Clinical and Molecular Genetics of Inherited Hydrocephalus*

Cong. Anom., 34: 303-3 10, 1994 Review

Norio SAKURAGAWA' and Yasunobu YOKOYAMA'?* 'Department of Inherited Metabolic Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1, Ogawahigashi, Kodaira. Tokyo 187, Japan and 2Department of Chromosome Analysis, Center for Molecular Genetics and Cytogenetics, SRL Inc., 153 Komiya-cho, Hachioji, Tokyo 192, Japan

ABSTRACT Congenital hydrocephalus has a broad spectrum of etiology and has not been elucidated in terms of pathogenesis or mechanism of hydrocephalus. Recent advance of molecular genetics disclosed the genetic defects of X-linked hydrocephalus (HSAS), MASA syndrome and X-linked spastic paraplegia (SPG I), characterized by mutations in LICAM, a gene coding for a neural cell adhension molecule. Meanwhile, genetic heterogeneity in X-linked hydrocephalus has been pointed out; linkage to markers within Xq27.3. We reviewed inherited hydrocephalus and chromosomal aberrations with hydrocephalus because fibroblasts from patients with these disorders may be useful for a molecular genetic approach to inherited hydrocephalus. Animal models of congenital hydrocephalus are also very important for a comparative mapping analysis between mice and human. We have tried to clone a candidate gene of hydrocephalus using fibroblasts which show a de nove chromosomal aberration [t(4;16)(q35;q22. I)], because the synteny conservation locus for mouse hydrocephalus-3 (hy-3) gene is suggested to be located in the long arm of human chromosome 16. A rearranged band of 1.2 Mb was detected by Pulse-Field Gel Electrophoresis (PFGE) with Not I digestion using the calretinin probe. It may exist within 1.2 Mb distal apart from calretinin gene. Further analysis is carried out in order to clone this candidate gene of congenital hydrocephalus. Key words: inherited hydrocephalus, molecular genetics, animal model, chromosome aberration

Hydrocephalus is defined as an abnormal accumulation of intracranial cerebrospinal fluid (CSF) due to overproduction or disturbed absorption of CSF. Abnormal circulation of CSF is also one of the common

Received September 2, 1994 * Presented in the Symposium on "Developmental Anomalies of the Central Nervous System: Basic and Clinical Research" at the 34th Annual Meeting of the Japanese Teratology Society, Kochi, July IS, 1994. &fllE%\ItlG!$O)C . It#$.%+ i 9 -w&3wFfi ~Tmv="-hs 5 3, TI87 ~~-4i6.I\.ii-m/l~Jll~~4-I-I E6llJZl@. lFll.%LzF xx7=-1l,x1!, BK3~%l3f&%+t+i9-'%.$f5?3, T I92 ~~83~\3~ffi~J\~Ul53 304 N. Sakuragawa and Y. Yokoyama causes of hydrocephalus such as stenosis of aqueduct of Sylvius. Most cases are sporadic but a small propor- tion is inherited in either an autosomal recessive or X-linked fashion. Clinical heterogeneity in familial hydrocephalus has been pointed out since there exist several reported families of an autosomal recessive trait (Willems, 1988). The X-linked hydrocephalus with stenosis of the aqueduct of Sylvius (HSAS, McKusick No. 307000) is the most common genetic form of hydrocephalus. Inherited hydrocephalus with an autosomal trait has not been studied with mordern techniques of molecular genetics. Recently, HSAS has been reported to have aberrant splicing of LI mRNA or a duplication in the LICAM gene (Rosenthal et al., 1992; Jouet et al., 1993). First of all, clinical and genetic backgrounds of inherited hydrocephalus are described in this report. And we review animal models of congenital hydrocephalus, especially about mouse models, of which mutations have been reported to be on defined chromosomes. Then we will mension briefly our study on an approach to clone a candidate gene of congenital hydrocephalus using human fibroblasts showing reciprocal transloca- tion.

Table 1 Classification of familial hydrocephalus

~ ~ Disorder5 McKuwk No Gene anomaly

Syndromal

AD Apert’s syndrome (McKusick I0 1200) Basal cell nevus syndrome (McKusick 109400) Achondroplasia (McKusick 100800)

AR HARD +I- syndrome (McKusick 236670)

Meckel-Gruber syndrome (McKusick 249000)

Roberts syndrome (McKusick 268300)

Smith-Lemli-Opitz syndrome (McKusick 270400)

Nonsyndromal

AR Autosomal recessive nonsyndromal (McKusick 236600) hydrocephalus (Teebi and Naguib, 1988)

