms in the fetus, prenatal diagnosis of MCD relies on the identification of imaging features and genetic findings. Since next-generation sequencing (NGS) is usually performed only for patients at risk, the prerequisites for prenatal diagnosis of MCD are a family history or suspected abnormal findings. Previous studies have explored the imaging features of MCDs. Malinger et al. [4] reported the ultrasound findings and pathological results for 23 fetuses with MCD, and later on they described the prenatal sonographic diagnosis of different MCDs [5]. Pooh et al. [6] reported that an increased Sylvian fissure angle may be a sonographic sign of MCD. Several studies also tried to explore the early sonographic findings that are highly suggestive of the presence of MCD [7] [8] [9] [10] [11]. However, whether genetic MCD can be diagnosed once these imaging signs are identified remains to be determined.

Creating a connection between neuroimaging and genetic findings is critical for a more accurate prenatal diagnosis of MCD. On the basis of previous research and our clinical experience, we aimed to identify 10 sonographic signs suggestive of fetal MCD, and to analyze the association between these sonographic signs and fetal genetic abnormalities, which may play an important role in guiding prenatal diagnoses and consultation.

Methods

This retrospective study was conducted at our hospital and included data collected between January 2016 and December 2022. The study was approved by the Human Research Ethics Committee of our hospital (reference number: 2022–118). Neurosonography (NSG) was performed for fetuses over 18 weeks of gestation with a high risk of central nervous system abnormalities according to the ISUOG Practice Guidelines [12] [13]. Transvaginal examination was preferred.

Ultrasound examinations were conducted using a Voluson E8 or E10 ultrasound device (GE Healthcare Ultrasound, Milwaukee, WI). Transabdominal and transvaginal NSGs were performed by experienced doctors using 3–5 MHz, 1–7 MHz, 5–9 MHz and 6–12 MHz probes. All examinations included a detailed evaluation to identify other CNS and non-CNS abnormalities.

Based on previous studies [4] [5] [6] [7] [8] [9] [10] [11] [14] [15], mainly the study by Lerman-Sagie et al. [7], as well as our own clinical experience, fetuses were included if they presented at least one of the following ultrasound signs ([Table 1] and [Fig. 1]): sign A, abnormal development of the Sylvian fissure ([Fig. 1] A); sign B, delayed achievement of cortical milestones ([Fig. 1] B); sign C, premature or aberrant appearance of sulcation ([Fig. 1] C-1 and C-2); sign D, irregular border of ventricular walls or irregular shape of ventricles ([Fig. 1] D-1 and [Fig. 1] D-2); sign E, abnormal shape or orientation of the sulci and gyri ([Fig. 1] E); sign F, hemispheric asymmetry ([Fig. 1] F); sign G, non-continuous cerebral cortex or cleft ([Fig. 1] G); sign H, intraparenchymal echogenic nodules ([Fig. 1] H); sign I, persistent ganglionic eminence (GE) or GE cavitation ([Fig. 1] I-1 and I-2); and sign J, abnormal cortical lamination ([Fig. 1] J).

Table 1 The ultrasound signs suspicious for MCD, observations in the basic examination (routine transabdominal screening examination) and observations in NSG.

Ultrasound signs	Observations in basic examination	Observations in NSG
Abnormal development of the Sylvian fissure ([Fig. 1] A)	31	49
Delayed achievement of cortical milestones ([Fig. 1] B)	7	20
Premature or aberrant appearance of sulcation ([Fig. 1] C-1 and [Fig. 1] C-2)	0	10
Irregular border of ventricular walls or irregular shape of ventricles ([Fig. 1] D-1 and [Fig. 1] D-2)	0	34
Abnormal shape or orientation of the sulci and gyri ([Fig. 1] E)	0	7
Hemispheric asymmetry ([Fig. 1] F)	0	3
Non-continuous cerebral cortex or cleft ([Fig. 1] G)	0	4
Intraparenchymal echogenic nodules ([Fig. 1] H)	0	6
Persistent GE or GE cavitation ([Fig. 1] I-1 and [Fig. 1] I-2)	0	16
Abnormal cortical lamination ([Fig. 1] J)	0	2

