Genetics of human hydrocephalus

Congenital hydrocephalus is the more common of the two forms of hydrocephalus, and is probably the consequence of abnormal brain development and perturbed cellular function, which emphasizes the important roles that congenital hydrocephalus genes play during brain development. In general, the recurrence risk for congenital hydrocephalus excluding X-linked hydrocephalus is low. Empiric risk rates range from <1% to 4% [28‐30], indicating the rarity of autosomal recessive congenital hydrocephalus [10, 20, 31, 32]. However, multiple human kindreds with congenital hydrocephalus have been reported [10, 15, 20, 32‐43]. The loci or genes for human autosomal recessive congenital hydrocephalus have not yet been identified, but there is at least one locus for this trait. Furthermore, like animal models, since there is heterogeneity among clinical phenotypes, there may be more genetic loci in human autosomal recessive congenital hydrocephalus.

One kindred was reported in which congenital hydrocephalus was transmitted in an autosomal dominant fashion. This condition was associated with aqueductal stenosis but was not associated with mental retardation or pyramidal tract dysfunction. The lack of mental retardation and pyramidal tract dysfunction was in contrast to X-linked or recessive congenital hydrocephalus with stenosis of the aqueduct of Sylvius (HSAS), in which these abnormalities are commonly seen [44]. Another study identified a kindred with a microdeletion of 8q12.2-q21.2 which subsequently developed hydrocephalus. This trait was also transmitted in an autosomal dominant fashion [45]. Molecular genetic studies have revealed that the responsible gene for X-linked human congenital hydrocephalus is at Xq28 encoding for L1CAM (L1 protein) [46]. The mutations are distributed over the functional protein domains. The exact mechanisms by which these mutations cause a loss of L1 protein function are still under investigation.

Another form of this disorder, acquired or adult-onset hydrocephalus is mostly sporadic and characterized by ventricular enlargement in the absence of significant elevations of intracranial pressure; therefore this form is termed normal pressure hydrocephalus (NPH). Definite changes in CSF flow, resorption, and associated dynamics have been found in NPH patients, and these changes may represent a pathogenic mechanism or a secondary phenomenon [47]. Adult-onset hydrocephalus may develop either as a result of decompensation of a “compensated” congenital hydrocephalus, or it may arise de novo in adult life secondary to an acquired disturbance of normal CSF dynamics. The latter may be due to late-onset aqueductal stenosis or disruption of normal CSF absorptive pathways [11, 48]. Acquired (adult-onset, or NPH) form of inherited hydrocephalus is very rare. Recently, an X-linked adult-onset NPH [49] and a form of familial NPH that is transmitted in autosomal dominant fashion [50] have been reported, but detailed genetic linkage studies have not been carried out yet. The genetic etiology of this form is therefore totally unknown.

Hydrocephalus has been observed in many mammals [51‐59]. Animal hydrocephalus models have many histopathological similarities to humans and can be used to understand the genetics and pathogenesis of brain damage [59‐64]. It has been well documented in the animal models that in the majority of cases, congenital hydrocephalus is a genetic disease. Furthermore, many congenital hydrocephalus loci have been mapped and identified in the animal models.

Hydrocephalic Texas strain (HTX) rat model of inherited congenital hydrocephalus is characterized by onset in late gestation, a complex mode of inheritance, and ventricular dilatation associated with abnormalities in the cerebral aqueduct and subcommissural organ (SCO), a structure that is important for the patency of the aqueduct of Sylvius and normal CSF flow in the brain. Quantitative trait locus (QTL) genetic mapping has been performed from the progeny of a backcross of HTX rat with the non-hydrocephalic Fischer F344 strain. The disease has been linked with loci on chromosome (Chr) 9 (peak markers D9Rat2), 10 (between markers D10Rat136 and D10Rat135), 11 (peak markers D11Arb2 and D11Rat46) and 17 (peak markers D17mit4 and D17Rat154) respectively. The severity of hydrocephalus in HTX rat seems to be influenced by different genetic loci [65‐68]. Another study suggested that the HTX strain is homozygous carrier of an autosomal recessive hydrocephalus gene with incomplete penetrance [69]. The genetics of another hydrocephalus inbred strain, Wistar-Lewis rats (LEW/Jms) which demonstrate inherited congenital hydrocephalus, is less clear with possible traits as an autosomal recessive [70] or semidominant or multigenic (possible QTL) with a possible locus on sex chromosomes [71], but none of the loci has been localized.