Autosomal recessive hydrocephalua 3rd (McKusick 236600) ventricle obstruction (Chow et al., 1990)

Hydrocephalus with associated (McKusick 236640) malformations (Game el al., 1989)

X-linked Hydrocephalus due to congenital stenosis (McKusick 307000) Point mutations or of aqueduct of Sylvius (HSAS I ) duplication of LlCAM

MASA syndrome (McKusick 303350)

X-linked hydrocephalus linked to markers Linkage to markers within within Xq27.3 (Strain et al., 1994) Xq27.3

AD, autosomal dominant; AR, autosomal recessive Genetics of inherited hydrocephalus 305

1. Clinical heterogeneity of familial hydrocephalus Familial hydrocephalus presents considerable morphological and etiological heterogeneity. which is de- vided into both syndromal and nonsyndromal entities (Teebi et al., 1988). Hydrocephalus is a manifestation of several autosomal dominant or recessive syndromes, which occurs as part of a syndrome (Table I). The nonsyndromal forms include autosomal recessive or X-linked types. Regarding autosomal recessive forms, Teebi and Naguib (1988) described the consanguineous family with four hydrocephalic sibs not associated with spina bifida. Chow et al. (1990) reported a brother and sister who presented in the neonatal period with hydrocephalus. Game et al. ( 1989) described a new syndrome, characterized by fetal growth retardation, hydrocephalus, hypoplastic multilobed lungs and other anomalies in four sibs. These autosomal recessive forms have not been studied genetically. The X-linked recessive form of congenital hydrocephalus (HSAS) is clinically characterized by mental retardation, enlarged cerebral ventricles, spastic paraparesis and adducted thumbs and, occasionally, visual defects or seizures. The most severe cases die pre- or perinataly with gross hydrocephalus and enlarged head circumference. Its overall frequency has been estimated at 1/30,000 male births. While, MASA syndrome is the clinical phenotype of another Xq28-linked disorder, manifested with mental retardation, aphasia, shuffling gait and adducted thumbs. Some patients with MASA syndrome show enlarged lateral ventricles detected by CT scanning. Since the wide variation in the severity and symptoms are associated with HSAS and some of HSAS are overlap with the clinical phenotype of MASA syndrome, it has been suggested that HSAS and MASA have variable manifestations of the same disorder (Winter et al., 1989).

2. Molecular genetics of X-linked hydrocephalus Positional cloning has been used to investigate the disorder and genetic linkage analysis of gene located in Xq28. Willems et al. (1990) localized HSAS to chromosome Xq28 using a linkage analysis in four families, Analysis of rare recombination events in HSAS families has indicated that HSAS lies within a 2 mega- base (Mb) region between polymorphic markers for St14 (DXS.52) and coagulation factor VIII (F8) withir. the Xq28 locus (Willems et al., 1992). Because LICAM was the only known Xq28 gene with a function in neurological tissue, this gene has been considered as a candidate gene for HSAS (Willems et al., 1992). Eventually, Rosenthal et al. (1992) have found aberrant splicing of neural cell adhension molecule LI mRNA in a family with HSAS. Subsequently they observed a G to A transition in the patient at nucleotide position 791 of the cDNA sequence (Jouet et al., 1993). While, Camp et al. (1993) conducted a mutation analysis of LICAM in 25 HSAS families. They found 1.3 kb genomic duplication in one family, cosegregating with HSAS. Interestingly, Vits et al. (1994) reported that 3 different mutations were identified from 8 unrelated patients with MASA syndrome which is another X-linked hydrocephalus. Furthermore, Jouet et al. (1994) described the mutations of the LI gene in MASA syndrome, SPGl and HSAS syndrome. They concluded that these three syndromes form part of a clinical spectrum resulting from a heterogeneous group of mutations in the LI gene. The function of LICAM is not yet understood completely but it is involved in neuronal cell migration, fasciculation, outgrowth and regeneration (Lemmon et al, 1989; Miura et al.. 1992; Sonderegger and Rathjen, 1992, Williams el al., 1992). Although detailed genotype-phenotype correlation has not yet been cleared, it is also speculated that mutations in LICAM are responsible for a specific X-linked mental retardation, because some patients belonging to HSAS or MASA families show no hydrocephalus, spastic paraparesis or adducated thumbs, but only mental retardation (Vits et al., 1994). On the other hand, Strain et al. (1994) investigated a family with typical X-linked aqueductdl stenosis, in which no linkage to DXS52 and F8C 306 N. Sakuragawa and Y. Yokoyama markers was present. They demonstrated the X-linked aqueductal stenosis may result from mutations at two different loci on the X chromosome because close linkage was established to the DXS548 and FRAXA loci in Xq27.3. It is also expected that secondary factors, other than the LICAM mutations, might contribute to the phenotype as a large intrafamilial variability is observed (Vits et al., 1994). We have been investigated a Japanese family with X-linked hydrocephalus, who did not show a duplication in the LICAM gene.