Fig. 1 A Case 23 (28 weeks): bilateral asymmetric malformation of the Sylvian fissures (arrows). B Case 27 (33 weeks): abnormal morphology of bilateral Sylvian fissures (arrows), the smooth surface of the whole brain without obvious sulci, with the brain contour resembling an “8”. C-1 Case 2 (19 weeks): premature and aberrant sulci (arrows) in the bilateral occipital lobes. C-2 A 25-week-old fetus with schizencephaly: aberrant abnormal sulci (arrows) on the surface of the schizencephaly. D-1 Case 24 (25 weeks): abnormal angulation of the superior wall of the lateral ventricle (arrow). D-2 Case 7 (31 weeks): 2 nodular bulges arranged like beads on the inferior wall of the lateral ventricle (arrow), which represent paraventricular heterotopia. E A 36-week-old fetus: no cingulate sulcus above the corpus callosum, and the sulci above the corpus callosum were arranged radially (arrow). F A 23-week-old fetus: obviously enlarged right hemisphere relative to the left side, with multiple small and irregular sulci (arrow) on the surface of the right frontal lobe. (L: left hemisphere; R: right hemisphere). G A 29-week-old fetus: a cleft in the cerebral cortex (arrow), which was covered by gray matter, connecting the posterior horn of the lateral ventricle and subarachnoid space. H Case 17 (29 weeks) with cardiac rhabdomyoma: intraparenchymal echogenic nodules (arrow) in the bilateral occipital lobes. I-1 Case 13 (34 weeks): obvious hypoechoic foci surrounding hyperechoic lines (hand mark) in the lower area of the anterior horns of the bilateral lateral ventricles, which represents bilateral persistent ganglionic eminence. I-2 Case 25 (26 weeks): thick-walled cysts (arrows) below the anterior horns of bilateral lateral ventricles, which represent the bilateral cavitations of the ganglionic eminence. J A 32-week-old fetus: abnormal echogenic layers (short white arrows) in the bilateral brain parenchyma outside the lateral ventricles. The fetus also had a hypoechoic subplate (short black arrow) and big cysts in bilateral lateral ventricles (long black arrow). CMV-PCR in amniotic fluid is negative. The fetuses of Figure C-2, Figure E, Figure F, Figure G, and Figure J didn’t appear in [Table 4] and [Table 5], because there were no pathogenic results by CMA+WES or the mothers declined genetic examinations.

When NSG findings led to a suspicion of MCD, magnetic resonance imaging (MRI) was performed using either a 1.5 Tesla MR scanner (Aera; Siemens, Germany) or a 3.0 Tesla MR scanner (Achieva; Philips, The Netherlands) to confirm the ultrasound diagnosis and obtain additional information.

Karyotype and chromosomal microarray (CMA) were obtained from all available samples of amniotic fluid or neonatal blood. If these were considered normal, trio exome sequencing (ES) was performed. Data were analyzed using Cytogenomics (version 5.0.2.5) software. Library preparation was performed using Illumina Library Amplification, and HiFi HotStart ReadyMix (KAPA) was used for library amplification. The Novaseq6000 platform (Illumina, USA), using 150-bp pair-end sequencing mode, was used to sequence the genomic DNA of the family. Raw data were analyzed using the NextGENe software (version 2.4.2.3, SoftGenetics, USA). The GRCh38 genome was used for annotation. All identified variants were further analyzed with reference to public databases, including ClinGen, DGV, gnomAD, the 1000 Genome Project, DECIPHER, ClinVar, OMIM, and a comprehensive review of the literature from PubMed was performed to determine the clinical significance. The variants were classified according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) [16] [17] for interpretation of genetic variants. Only pathogenic and likely pathogenic variants were included in the study.

Pregnancy outcome data were recorded for all patients. Requests for termination of pregnancy (TOP) were discussed and approved by the multidisciplinary team. The delivered babies were followed up by pediatric neurologists and physicians, and any abnormalities or delayed development was reported.

Results

A total of 95 fetuses (93 cases were singleton pregnancies; 2 cases were twins, both dichorionic diamniotic) with any of the 10 signs described earlier were included in this study.

