Malformations of cortical development: Fetal imaging and genetics

Abstract

Background

Malformations of cortical development (MCD) are a group of congenital disorders characterized by structural abnormalities in the brain cortex. The clinical manifestations include refractory epilepsy, mental retardation, and cognitive impairment. Genetic factors play a key role in the etiology of MCD. Currently, there is no curative treatment for MCD. Phenotypes such as epilepsy and cerebral palsy cannot be observed in the fetus. Therefore, the diagnosis of MCD is typically based on fetal brain magnetic resonance imaging (MRI), ultrasound, or genetic testing. The recent advances in neuroimaging have enabled the in-utero diagnosis of MCD using fetal ultrasound or MRI.

Methods

The present study retrospectively reviewed 32 cases of fetal MCD diagnosed by ultrasound or MRI. Then, the chromosome karyotype analysis, single nucleotide polymorphism array or copy number variation sequencing, and whole-exome sequencing (WES) findings were presented.

Results

Pathogenic copy number variants (CNVs) or single-nucleotide variants (SNVs) were detected in 22 fetuses (three pathogenic CNVs [9.4%, 3/32] and 19 SNVs [59.4%, 19/32]), corresponding to a total detection rate of 68.8% (22/32).

Conclusion

The results suggest that genetic testing, especially WES, should be performed for fetal MCD, in order to evaluate the outcomes and prognosis, and predict the risk of recurrence in future pregnancies.

INTRODUCTION

The brain plays a central role in regulating most of the bodily functions, including cognitive, behavioral and social functions. During human embryogenesis, the formation of the neural tube on the 22nd day after fertilization represents the initiation of the development of the central nervous system (Devakumar et al., 2018). With further growth, the anterior portion of the neural tube swells to form the cerebral vesicle, which eventually develops into the brain.

Human brain development is a complex process that involves the expression and regulation of more than 1000 genes (Oegema et al., 2020). The term “malformations of cortical development” (MCD) refers to a group of congenital disorders caused by abnormal brain development. The etiology of MCD includes gene mutations, infection, ischemia, and toxicity. There are different types of MCD, including heterotopia, polymicrogyria, lissencephaly, schizencephaly, and dysgyria and/or simplified gyral pattern. The typical manifestations include refractory epilepsy, intellectual and motor developmental delay, autism, and other neurocognitive disorders (Severino et al., 2020).

The development of the fetal cerebral cortex involves three overlapping stages: proliferation of neural progenitor cells, migration, and organization. The present classification of MCD is mainly based on the disruption of these three stages of cortical development. Neuronal perturbation during the proliferation, migration, or organization stage leads to the malformation of the cerebral cortex. For example, abnormal proliferation can lead to primary microcephaly, or hemimegalencephaly (HME). The failure of migration leads to periventricular heterotopia, or lissencephaly. Furthermore, the excessive migration of neurons can cause cobblestone malformation (Barkovich et al., 2012). Disrupted postmigrational cortical organization leads to focal cortical dysplasia, polymicrogyria, or schizencephaly (Selvanathan et al., 2023).

Fetal MCD is detected by prenatal ultrasound or magnetic resonance imaging (MRI). Certain subtypes of MCD can be initially suspected by fetal ultrasound assessment at the gestational age of 20–22 weeks. However, most types of MCD, including HME, polymicrogyria and heterotopia, are difficult to diagnose by ultrasound alone. In utero MRI is an alternative modality to confirm and characterize brain abnormalities. This allows for the better differentiation of the type, severity and localization of cortical malformations, thereby providing a distinct leverage in the diagnosis and timely counseling of parents (Khandelwal et al., 2022). In addition, fetal imaging can detect additional brain malformations including subependymal cysts, cerebellar malformations, hydrocephalus/ventriculomegaly, corpus callosum abnormalities and leukomalacia, and other structural abnormalities that may be associated with adverse prognosis.

