Clinical manifestations

Presentation and course

Infants with Walker-Warburg syndrome are symptomatic from birth. Manifestations at birth include generalized hypotonia and severe generalized paresis, macrocephaly due to hydrocephalus (occasionally microcephaly), and eye abnormalities that encompass anterior as well as posterior chamber abnormalities.

Epilepsy is not a complication in all patients, but at least one third develop seizures during infancy or early childhood. Seizures may be generalized major motor, partial, myoclonic, or a mixture of types.

Ultrasonography, CT, or MRI will reveal gross hydrocephalus, together with partial aplasia of the cerebellar vermis, hypoplasia of the cerebellar hemispheres, and abnormal structure of the brainstem. In one third of the patients, posterior encephalocele or occipital meningocele is encountered. MRI is superior in demonstrating all the cerebral abnormalities of which the disease is composed (53; 15; 30). The usual MRI findings include a smooth neocortical surface, an irregular inferior cortical margin, hypoplasia or partial aplasia of the cerebellar vermis, and hypoplasia of the pons.

Further clinical and neuropathologic contributions provide ample documentation of a distinct and well-recognizable profile (13; 10; 72; 57). Ophthalmological findings include microphthalmia and cataracts.

Most patients die before their second birthday, but survival may be more prolonged (70).

Besides cobblestone lissencephaly and hydrocephalus, the findings in the brain include cerebellar cortical dysplasia with apparently fused cerebellar folia (39) and diminution in size of the ventral pons.

A large neuropathological material based on autopsy of affected fetuses correlated the severity of cobblestone lissencephaly with mutated genes affecting dystroglycan glycosylation (18). Cobblestone lissencephaly was graded according to the ratio between the extracerebral (ie, ectopic) part of the cortex and the true cortex. In the most severe cases, the distinction between the 2 is almost completely lost. The most severe cases were affected by mutations in POMT1, POMT2 and FKRP, whereas the milder cases were affected by mutations in POMGNT1, and intermediary cases by mutations in LARGE. In 34% no mutation was identified.

Jissendi-Tchofo and colleagues put emphasis on characteristic MRI abnormalities of the midbrain and hindbrain (30). Kinking of the brainstem in sagittal images is a characteristic finding; however, this abnormality is also observed in tubulinopathies (28). Rarely, cerebellar cysts may be found (52). Serum creatine kinase is usually elevated. Muscle abnormalities consist of increased variability in diameters, affecting both fiber types: small fibers, with regenerated (basophilic fibers), internal nuclei and rounded fibers, with increase of endomysial connective tissue and increase of fat tissue. Most early descriptions refer to paraffin sections (17; 61; 72). Immunohistochemical refinement is achieved by finding negative staining for alpha-dystroglycan (43).

Table 1. Clinical Findings of Walker-Warburg Syndrome

Prognosis and complications

Diagnosis is uniformly lethal at an early age in typical Walker-Warburg syndrome. Although some patients may reach childhood, most will die before their second birthday, and some will not survive the neonatal period because of respiratory problems. Survival in muscle-eye-brain disease and Fukuyama syndrome is much longer, and puberty may be reached.

Clinical vignette

A female patient was born as the third of nonconsanguineous parents who previously had 2 healthy children. She presented at birth with macrocephaly, 42 cm (+ 6 standard deviation), and normal 3530 g body weight. Advanced hydrocephalus was demonstrated by immediate ultrasonography. She had no other outward stigmata. She had mild flexion contractures of elbows, hips, and knees without deformations. The muscles felt tense, she had areflexia, and her creatine kinase was 3460 IU at 4 days. MRI showed a serrated inferior margin of the neocortex suggesting type 2 or cobblestone lissencephaly, apparently fused frontal lobes, hypoplastic ventral pons, and a hypoplastic cerebellar vermis. Ophthalmoscopy revealed ablatio retinae on the right side. A muscle biopsy from the femoral rectus muscle revealed increased diameter variation, acting on both types, endomysial fibrosis, and some regenerating fibers. Immunohistochemistry showed absence of staining of alpha-dystroglycan. Electron microscopy revealed a loss of normal sarcomere structure in the majority of the fibers. A diagnosis of Walker-Warburg syndrome was made. She received ventriculoperitoneal drainage that had to be changed twice because of obstruction. She deteriorated progressively, needing constant gavage feeding from 3 months. She had bouts of aspiration pneumonia and died at 4 months.