The indications for referral for NSG are listed in [Table 2]. The 10 ultrasound signs found during the basic morphology scan and the NSG examinations of all 95 fetuses are listed in [Table 1].

Table 2 The reasons for referral of 95 enrolled cases.

Referral reasons	Case number	Referral reasons	Case number
Lateral ventriculomegaly	39	Microcephaly	12
Fetal cardiac rhabdomyoma	7	Short femur	6
Polyhydramnios	7	Mega Cisterna Magna	4
Cyst in the fetal brain	6	Macrocephaly	2
Suspected abnormal development of the Sylvian fissure	1	Suspected agenesis of the corpus callosum	1
Fetal growth restriction	2	Polydactyly	1
Abnormal fetal skull morphology	1	Family history of central nervous system malformation	2
Fetal tetralogy of Fallot	1	Micrognathia	1
Suspected fetal intraparenchymal calcification	1	Fetal duodenal atresia	1

Following our investigation protocol ([Fig. 2]), 3 fetuses with one or more of these signs were diagnosed with an intrauterine infection (cytomegalovirus, 2 cases; syphilis, 1 case). Chromosomal microarray was recommended for the remaining 92 cases and was performed for 61. Eight patients requested TOP and 23 were lost to follow-up. Pathogenic/likely pathogenic CMA results were observed for 9 cases, and all mothers opted for TOP. CMA findings were considered benign, likely benign or with a variant of uncertain significance in the remaining 52 cases. ES was offered to this group and performed in 31 cases, the remaining 21 opted for TOP.

Fig. 2 Participant flowchart.

Following ES, pathogenic/likely pathogenic variants were diagnosed for 22 fetuses, and 19 mothers opted for TOP while 3 continued their pregnancy. These 3 children showed delayed development after birth. One child (case 7) was diagnosed with periventricular nodular heterotopia and died of heart failure at 2 years of age. Another child (case 9) was 28 months old at the end of our study and was abnormally taller than the average height of her age, while also presenting delayed development of learning and cognitive abilities. The third child (case 10) was 18 months of age at the end of the study and her mental development index and psychomotor development index were <50 when assessed by Bayley Scales of Infant Development (First Edition), indicating severely delayed development.

ES findings were considered benign, likely benign, or with a variant of uncertain significance for the remaining 9 fetuses. Only 2 were eventually delivered.

Ultrasound findings

Genetic abnormalities associated with each sign in all enrolled fetuses are listed in [Table 3]. The clinical details of the 31 fetuses diagnosed with germline genetic MCD (mean gestational age: 27.4 weeks; range: 19–38 weeks) are shown in [Table 4] and [Table 5]. [Table 4] presents 22 fetuses with pathogenic/likely pathogenic variants detected by ES. They all showed abnormalities other than the 10 studied ultrasound signs. [Table 5] shows 9 fetuses diagnosed with MCD, carrying pathogenic copy number variants found in CMA. Seven of them showed associated anomalies.

Table 3 Detection rate of fetal genetic abnormalities in 95 fetuses according to the presence of specific US signs.