To date, more than 100 genes associated with MCD have been described (Guerrini & Dobyns, 2014). A number of these genes are linked to pathways that are known to regulate corticogenesis. Therefore, the present classification of MCD mainly relies on genetic etiology. Several studies have unraveled the relationship between gene mutations and MCD in pediatric or adult patients. However, studies that investigate the frequency of gene mutations in fetal MCD are rare. Compared to imaging examinations, genetic tests can enable the early diagnosis of fetal MCD.

In previous studies, the overall diagnosis rate of prenatal whole-exome sequencing (WES) ranged from 6.2% to 80.0% (Best et al., 2018; McMullan et al., 2017; Vora et al., 2017). However, in some large-scale prospective studies, the added value of prenatal WES in the diagnosis of fetal structural abnormalities was reported to be 8%–38% (Gabriel et al., 2022; Lord et al., 2019; Petrovski et al., 2019). The wide variability of detection rates in different studies may be related to the differences in the distribution of fetal anomalies in the study populations. For fetuses with abnormalities of the central nervous system, the diagnosis rate of WES can range from 4% to more than 50% (Lord et al., 2019; McMullan et al., 2017; Petrovski et al., 2019).

OBJECTIVES

Based on previous studies that revealed that exome sequencing (ES) can improve the diagnosis rate of fetal anomalies, the present study aimed to evaluate the role of WES in the molecular diagnosis and management of fetal MCD.

MATERIALS AND METHODS Subjects

The present study retrospectively reviewed 32 pregnant women who visited the Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, China, between December 2017 and January 2023. The fetal MCD was identified by ultrasound and/or MRI at the gestational age of 22–33 weeks. Then, the MCD were diagnosed by fetal medicine specialists and radiologists, and classified based on the MCD classification of Barkovich et al. (2012). The fetal samples included the amniotic fluid, cord blood, muscle tissue, or affected brain tissue. After the diagnosis of fetal MCD, amniotic fluid or cord blood specimens were collected to perform the karyotype and/or single nucleotide polymorphism (SNP) array. If the karyotype and/or SNP array results were negative, WES was performed.

Radiological evaluation

For the imaging diagnosis of fetal MCD, an MRI was performed using the Philips Achieva MRI 1.5 T (Philips Medical Systems, Best, The Netherlands) with an 8-element phased-array surface coil. Then, the conventional transverse, sagittal, and coronal MRIs were obtained. The scanning parameters for the various sequences were as follows: 2D small angle T1-weighted imaging (T1WI) (repetition time [TR], 122 ms; echo time [TE], 4.6 ms; field of view [FOV], 400 mm; matrix, 272 × 200; number of average signals [NAS], 2); T2-weighted imaging (T2WI) sequence (TR, 1200 ms; TE, 80 ms; FOV, 350 mm; matrix, 204 × 182); diffusion-weighted imaging (DWI) b values of 0 and 800 s/mm²; balanced rapid gradient echo (TR, 10 ms; TE, 5 ms; FOV, 350 × 350 mm; matrix, 256 × 256; NAS, 1; layer thickness, 5 mm; layer interval, 1 mm); semi-Fourier acquisition single excitation rapid spin echo (TR, 3500 ms, TE, 60 ms, FOV, 350 × 350 mm; matrix, 256 × 256; NAS, 1; layer thickness, 5 mm; layer interval, 1 mm). The fetal ultrasound and MRI signs suggestive of MCD include delayed cortical development, dysgenesis of the Sylvian fissure, irregularity of the ventricular wall, discontinuous cortex, absence or abnormal appearance of fissures, and abnormal or asymmetric gyri.