In mouse models, three QTL loci associated with congenital hydrocephalus have been identified and labeled as Vent8a, Vent4b, and Vent7c. As a major QTL controlling variance in ventricular size, Vent8a is located on Chr 8 (near the markers D8Mit94 and D8Mit189). The Other two loci, Vent4b and Vent7c, show strong epistatic interactions affecting ventricular size in the developing embryo. Vent4b is located on Chr 4 (near D4Mit237 and D4Mit214), and Vent7c is located on Chr 7 (between D7Mit178 and D7Mit191) [72].

The autosomal recessive congenital hydrocephalus-1 (hy1) mouse has been characterized phenotypically by a dome-shaped head that is sometimes seen at birth or develops during the first 2 weeks. Internally, dilatation of the entire ventricular system is observed [73, 74]. A more severe phenotypic form, hydrocephalus-2 (hy2) mouse [75, 76], and an obstructive hydrocephalus (oh) mouse with communicating hydrocephalus and secondary aqueductal stenosis have also been described [77, 78]. Unfortunately subsequent efforts to identify genetic loci have not been done on these non-inbred mouse strains.

In mouse targeted insertional mutagenesis, the accidental insertion of a transgene into a crucial genomic locus could yield important information, which has happened twice in hydrocephalus genetic studies. The transgenic mouse line OVE459 demonstrates autosomal recessive congenital hydrocephalus. This is caused by a Bdnf transgene-induced insertional mutation on a single locus on mouse Chromosome 8 (near marker D8Mit152). The OVE459 insertion locus is overlapped with that of autosomal recessive hydrocephalus-3 (hy3) mouse that phenotypically shows lethal communicating hydrocephalus with perinatal onset [79, 80]. The transgene insertion resulted as a rearrangement of Hydin exons in OVE459 mice. Subsequently, a single CG base-pair deletion in exon 15 of Hydin was also discovered in hy3 mice carrying the spontaneous hy3 mutant allele [81, 82].

In another targeted insertional mutagenesis resulting in congenital hydrocephalus, the CYP2J2 transgene interferes with the expression of a brain-specific isoform of the regulatory factor X4 (RFX4), which belongs to the winged helix transcription factor family. This brain specific isoform is called variant transcript 3 or RFX4_v3 and is crucial for normal brain development as well as for the genesis of the SCO. Loss of a single allele prevents formation of the SCO and leads to an autosomal dominant congenital hydrocephalus. This obstructive hydrocephalus appeared to be secondary to failure of development of the SCO [83].

The autosomal recessive congenital hydrocephalus (ch) mouse was reported decades ago [79]. Recently this mouse has been shown to have a mutation on another winged helix/forkhead transcription factor gene, Foxc1 (Mf1) on mouse Chromosome 13 [84, 85]. There is a recent report of 6 children with hydrocephalus from 3 different families with subtelomeric deletions from chromosome 6p. Three forkhead genes within this region (FOXF1 and FOXQ1) or proximal to it (FOXC1) were evaluated as potential candidate disease genes but no disease causing mutations were identified [86].

The autosomal recessive hydrocephalus with hop gait (hyh) mouse exhibits dramatic dilation of the ventricles at birth and invariably develops hopping gait. The hyh mouse shows a markedly small cerebral cortex at birth and dies postnatally from progressive enlargement of the ventricular system. The small cortex in hyh mouse reflects altered development of the neuronal cells. In this mouse, it is postulated that neural progenitor cells withdraw prematurely from the cell cycle, producing more early-born, deep-layer cerebral cortical neurons but depleting the cortical progenitor pool, and creating a small cortex. Genetic linkage analysis localized the hyh locus between markers D7Mit75 and D7Mit56 on mouse Chr 7. Later, the hyh gene was identified as α-SNAP (soluble NSF attachment protein α) [87]. Homozygous mutant mice harbor a missense mutation M105I in a conserved residue in one of the α-helical domains. The hyh mutant was not a null allele and is expressed; however, the mutant protein is 40% less abundant in hyh mice.