Biological basis

Etiology and pathogenesis

Walker-Warburg syndrome is an autosomal recessive disorder with the combined features of a congenital muscular dystrophy and lissencephaly type 2 (cobblestone lissencephaly). It belongs to the group of alpha-dystroglycanopathies caused by deficient glycosylation of alpha-dystroglycan. Dystroglycan is a member of the dystrophin-associated glycoprotein complex that links the muscle cell cytoskeleton to the extracellular matrix via dystrophin. An analogous situation in the brain causes a neuronal migration disorder (42). Dystroglycan is synthesized as a precursor molecule that is post-translationally cleaved into alpha and beta subunits. The alpha subunit, which is of primary concern here, is located outside the cell membrane and binds the extracellular matrix protein laminin (27). Dystroglycanopathies other than Walker-Warburg syndrome include Fukuyama syndrome (31) and muscle-eye-brain disease, a disorder first identified in Finland (51; 56; 62; 26). The first gene defect causing a dystroglycanopathy was detected in Fukuyama syndrome by Kobayashi and colleagues, who identified the ancient transposon insertion in the untranslated part of the gene, henceforth called Fukutin (FKTN) (35). The first gene to become associated with Walker-Warburg syndrome was POMT1, an O-mannosyltransferase gene (08). Defects in functionally related genes: POMGnT1, POMT2, FKTN (fukutin gene), FKRP, and LARGE have all been implicated in 1 or more alpha-dystroglycanopathy deficiency phenotypes: Walker-Warburg syndrome, Fukuyama syndrome (31), muscle-eye-brain disease, or congenital muscular dystrophy with mental retardation. In a series of 47 fetuses with Walker-Warburg syndrome from 41 families detected by fetal ultrasound, sequence analysis revealed mutations in POMT1 in 32%, POMGnT1 in 15%, and POMT2 in 7%. Screening for mutations in FKRP, fukutin, and LARGE was negative in the remainder (11). Severe homozygous mutations in FKTN, however, were the cause of Walker-Warburg syndrome in a Turkish consanguineous family (58) and in 4 Ashkenazi-Jewish families (14). Rare cases identified as Walker-Warburg syndrome were associated with mutations in LARGE (64) and FKRP (41; 66). A population study in Italy incorporating 81 patients with congenital muscular dystrophy who were expressing one or more signs of alpha-dystroglycanopathy produced homozygous or compound heterozygous mutations in 43 of the 81 patients, including patients who were expressing Walker-Warburg syndrome and muscle-eye-brain disease phenotypes (40). Six Walker-Warburg syndrome patients in this study were reported to harbor mutations, including POMT1, POMGnT1, and LARGE. Thirteen of 24 patients with muscle-eye-brain disease or Fukuyama syndrome had mutations in POMGnT1, FKRP, POMT1 or POMT2.

Besides Walker-Warburg syndrome, Fukuyama syndrome, and muscle-eye-brain disease, atypical syndromes with alpha-dystroglycan hypoglycosylation manifest with muscular dystrophy, variable mental deficiency, microcephaly, and absent to moderate abnormalities visible on MRI. Mutation analysis may reveal abnormalities in the above-mentioned genes, eg, POMT2 mutations (74).