US finding	Participants who declined genetic examination or were lost to follow-up (n=31)	Fetuses with P/LP result of CMA or karyotype analysis (n=9)	Fetuses with non-pathogenic results of CMA and karyotype analysis			Fetuses with P/LP CMA or ES results/all fetuses with the US sign N (%)	Fetuses with P/LP CMA or ES results/ fetuses with the US sign and performed CMA+ES N (%)
			Declined ES (n=21)	Fetuses with non-pathogenic ES result (n=9)	Fetuses with P/LP ES result (n=22)
US: ultrasound. Non-pathogenic: benign/likely benign/variants of uncertain significance. (A) abnormal development of the Sylvian fissure, (B) Delayed achievement of cortical milestones, (C) Premature or aberrant appearance of sulcation, (D) Irregular border of ventricular walls or irregular shape of ventricles, (E) Abnormal shape or orientation of the sulci and gyri, (F) Irregular, abnormal, asymmetric, and enlarged hemisphere, (G) Non-continuous cerebral cortex or cleft, (H) Intraparenchymal hyperechogenic nodules, (I) Persistent ganglionic eminence (GE) or GE cavitation, (J) abnormal cortical lamination
A	23	7	7	1	11	18/49 (36.7)	18/19 (94.7%)
B	3	6	4	1	6	12/20 (60.0)	12/13 (92.3%)
C	2	0	1	0	7	7/10 (70.0)	7/7 (100%)
D	7	3	8	5	11	14/34 (41.2)	14/19 (73.7%)
E	4	1	0	1	1	2/7 (28.6)	2/3 (66.7%)
F	2	0	1	0	0	0/3 (0)	0/0 (0)
G	4	0	0	0	0	0/4 (0)	0/0 (0)
H	4	0	0	0	2	2/6 (33.3)	2/2 (100%)
I	5	1	2	3	5	6/16 (37.5)	6/9 (66.7%)
J	1	0	0	1	0	0/2 (0)	0/1 (0)

Table 4 Imaging signs and ES results of fetuses with pathogenic or likely pathogenic ES results.