The autosomal recessive hemorrhagic hydrocephalus (hhy) homozygous mutant mouse has dilated lateral ventricles and a patent aqueduct, with no histological abnormalities either in the subarachnoid space or in the choroid plexus. Multiple hemorrhages in the meninges and throughout the brain parenchyma can be observed in the advanced stages of hydrocephalus. The hhy locus has been localized on mouse Chr 12 [88].

Recently, several new congenital hydrocephalus models have emerged in zebra fish mutagenesis screening. These models have been shown to have the defects in embryogenesis and early development leading to enlarged brain ventricles. However, genetic loci for these models have not been identified yet [89, 90].

Genetic studies in animal models have started to open the way for understanding the underlying pathology of hydrocephalus. In contrast to research with animal models, human hydrocephalus genetic research has lagged far behind. To date, at least 43 mutants of hydrocephalus have been described, and 10 congenital hydrocephalus genes have been identified. Among them, only one hydrocephalus gene has been identified in humans (see Table 1).

The neuropathology of hydrocephalus has been adequately elucidated. Cerebral ventricle dilatation secondary to disturbed CSF flow has been observed as an inheritable trait in a variety of laboratory animals (as well as in humans). In most cases, defective development of the cerebral aqueduct or the subarachnoid space has been observed [61]. Affected individuals may have severe developmental delay and radiographic findings of hydrocephalus [91].

The morphological and developmental changes in the ventricular system have been well studied in three major rat models of congenital hydrocephalus: 6-aminonicotinamide (6-AN)-induced, LEW/Jms and HTX mutant rats. Comparative morphological studies revealed that 6-AN-induced hydrocephalus was comparable to the Dandy-Walker syndrome. The LEW/Jms and HTX mutant models were identical with regard to the form of presentation and progression of hydrocephalus in the postnatal period; but the pathogenesis of these two conditions in the fetal period was different. The LEW/Jms rats showed primary congenital aqueductal stenosis in early prenatal life and the hydrocephalic state appeared before pulmonary maturation was completed. However, although the model has been considered to be of congenital communicating hydrocephalus [64], the HTX fetuses demonstrated secondary closure of the aqueduct in the perinatal period. This secondary closure of the aqueduct in HTX rats is believed to be due to retrograde degeneration of the thalamus caused by apoptotic cell death [92, 93] and failure in cell proliferation [94, 95]. The HTX rat also shows a reduction in the secretory cells of the SCO. Regarding the role of the SCO in hydrocephalus pathogenesis, serial brain sections through aqueduct regions containing the SCO from HTX rats, in comparison with normal Fischer F344 strain, have been studied and found that reduced SCO glycoprotein immunoreactivity precedes both aqueduct closure and expansion of the lateral ventricles in the HTX rate (as it’s redundant) [96, 97].

Although some studies have addressed the activation of macrophages and microglia (the resident mononuclear phagocytes of the brain) within the brain in animal hydrocephalus models, little is known of their state of activation or regional distribution in human congenital hydrocephalus. In one experiment, brain tissue samples of 10 human fetal cases with hydrocephalus and 10 non-hydrocephalic controls were stained immunohistochemically with antibodies directed against MHC class II and CD68 antigens, and lectin histochemistry was done with tomato lectin. Hydrocephalus cases showed focal collections of CD68 and tomato lectin-positive macrophages along the ependymal lining of the lateral ventricles, particularly within the occipital horn. By comparison, brain tissue samples from controls showed few or no ependymal or supraependymal macrophages and the few macrophages that were present were not as intensely immunoreactive as in the hydrocephalus cases. The macrophage response detected at the ependymal lining of the ventricles and within the periventricular area in hydrocephalus may be related both to the severity of hydrocephalus and the age of the fetus [98]. Microglia that are normally interspersed throughout the intermediate zone and circumscribing the basal ganglia were within normal confines in all cases examined. Unexpectedly, hydrocephalic cases also showed focal regions of hypovascularization or alterations in the structure and orientation of capillaries within periventricular areas, compared with controls [98].