In 2012 another gene, ISPD, encoding “isoprenoid synthase domain containing”, became associated with Walker-Warburg syndrome. It was discovered simultaneously by 2 groups (55; 71). ISPD mutations were found to be the second cause in frequency associated with Walker-Warburg syndrome. ISPD is involved in dystroglycan glycosylation. A zebrafish knockout of ISPD was shown to cause muscle degeneration and hypoglycosylation of alpha-dystroglycan as well as hydrocephalus and reduced eye size (55). Mutations in B3GALNT2 encoding the enzyme beta-1,3-N-acetylgalactosaminyltransferase 2 were found to cause muscular dystrophy and structural brain defects highly similar to muscle-eye-brain disease (59). Recessive mutations in LAMB1 encoding laminin subunit beta-1 were found to cause all the features of cobblestone lissencephaly with minimal involvement of eye and muscle (50). Homozygous loss-of-function frameshift mutation in the DAG1 gene resulted in complete absence of both alpha- and beta-dystroglycan (54).

As of January 2018, the following genes affecting dystroglycan glycosylation have been associated with Walker-Warburg syndrome: POMT1; POMT2; FKPR; FKTN; ISPD; POMGNT2 (GTDC2); POMGNT1; TMEM5; B3GALNT2 (G3GALNT2); GMPPB; B3GNT1; POMK (SGK196); LARGE; DAG1 (09; 22; 32; 54).

Between 2010 and 2012, other genes were added to the spectrum, not involved in dystroglycan glycosylation, but causing phenocopies. In a study by Labelle-Dumais and colleagues, it was shown that heterozygous mutations in COL4A1 may cause Walker-Warburg like syndrome (36). COL4A1 encodes a basement membrane protein and is not involved in alpha-dystroglycan glycosylation. Previously, this gene became associated with vascular disorders including hemorrhage as well as thrombosis at all ages. It was also shown that heterozygous Col4a1-mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of muscle-eye brain disease/Walker-Warburg syndrome.

Powis and colleagues reported another mutation in a gene not involved in dystroglycan glycosylation (48). They found a TUBB3 de novo missense alteration (in a completely conserved amino acid position) in a deceased infant with a Walker-Warburg phenotype (hydrocephalus, brainstem kinking, optic atrophy, elevated CK). Previous molecular genetic testing of 17 genes associated with congenital muscular dystrophies failed to provide a diagnosis.

Another partial phenocopy is due to mutations in the GPR56 gene (06). Features shared between alpha-dystroglycanopathies and homozygous mutation in GPR56 are polymicrogyria, patchy white matter abnormalities, overmigration with features of cobblestone lissencephaly (on neuropathological examination), and cerebellar hypoplasia, especially affecting the vermis and the finding of cerebellar cysts. Indeed, a fetal case initially suspected of Walker-Warburg syndrome was shown to be due to GPR56 mutation. GPR56 encodes a transmembrane protein. Mutations affect the integrity of the pial membrane.

A pathomechanism is shared between Walker-Warburg syndrome, Fukuyama syndrome, and muscle-eye-brain disease. These 3 syndromes constitute a spectrum of congenital muscular dystrophies with overlapping features of central nervous system malformations. All 3 share a deficiency in alpha-dystroglycan glycosylation. The defective gene in Fukuyama-type congenital muscular dystrophy, called fukutin, is a 461-amino-acid protein with the structure of a glycosyltransferase. Remarkably, 87% of Fukuyama-type congenital muscular dystrophy cases result from a single ancestral founder and are caused by a 3 kb-retrotransposal insertion into the 3' untranslated region of this gene. This causes a reduction of the normal amount of the encoded protein. Fukuyama congenital muscular dystrophy is the first human disease to be caused by an ancient retrotransposal integration. Japanese patients with Fukuyama syndrome either carry the founder mutation on one allele and a non-founder mutation on the other or carry the founder mutation on both alleles. The latter situation leads to a milder phenotype (76). The Japanese founder mutation has been identified outside Japan in a Chinese patient on one allele of the FKTN gene, the other allele carrying another mutation (73). The first patient of non-Japanese origin (Turkish) with a different mutation in the same gene (a 1 bp insertion) was identified in 2003 (58). Interestingly, this patient with a homozygous nonsense mutation in the fukutin (FKTN) gene had the phenotype of Walker-Warburg syndrome.