Case	Suspected MCD signs on ultrasound	Other abnormalities	Imaging signs on fetal MR	GA at diagnosis	Gene mutation	Mutation site	Changes in amino acids	Zygotic state	Pattern of inheritance	Syndrome/disease	Outcome
(A) Abnormal development of the Sylvian fissure, (B) Delayed achievement of cortical milestones, (C) Premature or aberrant appearance of sulcation, (D) Irregular border of ventricular walls or irregular shape of ventricles, (E) Abnormal shape or orientation of the sulci and gyri, (F) Irregular, abnormal, asymmetric, and enlarged hemisphere, (G) Non-continuous cerebral cortex or cleft, (H) Intraparenchymal hyperechogenic nodules, (I) Ganglionic eminence thickening or cavitation, (J) Abnormal cortical lamination .# FLNA/ARX/USP9X are all X-linked pathogenic genes. Case 7 was a male, so the zygotic state was hemizygote. Case 8, Case 9, and Case 11 were all female, so the zygotic states were heterozygotes, ^: ARX is a pathogenic gene for 6 diseases, including developmental and epileptic encephalopathy 1, intellectual developmental disorder, X-linked 29, hydranencephaly with abnormal genitalia, Partington syndrome, Proud syndrome, and developmental and epileptic encephalopathy 1. MPPH: Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome, TSC: tuberous sclerosis complex, DCC: dysgenesis of the corpus callosum, VM: lateral ventriculomegaly, HC: head circumference, SD: standard deviation, AW: atrial width of lateral ventricle, L: left, R: right
1	C D	long bone dysplasia, HC > + 2SD		20	FGFR3	c.742 C > T	p.R248C	Heterozygote (de novo)	AD	Thanatophoric dysplasia	termination
2	C	long bone dysplasia, HC > + 2SD		19	FGFR3	c.1118A>G	p.Y373C	Heterozygote (de novo)	AD	Thanatophoric dysplasia	termination
3	C	long bone dysplasia, HC > + 2SD bulging forehead, thoracostenosis		23	FGFR3	c.1118A > G	p.Y373C	Heterozygote (de novo)	AD	Thanatophoric dysplasia	termination
4	A C D I	postaxial polydactyly HC > + 2SD	A C D I	29	CCND2	c.841C > T	p.P281S	Heterozygote (de novo)	AD	MPPH syndrome 3	termination
5	B C D	bilateral VM (AW: L12.5mm, R10.5mm)	B C D J	27	GRIN1	c.1666C > A	p.Q556K	Heterozygote (de novo)	AD	Neurodevelopmental disorder with or without hyperkinetic movements and seizures，autosomal dominant	termination
6	A C D I	DCC, HC＞+2SD	A C D I	26	RHOBTB 2	c.2120G > A	p.R707H	Heterozygote (de novo)	AD	Developmental and epileptic encephalopathy 64	termination
7	D	mega cisterna magna, bowel dilatation，webbed fingers	D	28	FLNA	c.7940_c.7941 in sGCGTTGTGG TGCC	p.P2647Pfs* 107	Hemizygote (maternal)#	XLD	periventricular nodular heterotopias 1	Delivery Gestational week: 40 weeks, birth weight: 3670g. Died at 2 years old.
8	A B D E I	mega cisterna magna, DCC, aberrant right subclavian artery	A B D E I	25	FLNA	c.1632delC	p.M545Wfs*24	Heterozygote (maternal)#	XLD	Periventricular nodular heterotopias 1	termination
9	D	bilateral VM (AW: L13.8mm, R14.0mm)	D	29	USP9X	c.5632G > T	p.G1878W	Heterozygote (de novo)#	XLD	Intellectual developmental disorder, X-linked 99, syndromic, female-restricted	Delivery gestational week: 39 weeks, birth weight: 3057g Delayed development (follow-up period: 28 months after birth)
10	D	brachydactyly, left VM (AW:12.2mm) HC > + 2SD	D	31	NFIX	c.370C > T	p.R124W	Heterozygote (de novo)	AD	Marshall-Smith syndrome	delivery gestational week: 41 weeks, birth weight: 3573g, delayed development (follow-up period: 18 months after birth)
11	D	left choroid fissure cyst, absence of the right septum pellucidum	D	29	ARX	c.1278_1279insCACCC	p.D427Hfs*38	Heterozygote (de novo)#	XLR/XL	Developmental and epileptic encephalopathy 1, et al. Intellectual developmental disorder, X-linked 29; Partington syndrome; Proud syndrome	termination
12	A	DCC, HC< -2SD	A	28	ASPM	c.4195dup	p.T1399Nfs*20	Homozygote (paternal and maternal)	AR	Autosomal recessive primary microcephaly 5	termination
13	C I	mega cisterna magna, postaxial polydactyly, bilateral VM (AW: L: 13.2mm, R: 13.3mm), HC > + 2SD	C I	33	CCND2	c.812C>A	p.S271X	Heterozygote (de novo)	AD	MPPH 3	termination
14	A B D	Blake’s pouch cyst, unilateral cerebellar hypoplasia, bilateral VM (L: 18.1mm, R: 13.9mm)	A B D	29	TUBB3	c.5G>A	p.R2K	Heterozygote (de novo)	AD	Cortical dysplasia, complex, with other brain malformations 1	termination
15	A B D I	DCC, bilateral VM (AW: L: 13.1mm, R: 10.6mm)	A B D I	27	TUBB2B	c.517C>T	p.P173S	Heterozygote (de novo)	AD	Cortical dysplasia, complex, with other brain malformations 7	termination
16	H	cardiac rhabdomyoma	H	30	TSC2	c.1372C>T	p.R458X	Heterozygote (de novo)	AD	TSC	termination
17	H	cardiac rhabdomyoma	H	29	TSC2	c.5024 C>T	p.P1675L	Heterozygote (de novo)	AD	TSC	termination
18	A B	Hyperechoic band on the cortical surface, reduced cortical thickness, bilateral VM (AW: L: 15.9mm, R: 18.1mm), absence of septum pellucidum, kinked brainstem, meningocele	A B	21	POMT2	c.2070G>A	p.W690X	Heterozygote (maternal)	AR	Walker-Warburg syndrome	termination
						c.1654–1 G>C	p?	Heterozygote (paternal)
19	AB	bilateral VM (AW: L: 21.2mm, R: 18.9mm), dilation of the third ventricle, DCC, absence of vermis of cerebellum, kinked brainstem, reduced cortical thickness	A B	23	FKTN	c.139C＞T	p.R47X	Heterozygote (paternal)	AR	Walker-Warburg syndrome	termination
						ex.3–4del	p?	Heterozygote (maternal)
20	A	HC＜-2SD	A	29	MCPH1	c.22+1G>A	p?	Heterozygote (maternal)	AR	Microcephaly 1, primary, autosomal recessive	termination
						Ex.6–9del		Heterozygote (paternal)
21	A	right VM (AW: 10.2mm)	A	27	DYNC1H1	c.9961_9967delinsTCGTCTT	p.D3221_R3223delinsSSW	Heterozygote (de novo)	AD	Mental retardation autosomal dominant 13	termination
22	A	micrognathia, Blake’s pouch cyst		21	NIPBL	c.7330del	p.E2444Kfs*6	Heterozygote (de novo)	AD	Cornelia de Lange syndrome 1	termination

Table 5 Imaging signs and CMA results of fetuses with pathogenic copy number variants.