Whole-exome sequencing

WES was performed using the DNA extracted from amniotic fluid, cord blood, or fetal tissues with the Agilent SureSelect Human All Exon V5 Kit (Agilent Technologies, Santa Clara, CA, USA) for the target capture. The Hiseq2500 platform (Illumina, San Diego, CA, USA) was used for library sequencing. Next, greater than 99% of the reads were mapped to the genomic targets, with 20× coverage for >97% of the bases. Then, the sequence reads were aligned to the human genome sequence GRCh37 using the Burrows–Wheeler Aligner (BWA) software. A custom pipeline mainly built on the Genome Analysis Toolkit (GATK) was used for the sequence data analysis and annotation (McKenna et al., 2010). Causal variants were identified by the TGex software (LifeMap Sciences, USA), and these were used to annotate the selected single-nucleotide variants (SNVs) and indels. Then, the detected variants were validated by Sanger sequencing, and the pathogenicity was classified according to the guidelines of the American College of Medical Genetics and Genomics/American Association of Molecular Pathology (ACMG/AMP).

Statistical analysis

SPSS 16.0 was used and Pearson χ² test was used for statistical analysis of the data. p < 0.05 was considered statistically significant.

RESULTS

The samples obtained from 32 fetuses with MCD of suspected genetic etiology, with or without other abnormalities, were analyzed in the present study (Figure 1).

FIGURE 1. Study profile: +, pathogenic finding, −, normal result; N, number; TOP, termination of pregnancy.

Out of the 32 cases, 11 cases had MCDs combined with additional brain malformations or other structural abnormalities and 21 cases had isolated MCDs (iMCDs). The rates of pathogenic CNVs and SNVs in fetuses with iMCDs and fetuses with MCDs combined with other abnormalities were 61.9% (13/21) and 81.8% (9/11), respectively, with no significant difference (p > 0.05). The abnormality detection rate of the SNP array and WES were 9.4% (3/32) and 59.4% (19/32) in all MCDs cases (p < 0.05), corresponding to a total detection rate of 68.8% (22/32).

Brain pathological examination was conducted in 11 cases, and in these cases, the pathological findings were in agreement with the imaging findings.

SNP array results

The SNP array detected a pathogenic copy number variant (CNV) in three cases. Case no. In total, 5 had 17p13.3p13.2 microdeletion de novo, which involved the LIS1 gene and was correlated to the Miller–Dieker lissencephaly syndrome. The imaging findings were agenesis of the corpus callosum, lissencephaly, and tetralogy of Fallot. Case no. In total, 26 also had 17p13.3p13.2 microdeletion and Miller–Dieker syndrome. The imaging findings were macrogyria, microgyria, and ependymal cysts. The karyotype for case no. In total, 7 was 10 p15.3p11.21 unbalanced translocation. The SNP array indicated 10p15.3p11.21 (135708–35,406,626) × 3, 35 Mb duplication, which was correlated to the trisomy 10p syndrome. The imaging findings revealed microcephaly, increased cortical thickness, and fetal growth restriction.

Whole-exome sequencing results

The WES identified pathogenic mutations in 13 genes, which included ASPM, DCX, TUBA1A, FKTN, mTOR, ADGRG1, PDHA1, PIK3R2, TUBB3, PPIL1, NIPBL, PEX6 and SAMHD1. The detection rate of WES in the present cohort was 59.4% (19/32, Table 1).

TABLE 1 Summary of the imaging findings, genetic findings, and outcomes in the cohort.

