Spina bifida (SB) is the most common birth defect of the central nervous system (CNS) and is a member of a larger group of congenital malformations known as neural tube defects (NTDs) [1]. NTDs affect approximately 4 people per 10,000 live births in the United States, but worldwide, it is about 5 times more likely to occur [2][3]. The probability of an individual being affected by SB has been shown to be different between ethnic groups, such as Caucasians being less likely than Hispanics to experience SB but more likely than African Americans in the United States [4][5]. While compatible with life, SB contributes to increased mortality rates for infants and lifelong disability for those who survive [1][6].

NTDs are caused by a disruption of the normal process of neurulation, the severity and type of which depend on the location of this disruption. SB has three major forms: spina bifida occulta, closed neural tube defects, and spina bifida aperta. Spina bifida occulta is the least severe form, with no visible external malformation, only vertebral malformation. Closed neural tube defects cover a range of conditions that usually require corrective surgery and involve malformations of the bone, fat, and connective tissue of the spine and spinal cord. Spina bifida aperta refers to two distinct subtypes of SB, namely meningocele and myelomeningocele (MMC). Meningocele is a significant malformation of the vertebral column resulting in the meninges protruding from the spine and forming a sac filled with cerebrospinal fluid. MMC is the severest and the most clinically relevant form of SB, which is presented by a portion of the spinal cord or cauda equina extending out of a lesion in the vertebral column, which may or may not be covered by meninges [1][4][7].

SB etiology is complex and includes both genetic and nongenetic factors. As to the former, despite the SB inheritance pattern in humans being quite diverse, there is strong evidence that multiple genes may be involved, with some of the prominent genes—VANGL1, VANGL2, FUZ, CELSR1, and TBXT—having autosomal dominant inheritance [1]. Yet the SB recurrence patterns provide a clear indication that the SB trait is polygenic because it is underpinned by a combination of multiple genes and their respective multiple variants [8]. The most important nongenetic SB risk factors include maternal diabetes, obesity, and hyperthermia, deficiencies in folate, zinc, and B12, as well as certain antiepileptic drugs such as valproic acid [1].

MMC’s almost exclusive concurrence with hydrocephalus (HC) is one of its most notable features, as HC was present in most SB aperta (MMC) cases and absent in all SB occulta cases. [9]. Such MMC and HC concurrence has a strong clinical significance because when assessed independently, the debilitating threat of HC was comparable to that of MMC [10]. Despite a significant impact of concurrent MMC and HC on the patients’ well-being, where HC alone could be an acutely life-threatening condition [11][12], little is known about the genetic underpinnings of MMC and HC concurrence. To address this concurrent presentation, we performed a postmortem genetic screening of a 55-year-old female with a history of MMC and HC using whole exome sequencing of the respective DNA on the next-generation sequencing (NGS) platform. The results obtained highlight ALDH1L1 and DNAAF1 as potential genetic risk factors for the MMC and HC concurrence in humans.

Based on the medical history of the donor and the results of the respective anatomical examination, the present case could be described as NTD/MMC concurrent with HC. The genetic screening by WES provided important insights into possible molecular mechanisms underlying NTD/MMC and HC concurrence in humans. The functional annotation of 84 genes with rare variants revealed three genes being linked to NTDs: ALDH1L1 [15][16], DNAAF1 [17], and DUOX2 [18][19], whereas four genes have been associated with HC: ALDH1L1 [20], CCDC151 [21], DNAAF1 [22], and MOK (GeneCards). Intriguingly, ALDH1L1 and DNAAF1 appear to be pleiotropic, as they were associated with both NTDs and HC traits. Therefore, a pleiotropic genetic component may underlie the NTD/MMC and HC concurrence in humans.

The mechanism(s) governing such concurrence could be linked to impaired folate metabolism (ALDH1L1) and/or disturbed motile cilia function (DNAAF1), with both respective pathologies engaging cerebrospinal fluid (CSF) as their mediator. Indeed, ALDH1L1 is known as one of the key enzymes in folate metabolism, also serving as a folate transporter from CSF to cortical neurons and astrocytes [23]. The important role of ALDH1L1 in the control of NTDs is underscored by the facts that folate supplementation during the post-conceptional period is known to prevent nearly 70% of NTDs in humans and by the ALDH1L1 expression pattern during early CNS development in mice, where it was exclusively present in the midline [24]. Yet the CSF from rats with congenital HC was an ALDH1L1-deficient, which allowed identification of this enzyme as a specific HC marker [20].

DNAAF1, on the other hand, encodes a protein that is an essential structural component of motile cilia, and its impairment serves as a causative factor in a pathological condition known as primary ciliary dyskinesia [25]. More importantly, recently obtained evidence linked some of the DNAAF1 mutations to NTDs in humans [17] as well as to HC development in mice [22]. These data were consistent with a key role of motile cilia in neurodevelopment via regulation of CSF flow [26][27]. The presence of two putative mechanisms underlying NTD/MMC and HC in the present case, one mediated by folic acid deficiency and the other by ciliopathy, raises the question of their mutual dependence. One possible answer to this question could be found in the most recent publication [28], where it has been proposed that folic acid could regulate ciliogenesis, and thereby NTDs formation, by modulating the activity of reactive oxygen species-sensitive GTPase, which is required for cilia development.

The concurrence of MMC and HC could be underlined, at least in part, by pleiotropic genes responsible for both traits, with folic acid being a key mediator in the respective pathological processes.

The prenatal genetic screening for NTD/MMC and HC could benefit from the inclusion of ALDH1L1 and DNAAF1 genes as potential risk factors for NTD/MMC and HC concurrence.

Since the current report was based on a postmortem, single-patient study, the respective results should be viewed as a prerequisite to an antemortem clinical research using a large cohort of patients diagnosed with concurrent NTD/MMC and HC.

We are grateful to all individuals and their families for their invaluable contribution to the SLU Gift Body Program. We also like to thank Dr. Paul Cliften (Genome Technology Access Center, Washington University in St. Louis, St. Louis, MO, USA) for his expert assistance with the bioinformatics analysis.

This study was supported by the Center for Anatomical Science and Education, SLU School of Medicine.

These data were presented in part as an abstract at the Anatomy Connected Meeting on March 25, 2024.

All authors have read and approved the final version of the manuscript.

Throughout the study, no living human subjects were used. The cadaveric body used in the study was received through the SLU Gift Body Program with signed informed consent from the donor. The SLU Gift Body Program abides by all rules set forth by the Uniform Anatomical Gift Act (UAGA). All work with the cadavers, as well as with any material procured from the deceased bodies, is exempt from the Institutional Review Board approval as long as the identity of the deceased individual is not revealed.