In summary, the pathological studies of hydrocephalus clearly indicate that impaired and abnormal brain development in the early development stage caused by altered neural cell fate and perturbed regulation of cellular proliferation and apoptosis. The abnormal brain development subsequently leads to the accumulation of the CSF in cerebral cavities. All these cellular and developmental events eventually lead to the congenital hydrocephalus accompanied by possible secondary inflammatory reaction and neurovascular pathogenesis.

One of the possible mechanisms leading to the pathogenesis of hydrocephalus is the disruption of neural cell membrane proteins that play an important function during brain development. The L1 protein coded by human X-linked hydrocephalus gene is a member of the immunoglobulin superfamily of neural cell adhesion molecules that is expressed in neurons and Schwann cells, and seems to be essential for the brain development and function. Hirschsprung’s disease (HSCR) is characterized by the absence of ganglion cells and the presence of hypertrophic nerve trunks in the distal bowel. There have been several reports of patients with X-linked hydrocephalus and HSCR with a mutation in the L1CAM gene. Therefore, decreased L1CAM may also be a modifying factor in the development of HSCR [99].

The gene carrying the mutation for autosomal recessive hydrocephalus in the hyh mouse codes α-SNAP protein. α-SNAP is essential for apical protein localization and cell fate determination in neuroepithelial cells [100]. α-SNAP plays a key role in a wide variety of membrane fusion events in eukaryotic cells. Membrane fusion is required for two main cellular functions: 1) the transport of molecules to distinct inter- and intracellular compartments and thereby maintenance of the functional and structural organization of eukaryotic cells; 2) the intercellular communication such as the regulated exocytosis (secretion) of neurotransmitters by neuronal cell, which occurs temporally and spatially as the precise sequential regulation events at the plasma membrane during the early brain development [87]. In the hyh mouse model, altered neural cell fate is also accompanied by abnormal localization of many apical proteins implicated in regulation of neural cell fate, including E-cadherin, beta-catenin, atypical protein kinase C (aPKC), inactivation-no-afterpotential D-like (INADL), SNAP receptor (SNARE), and vesicle-associated membrane protein-7 (VAMP-7) [100]. Furthermore, disturbed astrocyte metabolism in the early brain development in the kaolin-induced rat model of hydrocephalus has also been reported [101].

Hydrocephalus may also be caused by a malfunction of the ependymal cells [102‐105]. Within the neonatal brain of the mouse, the hy3 protein (hydin) is confined to the ciliated ependymal cell layer lining the lateral, third and fourth ventricles. Hydin is not closely related to any previously known protein, with the exception of a 314 amino acid domain with homology to caldesmon, an actin-binding protein, suggesting that hydin interacts with the cytoskeleton [81, 82]. The protein of axonemal heavy chain 5 gene (Mdnah5), dynein is also specifically expressed in ependymal cells, and is essential for ultra structural and functional integrity of ependymal cilia. In Mdnah5-mutant mice, lack of ependymal flow causes closure of the aqueduct and subsequent formation of triventricular hydrocephalus during early postnatal brain development. The higher incidence of aqueductal stenosis and hydrocephalus formation in patients with ciliary defects proves the relevance of this novel mechanism in humans [106].

Hydrocephalus may be caused by malfunction of mesenchymal cells. In mice, Msx1 is a regulatory gene involved in epithelio-mesenchymal interactions in limb formation and organogenesis. In the embryonic brain, the Msx1 gene is expressed along the dorsal midline. The most important features observed in homozygous Msx1 mutants were the absence or malformation of the posterior commissure (PC) and of the SCO, the collapse of the cerebral aqueduct, and the development of hydrocephalus. The heterozygous mutants developed an abnormal PC and smaller SCO, as revealed by specific antibodies against SCO secretory glycoproteins. About one third of the heterozygous mutants also developed hydrocephalus; therefore the phenotype may be determined by the Msx1 gene dosage during a specific developmental period [107, 108]. In the autosomal recessive congenital hydrocephalus (ch) mouse model, a truncated protein lacking the DNA-binding domain of the forkhead/winged helix gene, Mf1, was generated. Mesenchymal cells from Mf1lacZ embryos differentiate poorly into cartilage in micromass culture and do not respond to added bone morphogenetic protein 2 (BMP2) and transforming growth factor-beta 1 (TGFB1). The differentiation of arachnoid cells in meninges of the mutant mice is also abnormal. The levels of developmental growth factors such as TGFB1 and BMP2 are dramatically increased in the ch mouse, and it is possible that phenotypic hydrocephalus of Mf1 mouse is due to the secondary effect of these elevated growth factors. Corresponding to studies in the ch mouse, human patients with deletions in the region containing human Mf1 homolog FREAC3 were found to develop multiple developmental disorders, including hydrocephalus [84]. Another winged helix transcription factor causing congenital hydrocephalus when mutated, RFX4_v3 transcript, is dynamically expressed in the developing brain from the neural plate stages. The RFX proteins belong to the winged-helix subfamily of helix-turn-helix transcription factors, and bind to ‘X-boxes’ in target DNA sequences and regulate expression of the downstream target genes [83]. Disruption of both RFX4_v3 alleles by insertional mutagenesis severely alters early brain morphogenesis, reduces Msx2 expression, and causes a deficiency in WNT signaling [83]. This may suggest that RFX4_v3 is probably upstream of Mf1 in the signaling pathway during early brain development.