Case	Gestational week	Imaging findings	CNVs	WES results	Classification	Diagnosis	Origin	Pregnancy outcome
1	33	Lissencephaly, heterotopia	No clinically significant copy number changes	DCX, c.232C>T (p.Arg78Cys), het	Pathogenic	Lissencephaly, X-linked; Subcortical laminal heterotopia, X-linked; MIM: 300121	De novo	TOP
2	28	Microlissencephaly	No clinically significant copy number changes	ASPM, c.1789C>T (p.Arg597*), hom	Pathogenic	Microcephaly 5, primary, AR; MIM: 605481	Paternal and maternal	TOP
3	24	Hemimegalencephaly	No clinically significant copy number changes	mTOR, c.6644C>T (p.Ser2215Phe), het	Pathogenic	HME, AD; somatic mosaicism; MIM: 607341	De novo	TOP
4	23	Lissencephaly, hydrocephalus	No clinically significant copy number changes	FKTN,c.919C>T (p.Arg307*), c.1100delT (p.Phe369fs), compound het	Pathogenic	Muscular dystrophy-dystroglycanopathy, congenital brain and eye anomalies, type A, 4;AR; MIM: 607440	Paternal and maternal	TOP
5	25	Agenesis of corpus callosum, lissencephaly, Tetralogy of Fallot	arr[hg19]17p13.3p13.2 (18901–4605200) ×1	-	Pathogenic	Miller–Dieker lissencephaly syndrome; AD; MIM:247200	De novo	TOP
6	29	Microlissencephaly	Karyotype 46, XX	ASPM, c.1789C>T (p.Arg597*), hom	Pathogenic	Microcephaly 5, primary, AR; MIM: 605481	Paternal and maternal	TOP
7	33	Lissencephaly, FGR	Karyotype: 46,XX,der(14)t(10;14)(p11;p11); SNP array: arr[hg19]10p15.3p11.21 (135708–35,406,626) ×3	-	Pathogenic		De novo	TOP
8	30	Lissencephaly, hydrocephalus	No clinically significant copy number changes	TUBA1A, c.1204C>T (p.Arg402Cys), het	Pathogenic	Lissencephaly 3; AD; MIM: 602529	De novo	TOP
9	27	Polymicrogyria	No clinically significant copy number changes	No pathogenic mutation	-	-	-	TOP
10	24	Microlissencephaly	No clinically significant copy number changes	No pathogenic mutation	-	-	-	TOP
11	24	Heterotopia	No clinically significant copy number changes	No pathogenic mutation	-	-	-	TOP
12	29	Polymicrogyria	No clinically significant copy number changes	No pathogenic mutation	-	-	-	TOP
13	29	Microlissencephaly	No clinically significant copy number changes	ASPM, c.1789C>T (p.Arg597*), c.4019delA (p.Lys1340fs); compound het	Pathogenic	Microcephaly 5, primary, AR; MIM: 605481	Paternal and maternal	TOP
14	23	Subependymal cysts, subcortical band heterotopia	No clinically significant copy number changes	PDHA1, c.854_855insGACT (p.Tyr287fs); het	Pathogenic	Pyruvate dehydrogenase E1-alpha deficiency; AD; MIM:300502	De novo	TOP
15	22	Microlissencephaly	No clinically significant copy number changes	ASPM, c.1789C>T (p.Arg597*), c.7782_7783delGA (p.Lys2595fs), compound het	Pathogenic	Microcephaly 5, primary, AR; MIM: 605481	Paternal and maternal	TOP
16	25	Microlissencephaly	No clinically significant copy number changes	ASPM, c.6994C>T (p.Arg2332*), c.7782_7783delGA (p.Lys2595fs), compound het	Pathogenic	Microcephaly 5, primary, AR; MIM: 605481	Paternal and maternal	TOP
17	26	Microcephaly, pachygyria	No clinically significant copy number changes	No pathogenic mutation	-	-	-	TOP
18	26	Polymicrogyria	No clinically significant copy number changes	ADGRG1, c.707_708insG (p.Val1237fs), c.1387A>C (p.Ser463Arg), compound het	Pathogenic	Polymicrogyria, bilateral frontoparietal; AR; MIM:604110	Paternal and maternal	TOP
19	31	Polymicrogyria	No clinically significant copy number changes	No pathogenic mutation	-	-	-	Neonatal death
20	33	Polymicrogyria	No clinically significant copy number changes	PIK3R2, c.1117G>A (p.Gly373Arg)	Pathogenic	Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 1; AD; MIM:603387	De novo	TOP
21	24	Lissencephaly	arr[hg19]4p16.3 (75033–767,642) ×1, VUS	TUBB3, c.836A>G (p.Gln279Arg)	Pathogenic	Cortical dysplasia, complex, with other brain malformations 1; AD; MIM:614039	De novo	TOP
22	30	Agenesis of the corpus callosum, left cerebral hemisphere pachygyria	No clinically significant copy number changes	No pathogenic mutation	-	-	-	TOP
23	23	Right frontal lobe polymicrogyria	No clinically significant copy number changes	No pathogenic mutation	-	-	-	TOP
24	27	Pachygyria, agenesis of corpus callosum	No clinically significant copy number changes	PPIL1, c.392G>A (p.Arg131Gln), c.391C>T (p.Arg131*), compound het	Pathogenic	Pontocerebellar hypoplasia, type 14;AR;MIM:619301	Paternal and maternal	TOP
25	22	Pachygyria	No clinically significant copy number changes	DCX, c.523A>G (p.Asn175Asp)	Pathogenic	Lissencephaly, X-linked; Subcortical laminal heterotopia, X-linked; MIM: 300121	Maternal	TOP
26	37	Pachygyria, polymicrogyria	arr[hg19]17p13.3p13.2 (18901–4605200) ×1	-	Pathogenic	Miller-Dieker lissencephaly syndrome; AD; MIM:247200	De novo	TOP
27	28	Pachygyria	No clinically significant copy number changes	No pathogenic mutation	-	-	-	TOP
28	25	Pachygyria, cleft palate, absent radius, oligodactyly, single digit	No clinically significant copy number changes	NIPBL, c.3445C>T (p.Arg1149Ter)	Pathogenic	Cornelia de Lange syndrome 1;AD; MIM:122470	De novo	TOP
29	36	Polymicrogyria, hydrocephalus	No clinically significant copy number changes	PEX6, c.1313dupT (p. Glu439fs*5), hom	Pathogenic	Peroxisome biogenesis disorder 4A (Zellweger);AR; MIM:614862	Paternal and maternal	TOP
30	32	Pachygyria, Ventricular septal defect	No clinically significant copy number changes	No pathogenic mutation	-	-	-	TOP
31	30	Leukomalacia, cortical atrophy	No clinically significant copy number changes	SAMHD1, c.619G>A (p.Asp207Asn), hom	Pathogenic	Aicardi-Goutieres syndrome 5;AR; MIM:612952	Paternal and maternal	TOP
32	23	Lissencephaly	No clinically significant copy number changes	DCX, c.887C>T (p.Thr296Ile)	Pathogenic	Lissencephaly, X-linked; Subcortical laminal heterotopia, X-linked; MIM: 300121	De novo	TOP