3. Chromosome aberrations associated with hydrocephalus Hydrocephalus is manifested as a part of the phenotypes in some of chromosome aberrations, such as trisomy 13, trisomy 18 and triploidy. Also, patients with different chromosomal anomalies have been reported to show ventriculomegaly in addition to other clinical signs and symptoms (Borgaonkar, 1991) (Table 2). Fibroblasts from these patients presenting congenital hydrocephalus and minor anomalies are sometimes very important for cloning a candidate gene of congenital hydrocephalus. Especially de novo chromosomal anomalies are useful such as reciprocal translocations or deletions.

4. Mouse models of congenital hydrocephalus Hydrocephalus has been described in rodents, rabbits. cats, dogs and ungulates. Regarding rat models for congenital hydrocephalus, several strains have been reported such as HTX (Kohn et al., 1981), LEW/Jms (Sasaki et al., 1983). WIC/Hyd (Koto et al., 1987) and etc. The chromosomal location of mutations of several hydrocephalic mice (Table 3) were found such as hy-3 on chromosome 8, congenital hydrocephalus (ch) on chromosome I3 (Fig. 1 ), hydrocephaly with hop gait (hyh) on chromosome 7 and hydrocephalic-polydactyly (hop/lpy) on chromosome 6 (Bronson and Lane, 1990). Molecular genetic approaches for cloning of hydrocephalus gene could be designed using these animal models.

Table 2 Chromosome aberrations associated with hydrocephalus (Borgaonkar D.S., 1991)

Karyotype Clinical manifestations

46,XX,der(6)t(I ;6)(q42;q27)mat abnormal ears, club feet, failure to thrive, psychomotor delay 46,XY,del( l)(q43 + qtcr) craniofacial dysmorphism, preauricular pit 46,XX,de1(2)(p21p.22.2) incomplete midface development, cyclopia, proboscis 46,XX,der( 14)t(2;14)(q21;q32)mat partial trisomy 2p syndrome, hypospadias 46,XY,t(4;16)(q3S;q22.1) hypospadias, dysmorphic features 46,XY,dcr(S)t(5;7)(pIS;q32)mat multiple congenital anomalies, CHD, cat-like cry 46,XY,r(6)(p24q26) aniridia, congenital glaucoma, Rieger anomaly, CEU 46,XY,-6,+der(6)t(6:8)(6pter to 6q25::8q22 to myelomeningocele,congenital anomalies, clubfeet 8qter)mat 46,XY,der(6)t(6;22)(q27;q13)mat IUGR, cleft palate, genital-hypoplasia,dysmorphic faces 46,XX,de1(8)(q13q?l.2) dwarf? 46,XY,t( 1 I q; 18q) premature born, epidermolysis 46,XX,rec(13)dup P,inv( 13)(pl lq22)pat generalized edema, absent thumbs, talipes, low-set ears moa46,XY/46,XY,del(13)(q12.lq12.3) 4S,XY.t( 15; 1% I Sql5::19ql3 to19 pter) muscle hypotonia, multiple congenital anomalies nios46,XX(4)/47,XX,+9(6) single transverse kidney with two ureters mos46,XY/47,XY,+mar poor shaped ears, COXSK skin, pigmentation of face and body

CHD, congenital heart defects; CEU, congenital ectropion uveae; IUGR, intrauterine growth retardation Genetics of inherited hydrocephalus 307

Table 3 Mouse models presented with hydrocephalus

Gene symbol Gene name Chromosome

ch congenital hydrocephalus hop hPY hydrocephalic polydactyly hy- I hydrocephalus- 1 hv-2 hydrocephalus-2 hy-3 hydrocephalus-3 hyh hydrocephaly with hop gait oh obstructive hydrocephalus

Fig. 1 Congenital hydrocephalus (ch). The mutant mouse was stillborn. The domed head was brownish due to intracranial hemorrhage.