Muscle-eye-brain disease was initially found in patients with a mutation in POMGnT1, encoding the sugar component of the enzyme O-mannose beta-1,2-N-acetylglucosaminyltransferase (75).

Remarkable strides have been made in the understanding of Walker-Warburg syndrome and the closely related Fukuyama syndrome and muscle-eye-brain disease. A focal role is played by alpha-dystroglycan, a component of the dystrophin-glycoprotein complex, which can be found in various tissues including muscle and brain.

In muscle this complex binds the contractile elements of the muscle cell to the extracellular matrix via the dystrophin-glycoprotein complex. The dystrophin-glycoprotein complex is disrupted in different forms of muscular dystrophy, such as Duchenne muscular dystrophy (dystrophin), limb-girdle muscular dystrophies (sarcoglycans), and congenital muscular dystrophy with dysmyelination (merosin). Alpha-dystroglycan is affected in Walker-Warburg syndrome, Fukuyama syndrome, and muscle-eye-brain disease through deficient glycosylation of its protein structure, resulting in loss of its extracellular binding to laminin (43; 33).

Further analysis of patients with a mutation in the POMGnT1 gene revealed that those with mutations near the 5’ terminus had relatively more severe brain symptoms such as hydrocephalus, demonstrating that deficiency of this gene can cause either a Walker-Warburg syndrome or muscle-eye-brain disease phenotype dependent on the type of mutation (60).

Sequence homology to POMT1 led to the identification of POMT2 as another gene associated with Walker-Warburg syndrome (65). Mutations in FKRP (Fukutin-related protein) cause a phenotypic spectrum ranging between congenital muscular dystrophy with normal intellect and Walker-Warburg syndrome. A large intragenic mutation of the LARGE gene was found in a patient with Walker-Warburg syndrome (64).

Mutations causing Walker-Warburg syndrome, Fukuyama syndrome, and muscle-eye-brain disease only affect alpha-dystroglycan glycosylation, not the dystroglycan protein core itself. A possible exception is a patient with a heterozygous deletion that involved the dystroglycan gene; the patient had multiple cerebral white matter lesions and mild myopathy with normal alpha-dystroglycan staining (23).

Light has been shed on the cause of the neuronal migration disorder in the 3 disorders. In a mouse model with targeted disruption of brain alpha-dystroglycan, a neuronal migration disorder strikingly similar to Walker-Warburg syndrome was reproduced. Brain-selective deletion of dystroglycan caused Walker-Warburg-like lesions in these mice, including disarray of cerebral cortical layering, probably caused by discontinuities in the pial surface basal lamina (glia limitans) that normally anchors the radial glia to ensure well-patterned positioning of cortical neurons at the final stage of neuronal migration (42). Impaired neuronal migration is also involved in the pathogenesis of pontocerebellar hypoplasia as seen in Walker-Warburg syndrome and its allied disorders. Cerebellar external granule cells, pontine neurons, and neurons of the olivary complex all derive from the rhombic lips, a germinal structure that flanks the fourth ventricle. Neurons generated in this structure migrate to the cerebellar cortex, ventral pons, and ventral medulla oblongata by so-called tangential migration. Stalled migration in the ventral pons was observed in mice with mutations to the LARGE gene (49), a homologue of the human LARGE gene that was associated with Walker-Warburg syndrome.

Epidemiology

The prevalence of Walker-Warburg syndrome is not known. Reports indicate that it is present at least in Europe, in the western hemisphere, and in Japan. For Ashkenazi Jews, the carrier frequency for FKTN mutations was reported as 1:79 (29). Of the allied disorders, Fukuyama-type cerebro-muscular dystrophy appears to be exceptional outside Japan, whereas muscle-eye-brain disease has been reported outside Finland, the first country from which it was reported (63).

Prevention

Autosomal recessive inheritance entails a 25% risk of subsequent siblings to affected families. Antenatal diagnosis in the first half of pregnancy has been documented making use of ultrasonography (16; 24). Hydrocephalus and encephalocele are candidates for early detection. Identification of genes associated with Walker-Warburg syndrome now allows precise antenatal diagnosis in about 50% of affected families.