Hydrocephalus may be caused by perturbation of growth factor signaling [109, 110]. Developmental abnormalities in congenital hydrocephalus provide the clues for the perturbation of major signaling pathways in the development [111]. TGFB is an important cytokine and growth-signaling molecule in the brain. In mouse models, severe hydrocephalus has been observed in transgenic mouse overexpression of TGFB1 in astrocytes [112, 113]. In the HTX rat, increased level of TGFB3 may contribute to the development of hydrocephalus [114]. In mouse models, fibroblast growth factor-2 (FGF-2) seems to play a predominant role in the proliferation of neuronal precursors and in neuronal differentiation in the developing cerebral cortex even at relatively late stages of brain neurogenesis. Administration of FGF-2 to embryonic brain induces hydrocephalic brain morphology and aberrant differentiation of neurons in the postnatal cerebral cortex [110]. IGF binding protein-1 (IGFBP-1) modulates the cellular action of insulin-like growth factors (IGFs), some of which are expressed in the fetal brain. Hydrocephalus has been observed in mouse models that overexpress liver-specific IGFBP-1 during fetal life. The hepatic over-expression of IGFBP-1 may have endocrine effects on brain development and induction of congenital hydrocephalus [115]. Other studies have shown that up-regulation of certain growth factors in the brain could lead to altered brain fluid dynamics [116, 117]. SOCS7 is a member of the suppressor of cytokine signaling (SOCS) protein family. SOCS proteins have a similar structure: an N-terminal domain of variable length, a central Src homology-2 domain, and a C-terminal SOCS box. Biochemical and genetic studies have revealed that SOCS family members play an important role in the termination of cytokine and growth factor signaling. Homozygous Socs7 mutant mice were born in expected numbers, were fertile, and did not exhibit defects in hematopoiesis or circulating glucose or insulin concentrations. Strikingly however, these homozygous Socs7 mice were 7–10% smaller than their wild-type littermates, and within 15 weeks of age approximately 50% of the homozygous Socs7 mice died as a result of hydrocephalus. In situ hybridization studies in normal mice have revealed that Socs7 is prominently expressed in the brain, suggesting that SOCS7 plays an important functional role in early brain development [118]. We can therefore hypothesize that loss of SOCS7 function will lead to increased expression of cytokines resulting in developmental abnormalities and congenital hydrocephalus due to its inhibitory role in cytokine signaling.

Hydrocephalus may also be caused by the disruption of extracellular matrix (ECM). In the TGFB1 over-expression mouse model, the changing expressions of a remodeling protein - matrix metalloproteinase-9 (MMP-9) and its specific inhibitor- tissue inhibitor of metalloproteinases-1 (TIMP-1) were also found to be important factors in the spontaneous development of hydrocephalus by altering the ECM environment [119]. Furthermore, increased expression of cytokines such as TGFB1 might also reciprocally play an important role by disrupting the vascular ECM remodeling, promoting hemorrhages, and altering the re-absorption of CSF [120]. In another mouse model, ablation of the nonmuscle myosin heavy chain II-B (NMHC-B) results in severe hydrocephalus with enlargement of the lateral and third ventricles. These defects may be caused by abnormalities in the cell adhesive properties of neuroepithelial cells and suggest that NMHC-B is essential for both early and late developmental processes in the mammalian brain [121].