Case	Suspected MCD signs on ultrasound	Other abnormalities	Imaging signs on fetal MR	GA at diagnosis	Chromosome number	Band	Genomic coordinate (hg19)	CNV size (Mb)	Variant type	Syndrome/disease	Outcome
(A) Abnormal development of the Sylvian fissure, (B) Delayed achievement of cortical milestones, (C) Premature or aberrant appearance of sulcation, (D) Irregular border of ventricular walls or irregular shape of ventricles, (E) Abnormal shape or orientation of the sulci and gyri, (F) Irregular, abnormal, asymmetric, and enlarged hemisphere, (G) Non-continuous cerebral cortex or cleft, (H) Intraparenchymal hyperechogenic nodules, (I) Persistent ganglionic eminence (GE) or GE cavitation, (J) abnormal cortical lamination, DCC: dysgenesis of the corpus callosum, VM: lateral ventriculomegaly, HC: head circumference, SD: standard deviation, AW: atrial width of lateral ventricle, L: left, R: right.
23	A B	bilateral VM (AW: L: 10.6mm, R: 12.6mm)	A B I	28	Chr5	q35.2 q35.3	175576602–177422760	1.5	deletion	5q35 microdeletion syndrome	termination
24	A D E		A D E	25	Chr2	p25.3p23.1	17019_31664339	31.6	duplication	2p25.3 duplication syndrome	termination
					Chr9	p24.3p23	216640_13276052	13.1	deletion	9p deletion syndrome
25	A B I	cerebellar vermis cyst, DCC, bilateral VM (AW: L: 13.3mm, R: 16.3mm)	A B I J	26	Chr1	q43q44	240210001_249210000	9.0	deletion	1q43-q44 deletion syndrome	Termination
					Chr4	p16.3p15.1	10001_31851000	31.8	duplication	overlaps completely with an established dosage-sensitive region: 4p16.3 terminal region
26	A B		A B J	27	Chr17	P13.3	2286311–2494902	0.2	deletion	Miller-Dieker syndrome	termination
27	A B	right VM (AW: 15.3mm), HC＜-2SD	A B J	33	Chr17	p13.3p13.2	87009–2096909	3.3	deletion	Miller-Dieker syndrome	termination
28	A B	bilateral VM (AW: L: 10.4mm, R: 12.7mm)	A B J	31	Chr17	p13.3	1537730–2537850	1.0	deletion	Miller-Dieker syndrome	termination
29	D	skull morphological abnormality, facial abnormalities, bilateral VM (AW: L: 12.0mm, R: 11.9mm)	D	33	Chr2	q37.1q37.3	232665757–243041364	10.4	deletion	2q37 deletion syndrome	termination
30	A	fetal growth restriction, cerebellar hypoplasia, DCC bilateral VM (AW: L: 12.0mm, R: 12.8mm)	A	29	Chr6	q27	168508297–170914297	2.4	deletion	6q27 terminal deletion syndrome	termination
31	D	nasal bone hypoplasia, bowel dilatation, bilateral VM (AW: L: 12.2mm, R: 11.8mm)	D	27	Chr3	q24q25.32	147386258–157521352	10.1	deletion	overlaps 1.237Mb with the single dose-sensitive 3q24 (including ZIC1)	termination

The most common concomitant malformation in the 31 cases was ventriculomegaly, which was present in 16 cases. Other findings included posterior fossa anomalies (n=9), callosal dysgenesis (n=7), limb anomalies (n=7), facial dysmorphism (n=3), intestinal dilatation (n=2) and cardiac rhabdomyomas (n=2).