The h.~-3mice are usually detectable at 3-5 days of age, characterized by enlarged lateral and third ventricles (Fig. 2). Hydrocephalic mice die at approximately 3 weeks of age. The pathogenesis of this hydrocephalus seem to be due to cellular degeneration and atrophy of the meninges. Also a significant role for cerebral astrocytes and interastrocytic junctional complexes is indicated in the pathophysiology of this hydrocephalus (McLone et al., 1971). Ventriculomegaly in the ch strain begins prenatally at I1 days of gestation. Homozygotes die immediately postparatum. In cWch embryos, a demarcation between the arachnoid and the subarachnoid space is much less distinct and at 14 days the subarachinoid space is undeveloped and the lateral ventricles are greatly distended. The hyh is another lethal recessive mutation of mouse, characterized by a domed head and a hopping gait observable at 2 weeks of age and death between 4 and 10 weeks of age. Pathologically, mutant mice have 308 N. Sakuragawa and Y. Yokoyama

Fig 2 Hydroccphalus-3 (h\-3) mouse (left) has a frontal boss and is smaller than a control mowe (right)

dilated lateral ventricles, a large third ventricular cyst, cystic caudal aqueduct and no communication of the aqueduct with the fourth ventricle. It has been mapped to the proximal end of chromosome 7 close to the glucose phosphate isomerase- 1 locus (Bronson and Lane, 1990). The hophp-”is a recessive pleiotrophic mutant in the mouse, which shows a rabbit-like gait, both hindlegs tending to move in unison, associated with hydrocephaly and/or polydactyly (Hollander, 1966).

5. Cloning of candidate gene of congenital hydrocephalus In the comparative mapping analysis between mice and human about the synteny conservation loci, we found the fibroblasts from a patient with congenital hydrocephalus, which involve a de nnvo chromosomal aberration: t(4; 16)(q35;q22.1). The synteny conservation locus for mouse hy-3 gene, which is on mouse chromosome #8D-E (Mouse genome, 1994), is suspected to be located in the long arm of human chromosome 16. So we carried out the DNA analysis for cloning the candidate gene of human congenital hydrocephalus using the fibroblasts. Since haptoglobin and calretinin genes were located at both sides of its breakpoint, we used these genes as anchor points for genomic studies of human chromosome 16q22.1 region. A rearranged band of 1.2 Mb was detected by Pulse-Field Gel Electrophoresis (PFGE) with Not I degestion using the calretinin probe. PFGE using the haptoglobin probe did not show any rearranged band. It may exist within 1.2 Mb distal apart from calretinin gene. Further analysis is carried out in order to clone this candidate gene of congenital hydrocephalus.

CONCLUSIONS

Inherited hydrocephalus was reviewed from the clinical and genetical points of view. X-linked hydrocephalus and related diseases have been shown to be due to mutations in LlCAM. Since genetic heterogeneity is Genetics of inherited hydrocephalus 309 also pointed out, there must be several candidate genes involving pathogenesis of hydrocephalus. Molecular genetic approaches for congenital hydrocephalus will elucidate the basic mechanism of hydrocephalus and give an important suggestion to treat the patients with this disorder.

ACKNOWLEDGMENT

This study was supported by Grant-in-Aid for “intractable hydrocephalus” from the Ministry of Health and Welfare of Japan.