Hydrocephalus may also be caused by the disruption of major early brain developmental patterning molecules. Autosomal dominant hydrocephalus in Otx2 mutation mice is characterized by eminent dilatation of lateral ventricles and a ballooned cerebrum. Histopathology shows edematous change of the periventricular white matter, suggesting that Otx2 functions as a brain developmental organizer, and a disruption of this gene is a likely cause of hydrocephalus [122].

In conclusion, many genetic loci of hydrocephalus have been defined in animal models, which is building a foundation for better understanding of molecular etiology of hydrocephalus; however, genetic research of hydrocephalus in humans is limited. The histopathological similarities of animal models can be used to understand the genetics and pathogenesis of human hydrocephalus. For example, the histopathological and morphological appearance of hydrocephalic HTX rats is demonstrated in Fig. 1, in which hydrophilic HTX rat exhibits large cerebral ventricles, a progressively-thinned cortical mantle, and stretched internal capsule fibers[123]. Review of the molecular etiologies shows a very diverse set of pathogenetic mechanisms. Perturbation of almost any molecule that plays a crucial role in early brain development, and sequentially regulation of dynamics of cerebrospinal fluid, could lead to the pathogenesis of congenital hydrocephalus. The 10 known hydrocephalus genes mostly code for important cytokines, growth factors, or related molecules in the cellular signal pathways during early brain development.

It is essential to recognize that molecular genetics is the only current scientific approach that can be used to study hydrocephalus in which the usual concern about whether an observed phenomenon is a consequence or a cause is completely addressed.

Despite our knowledge of the genetics of hydrocephalus in animal models, we have very limited knowledge about the genetic and molecular mechanisms that cause human hydrocephalus. Without this knowledge, it is impossible to say whether the pathogenesis of human hydrocephalus is comparable to that seen in animal models, and impractical to extrapolate data gained from animal models to humans. In order to better understand human hydrocephalus and to develop more appropriate translational research, it will be necessary to conduct large-scale genetic studies of human hydrocephalus. If, and when, more heritable forms of human hydrocephalus are identified, and underlying genes and their functions are characterized, then this knowledge could be used to improve patient care in a variety of different ways such as prenatal diagnosis and new potential therapeutic approaches. Possible new mechanisms other than altered CSF circulation and resorption, if uncovered via the genetic research, may also help explain why patients with hydrocephalus may experience symptomatic progression despite functioning shunts.

Efforts to identify genetic variants associated with susceptibility to genetic diseases rely on three major approaches: pedigree and sib-pair linkage analysis and population association studies. The differences among these study designs reflect their derivation from biological versus epidemiological traits. Like most common diseases, it would be very difficult to identify and recruit large pedigrees in hydrocephalus that show hereditary transmission of the condition. Therefore, the last two approaches, sib-pair linkage analysis and population association studies, are the best options for the genetic study on this disease. For any study, but particularly in the case of genetic mapping for a common disease, a large sample size is crucial in achieving statistical significance. Recent advances in the genomics and statistical methodology in genetic mapping will certainly help in making well-powered studies more feasible, by reducing the number of genetic markers or workload required for these studies. For example, since many genes and loci response for hydrocephalus in animal models have been mapped, candidate genes approach will certainly be the very first choice to test the collected human hydrocephalus population for linkage and association analysis.

In collaboration with the Hydrocephalus Association (HA), our group has initiated a genetic study of human hydrocephalus. As part of a prospective study that has been approved by the Johns Hopkins Institutional Review Board, we are collecting blood samples from both congenital and acquired NPH hydrocephalus patients. The objectives of this study are to identify the genetic loci responsible for the development of hydrocephalus, to examine the relationship between genotype and phenotype and to define the functions of these genes during early development. This is the first large-scale research study of its kind and information gained from this study will undoubtedly provide invaluable information concerning the developmental mechanisms of this disease in humans. Such knowledge will hopefully lead to the more reasonable treatment schemes, the better diagnostic tools, and the more effective therapeutic modalities.