Abstract

Spina bifida (SB) is the most common congenital malformation of the central nervous system (CNS), which develops as a result of disrupted primary neurulation and neural tube closure. Myelomeningocele (MMC) is the most severe SB form and belongs to a larger group of congenital malformations known as neural tube defects (NTDs). MMC occurs when a portion of the spinal cord or nerves protrudes from an opening in the spine and may or may not be covered by meninges. MMC can be associated with hydrocephalus (HC), Arnold-Chiari II malformation, bowel and bladder dysfunction, lower limb paralysis, and pain syndromes. Despite many studies suggesting possible MMC causes such as folic acid deficiency, genetic disorders, ethnicity, and maternal health characteristics, the mechanism of MMC, particularly its concurrence with HC, is still unclear. To address such shortcomings, the whole exome sequencing (WES) of cadaveric DNA procured from the body of a 55-year-old female with concurrent MMC and HC was performed on the Illumina next-generation sequencing (NGS) platform. The bioinformatics analysis of WES data yielded 84 rare (minor allele frequency, MAF ≤ 0.01) pathologic/deleterious genetic variants, with few of the affected genes being previously linked to NTD/MMC or HC. The most interesting such genes were ALDH1L1 and DNAAF1 because they have been previously reported to be associated with both NTD/MMC and HC. The great extent of HC association with MMC raises a question of whether the ALDH1L1 and DNAFF1 mutations could, at least in some cases, underline the concurrent MMC and HC in humans.

Keywords

Spina Bifida, Myelomeningocele, Hydrocephalus, Whole Exome Sequencing, Next Generation Sequencing, Aldehyde Dehydrogenase 1 Family Member L1, Dynein Axonemal Assembly Factor 1

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1. Introduction

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.

2. Methods

2.1. Human Cadaveric Body Procurement

The body of a 55-year-old female was received through the Saint Louis University (SLU) Gift Body Program with signed informed consent. The self-reported medical history included surgical correction of open SB, urinary diversion (Kock pouch), kidney disease, HC (shunt removed at age 5), and seizures. The body was embalmed through the common carotid artery with a solution (1:1 ratio) of water and a mixture containing isopropyl alcohol (52%), ethylene glycol (24%), phenol (17%), formaldehyde (6%), and methanol (1%).

2.2. Anatomical Dissection

The cadaver was placed in the prone position, and a midline incision was made from the external occipital protuberance to the intergluteal cleft. The skin, subcutaneous fat, superficial and deep back muscles were dissected to expose the thoracic, lumbar, and sacral vertebrae. A laminectomy was then performed to expose the spinal canal expanding from the thoracic region as far inferiorly as possible. When supine, a midline abdominal incision and a transverse incision were made to reflect the anterior abdominal wall. Dissection of the abdominal cavity proceeded with cleaning of mesenteries, viscera, and scar tissue.

2.3. Genetic Analysis

The postmortem genetic screening by WES was performed by Omega Bioservices (Norcross, GA) on the Illumina NGS platform, and the respective bioinformatics analysis was conducted by the Genome Technology Access Center (GTAC, Washington University in St. Louis, MO) as previously described [13] [14]. Specifically, DNA was extracted from the tibia by the Paleo-DNA Laboratory (Lakehead University, Thunder Bay, Ontario, Canada), and DNA quality control prior to sequencing was performed using a TapeStation. WES was conducted by employing Illumina Nextera Rapid Capture Exome (45 Mb) on the Illumina HiSeq 2500 NGS platform with the PE 2 × 100 read format and 30× coverage depth (~4.7 GB per sample). The bioinformatics analysis was performed by GTAC using its standard data analysis pipeline, which included the identification of pathological/deleterious variants using three databases: SIFT, Polyphen-2, and Provean. To minimize false-positive detection of such variants by a single database, the rare genetic variants were further sequentially filtered through SIFT, Polyphen-2, and Provean, thereby taking advantage of these databases having unique data repositories along with their proprietary prediction algorithms for identifying pathological/deleterious genetic variants.

3. Results

3.1. Anatomical Examination

During the dissection of the deep back, a lack of posterior elements of the vertebral column was noted (Figure 1).

Figure 1. The lower vertebral column showcases the open midline vertebral defect associated with SB. Notice the spinal cord continues inferior to the LV2 level, which is inferior to the expected vertebral level where the conus medullaris is typically located. There was a lack of a distinct cauda equina, and it seemed that the spinal cord continued inferior to the L1/L2 level with adhesions between neural structures and the dura mater (Figure 2).

Figure 2. The lower vertebral column with the dural sac cut open. Notice the adhesion of the cauda equina to the dura.

Based on these observations and the individual’s self-reported history of SB, it was concluded that the individual had MMC. During the dissection of the abdomen, a Koch’s pouch was observed, consistent with the reported history of it being used in the treatment of neurogenic bladder (Figure 3). The left kidney was also noted to be absent, consistent with the medical history of kidney disease and it being removed in a prior surgery. The individual was also observed to have notably small hands and feet. Upon inspection of the head and face, the mandible was noted to protrude anteriorly in addition to a protruding forehead.

Figure 3. The abdominal cavity shows the reported history of urinary diversion. Notice the ureter directly connecting to the retracted cecum.

3.2. Genetic Screening

Following a very stringent filtering process [13] [14], 84 genes with rare (minor allele frequency, MAF ≤ 0.01) pathological/deleterious variants were identified (Table 1). Only three genes—HLA-DRB5, NPIPA5, and PRAMEF10—had homozygous variants, whereas the other genes had heterozygous ones.

Table 1. Genes with rare pathological/deleterious variants associated with the present case.

a. Gene-to-protein name conversion was performed by using GeneCards.

4. Discussion

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.

5. Conclusions

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.

Limitations

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.

Acknowledgements

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.

Funding

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

Disclosure

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

Author Contributions

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

Ethics Statement

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.

Conflicts of Interest

The authors declare that they have no competing interests.

References