Abbreviations: CNVs, copy number variants; het, heterozygous; hom, homozygous; TOP, termination of pregnancy; VUS, variants of uncertain significance; WES, whole-exome sequencing.

The homozygous or heterozygous mutations of the ASPM gene were the most common causes of fetal MCD in the present study. Pathogenic mutations in the ASPM were detected in five cases, accounting for 40% of all single-gene mutations. All fetuses with ASPM pathogenic mutations in the present study had microlissencephaly.

Representative case description

The ultrasound examination was performed for case no. In total, 3 at the gestational age of 24 weeks revealed the increased size of the right lateral ventricle (1.4 cm), but the left lateral ventricle was normal (Figure 2b). The fetal MRI confirmed the marked asymmetry of cerebral hemispheres (the right was larger than the left, Figure 2a). The right lateral ventricle was dilated, and a polymicrogyria cerebral cortex was observed in the same hemisphere. The left hemisphere was normal. These findings were consistent with the diagnosis of HME. A brain autopsy was conducted after delivery. On gross examination, the whole right hemisphere cortex exhibited polymicrogyria, while the left hemisphere was normal for a fetus at 24 weeks gestation (Figure 2c). On histological examination, the cortical cells on the right side (abnormal side) exhibited cellular polarity, while the cortical cells on the normal side showed no polarity and were poorly differentiated (Figure 2d). WES was performed using the tissues obtained from both hemispheres, the fetal cord blood, and the peripheral blood of the parents. One heterozygous pathogenic variant in the mTOR gene was detected (c.6644C>T, p.Ser2215Phe) in the cortical tissue of the abnormal brain hemisphere (Figure 2e). This variant was not detected in normal cortical tissues, fetal cord blood, or peripheral blood. Thus, this was a somatic variant that has been previously reported to be associated with focal cortical dysplasia (FCD) (Møller et al., 2016).