MRI findings

Fetal MRI was performed for 27 of the 31 cases with pathogenic/likely pathogenic variants, and it showed findings similar to the ultrasound signs in 21 fetuses. In addition, it revealed additional signs of abnormal lamination in 5 (Cases 5, 25, 26, 27 and 28) fetuses and persistent GE in one fetus (Case 23) ([Table 4], [Table 5]).

Correlations between ultrasound findings and genetic examination

[Table 3] shows the detection rates for fetal pathogenic germline genetic abnormalities associated with different ultrasound signs. We analyzed the incidence of germline genetic MCD among all patients who underwent complete clinical genetic tests (CMA+ES) in different groups with different ultrasound signs. All signs, except irregular, abnormal, asymmetric, enlarged hemisphere, non-continuous cerebral cortex, or cleft and abnormal cortical lamination, had an incidence of ≥ 66.7%. Only one of the fetuses with the sign of abnormal cortical lamination underwent complete clinical genetic tests. None of the fetuses with the sign of irregular, abnormal, asymmetric or enlarged hemisphere or non-continuous cerebral cortex or cleft received CMA+ES. Premature or aberrant appearance of sulcation and intraparenchymal hyperechogenic nodules were associated with genetic anomalies in all 7 cases and in 2 cases, respectively. Abnormal development of the Sylvian fissure and irregular borders of the ventricular walls/irregular shapes of ventricles were highly prevalent in cases with abnormal genetic findings, 18/19 (94.7%) and 14/19 (73.7%), respectively.

[Table 4] includes the ultrasound signs and ES results for fetuses with pathogenic or likely pathogenic genes found by ES. There were 22 fetuses diagnosed with germline genetic MCD involving 17 genes. The mutant gene was FGFR3 in 3 fetuses, CCND2 in 2, FLNA in 2, and TSC2 in 2. The ultrasound findings for these fetuses were consistent with these mutations. The other 13 fetuses with different gene mutations had a morphologically abnormal Sylvian fissure, irregular-shaped ventricles, persistent GE or GE cavitation.

[Table 5] lists the ultrasound signs and pathogenic copy number variants (CNVs) for the fetuses. Nine fetuses were diagnosed with germline genetic MCD with pathogenic CNVs involving over 300 genes. The pathogenic CNVs in these 9 fetuses played an important role in 7 genetic syndromes, including Miller–Dieker syndrome in 3 cases and 6q27 terminal deletion syndrome, 5q35 deletion syndrome, 2p25.3 duplication syndrome, 9p deletion syndrome, 1q43-q44 deletion syndrome, and 2q37 deletion syndrome in 1 case each. These cases had signs of abnormal development of the Sylvian fissures, delayed achievement of cortical milestones, or an irregular shape of the ventricles.

Discussion

Due to the lack of fetal clinical symptoms, the diagnosis of MCD in utero relies only on imaging signs, supported by genetic confirmation. The presence and description of these signs are crucial to interpret gene pathogenicity. Although more than 100 genes have been associated with MCD [18], the number of recognized putative mutations is rapidly increasing, necessitating frequent reanalysis of the data by clinicians.

Currently, it is understood that NSG should be performed by an experienced operator when an anomaly involving the CNS is suspected during the basic examination [12] [13]. The findings of the current study confirm this ([Table 1]). Basic examination of the fetal brain only includes axial planes, which might allow the detection of abnormal development of the Sylvian fissure and delayed achievement of cortical milestones, whereas for most of the other ultrasound signs, coronal and sagittal planes of NSG are essential. As shown by other groups, we demonstrated that the additional value of MRI is limited and when confronted with a suspicion of MCD during NSG, the next tier for diagnosis is NGS. A similar conclusion was proposed in a recent study that showed that ES was powerful for identifying the pathogenic genes of fetal MCD [19]. Contrary to our study, almost all cases with MCD were detected by MRI instead of ultrasound, and the expertise of the sonographers in the study was not mentioned. In another study, the aberrant sulci in some cases with corpus callosum agenesis were missed in the axial plane and detected on MRI [20]. Given the poor detection rate for MCD by routine ultrasound examination, the use of NSG should be promoted. MRI should be performed only after and as a supplement to NSG [13].