REFERENCES

Borgaonkar, D.S. (1991) Chromosomal Variation in Man. Koto, M., Miwa, M., Shimizu, A,, Tsuji, K., Okamoto, M. A Catalog of Chromosomal Variants and Anomalies. and Adachi, J. (1987) Inherited hydrocephalus in Csk: 6th ed., Alan R. Liss. Inc., N.Y. Wistar-Imamichi rats; Hyd strain: A new disease Bronson, R.B. and Lane, P.W. (1990) Hydrocephalus with model for hydrocephalus. Exp. Anim., 36: 157-162. hop gait (hyh): A new mutation on chromosome 7 in Lemrnon, V., Farr. K.L. and Lagenaur, C. (1989) the mouse. Dev. Brain Res., 54: 131-136. LI-mediated axon outgrowth occurs via a homophilic Camp, G.V., Vits, L., Coucke, P., Lyonnet, S., Schrander- binding mechanism. Neuron, 2: 1597-1 603. Stumpel, C., Darby, J., Holden, J., Munnich, A. and MacLone, D.G., Bondareff, W. and Raimondi, A.J. (1971) Willems, P.J. (1993) A duplication in the LICAM Brain edema in the hydrocephalic hy-3 mouse: Submi- gene associated with X-linked hydrocephalus. Nature croscopic morphology. J. Neuropathol. Exp. Neural., Genet., 4: 421425. 30: 627-637. Chow, C.W., McKelvie, P.A., Anderson, R.McD., Phelan, Miura, M., Asou, H., Kobayashi, M. and Uyemura, K. E.M.D., Klug, G.L. and Rogers, J.G. (1990) Autoso- (1992) Functional expression of a full-length cDNA ma1 recessive hydrocephalus with third ventricle coding for the neural cell adhension molecule LI obstruction. Am. J. Med. Genet., 35: 310-313. mediates homophilic intercellular adhension and Game, K., Friedmann, J.M., Paradice, B. and Norman, migration of cerebellar neuron. J. Biol. Chern., 267: M.G. ( 1989) Fetal growth retardation, hydrocephalus, 10752- 10758. hypoplastic multilobed lungs, and other anomalies in Rosenthal, A,, Jouet, M. and Kenwrick, S. (1992) Aber- 4 sibs. Am. J. Med. Genet., 33: 27&279. rant splicing of neural cell adhension molecule LI Hollander, M.D. ( 1966) Hydrocephalic-polydactyly, a mRNA in a family with X-linked hydrocephalus. recessive pleiotropic mutant in the mouse. Am. Zool., Nature Genet., 2: 107-1 17. 6: 588-589. Sasaki, S., Goto, H., Nagano, H., Furuya, K., Omata, Y., Jouet M., Rosenthal, A., MacFarlane, J., Kenwrick, S. and Kanazawa, K., Suzuki, K., Sudo, K. and Collmann, H. Donnai, D. (1993) A missense mutation confirms the (1983) Congenital hydrocephalus revealed in the LI defect in X-linked hydrocephalus (HSAS). Nature inbred rat, LWE/Jms. Neurosurgery, 13: 548-554. Genet., 4: 331. Sonderegger, P. and Rathjen, F.G. (1992) Regulation of Jouet, M., Rosenthal, A., Armstrong, G., MacFarlane, J., axonal growth in the vertebrate nervous system by Stevenson, R.A., Paterson, J., Metzenberg, A,, Iona- interactions between glycoproteins belonging to two sescu, V., Temple., K. and Kenwrick, S. (1994) subgroups of the immunoglobulin superfamily. J. Cell X-linked spastic paraplegia (SPG I ), MASA syndrome Biol., 119: 1387-1394. and X-linked hydrocephalus result from mutations in Strain, L., Gosden, C.M., Brock, D.J.H. and Bonthron, the LI gene. Nature Genet., 7: 402407. D.T. (1994) Genetic heterogeneity in X-linked hydro- Kohn, D.F., Chinokoswong, N. and Chou, S.M. (1981) A cephalus: Linkage to markers within Xq27.3. Am. J. new model of congenital hydrocephalus in the rat. Hum. Genet., 54: 236-243. Acta Neuropathol., 54: 21 1-218. Teebi, A.S. and Naguib, K.K. (1988) Autosomal recessive 310 N. Sakuragawa and Y. Yokoyaina

nonayndromal hydrocephalus. Am. J. Med. Genet., 31: Willems, P.J., Vits, L.. Raeymaeker, P., Beuten, J.. 467470. Coucke, P., Holder, J.J.A., Broekhove, V.. Warren, Vits, L., Camp. G.V., Coucke, P., Fransen, E., De Boulle, S.T., Sagi, M., Robinson, D., Dennis, N., Friedman K., Reyniers, E., Korn, B., Poustka, A., Wilson, G., K.J., Magnay, D., Lyonnet, S., White, B.N., Wittwer. Schrander-Stumpel. C., Winter, R.M., Schwaratz, C. B.H., Aylsworth, A.S. and Reicke, S. (1992) Further and Willems, P.J. (1994) MASA syndrome is due to localization of X-linked hydrocephalus in the chromo- mutations in the neural cell adhension gene LICAM. somal region Xq28. Am J. Hum. Genet., 51: 307-315. Nature Genet., 7: 408412. Williams, E.J. Doherty, P., Turner, G., Reid, R.A., Hemp- Willems, P.J. ( 1988) Heterogeneity in familial hydroceph- erly, J.J. and Walsh, F.S. (1992) Calcium influx into alus. Am. J. Med. Genet., 31: 471472. neurons can solely account for cell contact dependent Willeins, P.J., Dijkststa, I.. Van der Auwera, B.J., Vits, L., neurite outgrowth stimulated by transfected L I. J. Cell Coucke, P., Raeymaekers, P., Van Broeckhoven, C., Biol., 119: 883-892. Consalez, G.G., Freeman, S.B., Warren, S.T., Brouwer, Winter, R.M., Daview, K.E., Bell, M.V.. Huson, S.M. and O.F., Brunner, H.G., Renier, W.O., Van Elsen, A.F. and Patterson, M.N. (1989) MASA syndromes: Further Dumon, J.E. (1990) Assignment of X-linked hydro- clinical delineation and chromosomal localization. cephalus to Xq28 by linkage analysis. Genomics, 8: Hum. Genet., 82: 367-370. 367-370.