FIGURE 2. Case no. 3: Hemimegalencephaly (HME). (A) MRI image of the brain, the arrow indicates the HME-affected hemisphere. (B) Ultrasound image of the fetal head, the arrow indicates the hyperplasia cavum septum pellucidum. (C) Brain autopsy: gross morphology of the brain. (D) Histological examination: (a) HME-affected hemisphere; (b) non-HME-affected hemisphere (Hematoxylin and eosin staining). (E) Whole-exome sequencing results: mTOR p.Ser2215Phe. (a) father; (b) mother; (c) HME-affected brain tissue; (d) non-HME-affected brain tissue.

DISCUSSION

Molecular diagnosis can enable a better understanding of the biological pathways involved in the causation of MCD, and facilitate the development of new therapies (Desikan & Barkovich, 2016). The ideal classification of MCD would be based on the accurate characterization of the involved biological pathways. However, there are various gaps in our understanding of the genetic basis of MCD (Guerrini & Dobyns, 2014). At present, more than 100 genes have been implicated in the causation of MCD. Most of these genes encode proteins that play roles in neural development, such as cell cycle regulation, neuronal migration, and polarization. For example, tubulinopathies are caused by pathogenic mutations in genes that encode proteins involved in the formation of microtubules (TUBA1A, TUBB2A, TUBB2B, TUBB3, TUBB5, TUBG1 or TUBA8), and abnormalities in cortical development can manifest as lissencephaly and polymicrogyria (Maillard et al., 2023).

Autosomal recessive primary microcephaly (often referred to as microcephaly primary hereditary [MCPH]) is a rare disorder with a neonatal morbidity rate of approximately 1:30,000–1:250,000 (Zaqout et al., 2017). To date, at least 25 causative genes have been identified for MCPH, named MCPH1-MCPH25, in the order in which these were identified. These genes are highly conserved across species, and most of these are involved in mitosis and are expressed in the neuroepithelium during embryonic neurogenesis (Cox et al., 2006). MCPH is most commonly caused by mutations in abnormal spindle-like microcephaly associated (ASPM), which is also known as MCPH5, a gene that encodes the spindle pole protein, accounting for 68.6% of all cases of MCPH. During brain development, ASPM can positively regulate the Wnt signaling pathway, and participate in neurogenesis and neuronal migration (Khan et al., 2021; Zaqout et al., 2017).

The present study identified pathogenic mutations in ASPM in five cases, accounting for 62.5% (5/8) of cases with fetal microcephaly. All fetuses with ASPM pathogenic mutations had a head circumference (HC) of <median-3 standard deviation (SD), as well as lissencephaly or pachygyria. Therefore, for fetuses with microcephaly and lissencephaly or pachygyria, ASPM pathogenic mutations should be considered, and a genetic test should be offered. All ASPM mutations in the present study were inherited from the parents, indicating a potential recurrence rate of 25% in future pregnancies. This information would be helpful for the clinical decision of performing the preimplantation genetic test (PGT) or invasive prenatal diagnosis for the subsequent pregnancy.

The activation of the mammalian target of rapamycin (mTOR) results in the phosphorylation and activation of downstream ribosomal s6 kinases (S6K) and eukaryotic initiation factor binding protein l (4E-BPI). S6K and 4E-BPI are key regulators of protein translation, and their activation would eventually lead to cell proliferation (Memmott & Dennis, 2009). Furthermore, mTOR signaling affects cortical development. In addition, hyperactive mTOR is associated with the subtypes of MCD, such as FCD and HME (Crino, 2011; Lee et al., 2012).