There are a few studies on the ultrasonographic signs for prenatal diagnosis of MCD [4] [5] [6] [7] [8] [9] [10] [11]. In the present study we showed that the presence of different signs is associated with different probabilities of detecting a pathogenic/likely pathogenic genetic abnormality diagnosed by CMA or ES. The probability with most signs is greater than 60%. In particular, the probability with the following 4 signs is > 90%: premature or aberrant appearance of sulcation, intraparenchymal hyperechogenic nodules, abnormal development of Sylvian fissure, and delayed achievement of cortical milestones. However, more cases should be accumulated to elucidate the relationship between these signs and germline genetic abnormalities.

It must be remembered that not all MCDs have the same genetic background. The causes of lissencephaly and subcortical band heterotopia are genetic [18] [21] [22]. The causes of megalencephaly, hemi-megalencephaly, dysplastic megalencephaly, polymicrogyria, and focal cortical dysplasia can be somatic or germline mutations, and these diseases are often caused by mutations in genes affecting the phosphoinositide-3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) signaling pathway [18] [21] [22]. Children with MCD with somatic mutations may have a normal life following early local foci resection after birth [23]. The probability of a poor prognosis for fetuses with abnormal germline genes is significantly high. Therefore, the presence or absence of pathogenic/likely pathogenic germline gene mutations in a fetus with MCD is a crucial factor for assessing the prognosis of the fetus.

A few studies have clarified the relationship between some of these signs and specific genetic diseases. The same was observed in our study. The ultrasound findings in the fetuses with FGFR3, TCS, FLNA were as expected in these diseases [24] [25] [26]. Some ultrasound signs corresponded to abnormalities in the gene signaling pathway, such as fetal head oversize, overdevelopment of the sulcus, and GE thickening corresponding to CCND2 in the mTOR signaling pathway [27]. For fetuses with distinctive prenatal ultrasound features, prenatal Sanger sequencing, or disease-targeted gene panels can help reduce the examination time and costs.

However, most signs in this study were observed in association with different pathogenic genes. Abnormal development of the Sylvian fissure was found in cases with 7 monogenic diseases and 7 pathogenic CNVs. Fong et al. reported that the abnormal Sylvian fissure was detected in 7 fetuses with Miller-Dieker syndrome [28]. Moreover, Pooh et al. [6] also reported that 22 fetuses with an increased Sylvian fissure angle were diagnosed with schizencephaly, microlissencephaly, polymicrogyria, and MCD that could not be classified prenatally. Therefore, the same brain imaging abnormality may be caused by various gene mutations. Most ultrasound signs in the fetal period are not gene-specific [1] [2] [18] [29]. When these signs are detected by NSG, the next diagnostic modality is preferably CMA+NGS.

This study had some limitations. First, it was an exploratory study conducted with the aim of providing data that can be used by future prospective, formally powered studies. Second, TOP was performed in 48 cases in our study. Therefore, information about the postnatal development of children with these signs was scarce. More cases need to be collected to illustrate the relationships among the imaging signs, genetic abnormalities, and pathological results, and the clinical phenotypes of MCD. Third, some specific types of MCD with distinctive imaging features, such as thanatophoric dysplasia [30] and tuberous sclerosis [1], were included in our study. Future studies should concentrate on sonographic and genetic findings related to the conventional spectrum of MCD. Fourth, some MCDs are related to somatic mutations in brain tissue, while CMA + NGS reflect germline mutations rather than somatic mutations. Therefore, negative results in CMA + NGS cannot exclude MCD.

Conclusion

Several reliable signs for the diagnosis of germline genetic MCD can be identified using NSG, and the probability of germline MCD varies with different signs. Some signs are distinctly associated with pathogenic genes, and targeted gene testing may be performed when these signs are identified. Most signs are not associated with a specific gene. Therefore, CMA combined with NGS is preferred. The establishment of imaging signs for MCD diagnosis will help improve the prenatal gene-image database, while facilitating analysis of ES results.

Conflict of Interest

The authors declare that they have no conflict of interest.

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Publication History

Received: 14 May 2024

Accepted after revision: 12 November 2024

Article published online:
19 December 2024