In case no. 3, the fetal ultrasound revealed unilateral ventriculomegaly and dysplasia cavum septum pellucidum (Figure 2). Furthermore, the MRI revealed an abnormal cortex in the right hemisphere, confirming the diagnosis of HME. This case demonstrates the distinct leverage offered by MRI in evaluating some subtypes of MCD, since ultrasound may not detect cortical anomalies due to the fetal skull shadow or the position of the fetus. Thus, fetal MRI should be considered for cases with a high suspicion of brain abnormalities, based on the ultrasound findings (Williams & Griffiths, 2014). WES detected a pathogenic variant in the affected hemisphere but not in the normal hemisphere or cord blood. This finding indicates that the affected brain hemisphere might be the ideal tissue sample for genetic testing in some types of MCD, such as HME or FCD. This finding is supported by some previous studies (Bedrosian et al., 2022; Lai et al., 2022; Møller et al., 2016).

In our series, the abnormality detection rate in fetuses that had additional brain malformations or other structural abnormalities was higher than that in fetuses with iMCDs, but the difference was not statistically significant. This is not consistent with previous studies (Accogli et al., 2020). The statistically insignificant difference is likely attributable to the small sample size. Moreover, the subject characteristics (ethnicity and age) and detection approaches in our study may have differed from those in previous studies. Our study suggests that there is a high detection rate of pathogenic and/or likely pathogenic variants or SNVs in fetus with MCD, regardless of the presence of other structural abnormalities. We found a variable detection rate ranging from approximately 50% to 92.3%. The diagnostic yield in the fetuses with lissencephaly (92.3%) or pachygyria (55.6%) was higher than that in fetuses with polymicrogyria (50%) or heterotopia (50%), similar to previous studies (Accogli et al., 2020; Di Donato et al., 2018; Stutterd et al., 2021).

In the present study, prenatal WES achieved a detection rate of 68.8% in fetal MCD cases. On the other hand, the detection rate with the SNP array was 9.4%. Therefore, the findings suggest that WES and microarray can be offered as a first-line test for fetuses with MCD.

CONCLUSION

In the present study, a molecular diagnosis was established for 68.4% of fetal MCD cases that underwent prenatal whole-exome analysis. WES is a powerful tool for identifying the pathogenic genes of fetal MCD. This technique can increase the detection rate by an additional 57.9% when compared to the SNP array. Thus, the investigators recommend performing karyotype and WES for cases with fetal MCD. WES should be considered the first-line test in fetal MCD. In our study, ASPM pathogenic mutations are the most common cause of fetal microcephaly. Compared to ultrasound, MRI can enable better characterization of cortex morphology, regardless of the skull and fetal position. Thus, MRI should be used to confirm and characterize brain abnormalities and the subtypes of MCD.

AUTHOR CONTRIBUTIONS

Linlin Wang: Conceptualization, methodology, software, investigation, formal analysis, writing—original draft, funding acquisition. Pingshan Pan: Resources, supervision. Hui Ma: Visualization, investigation. Chun He: Visualization, investigation. Zai-Long Qin: Validation, investigation. Wei He: Data curation. Jing Huang: Data curation. Shu-Yin Tan: Data curation. Da-Hua Meng: Software, validation. Hong-Wei Wei: Visualization, writing—review & editing. Ai-Hua Yin: Conceptualization, resources, supervision, writing—review & editing.

ACKNOWLEDGMENTS

We thank all the individuals who participated in the study.

FUNDING INFORMATION

The study was supported by the Guangxi Science and Technology Project (Z-A20220302), Tho open topic of Guangxi Key Laboratory of Birth Defects and Stem Cell Biobank (Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region) (GXWCH-ZDKF-2023-08) and the “YUMIAOJIHUA” Project of Maternal & Child Health Hospital of Guangxi Zhuang Autonomous Region (GXWCH-YMJH-2018003).

CONFLICT OF INTEREST STATEMENT

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

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT

Guangxi Zhuang Autonomous Region Women and Children Care Hospital Ethics Committee approved the publication of this article.