In a pregnancy at risk (earlier sibling affected), a very early manifestation of a Walker-Warburg phenotype was achieved at 11 weeks of gestation using a high-resolution transvaginal ultrasound probe (01). Chorionic villous sampling confirmed a mutation in the Fukutin gene, the well-known Ashkenazi founder mutation c.1167_1168insA.

Differential diagnosis

The differential diagnosis of Walker-Warburg syndrome includes Fukuyama-type cerebromuscular dystrophy (the predominant muscular dystrophy in Japan) and muscle-eye-brain disease (first described in Finland). Since the discovery of the common role of alpha-dystroglycan in these disorders, the discovery of dystroglycan deficiency has taken prominence in diagnosis, and these disorders have become closely linked. Moreover, mutations in the same genes may cause overlapping features of these 3 disorders, together known as dystroglycanopathies.

Fukuyama-type cerebromuscular dystrophy, an autosomal recessive cerebral dysplasia with congenital muscular dystrophy, is the most prevalent form of congenital muscular dystrophy in Japan and has essential features in common with Walker-Warburg syndrome (31; 77). Similarly, there is overlap of findings in the 3 critical organs with the muscle-eye-brain disease, an autosomal recessive disorder prevalent in Finland (56). Clasped (hyperabducted) thumbs may occasionally be seen in Walker-Warburg syndrome. These, in combination with hydrocephalus in males, may suggest X-linked hydrocephalus. Meckel-Gruber syndrome has to be considered for differential diagnosis in the case of occipital encephalocele.

A combination of cerebral abnormalities and congenital muscular dystrophy is found in merosin-negative congenital muscular dystrophy, which features a severe cerebral myelin deficiency without gross functional deficiencies. Partial lissencephaly may be found in merosin-negative congenital muscular dystrophy, especially in the occipital regions (63). Overlapping morphological features exist between Walker-Warburg syndrome and the genetically related muscle-eye-brain disease and Fukuyama syndrome. In all 3, hypoplasia of the pons and hypoplasia/dysplasia of the cerebellum can be seen on MRI besides congenital muscular dystrophy with absent or reduced alpha-dystroglycan staining of biopsied muscle.

A mutation in the LARGE gene, the human homologue of a muscular dystrophy gene in a mouse, was found in a patient with profound mental deficiency and congenital muscular dystrophy, featuring mild pachygyria and a flat ventral pons on MRI (38). This gene has also become associated with classic Walker-Warburg syndrome (64).

The following table lists the main similarities and differences between Walker-Warburg syndrome, Fukuyama-type cerebro-muscular dystrophy, and muscle-eye-brain disease.

Table 2. Walker-Warburg Syndrome, Fukuyama-Type Cerebro-Muscular Dystrophy, and Muscle-Eye-Brain Disease

WWS: Walker-Warburg syndrome
F-CMD: Fukuyama-type cerebro-muscular dystrophy
MEB: muscle-eye-brain disease

A spectrum overlapping these features as revealed by MRI and including alpha-dystroglycan-deficient congenital muscular dystrophy is seen in mutations in POMT1, POMT2, POMGnT1, FKTN, FKRP, and LARGE (15). Between 2010 and 2012, heterozygous mutations in COL4A1 and homozygous mutations in ISPD were added to the causes of Walker-Warburg syndrome. A phenocopy with bifronto-parietal polymicrogyria and cerebellar hypoplasia is caused by homozygous mutations of GPR56.

Diagnostic workup

Examination of the eyes should include an active search for anterior chamber abnormalities in combination with symptoms of retinal dysplasia. MRI is the method of choice to delineate all aspects of the cerebral abnormalities (56; 45; 03; 02; 62; 26; 63; 07; 30). Besides the detection of cobblestone lissencephaly, midbrain-hindbrain involvement helps to differentiate cobblestone complex from classic lissencephaly. These findings include hypoplasia or dysplasia of the vermis and cerebellar hemispheres, small cerebellar cysts, midbrain hypoplasia or dysplasia, pontomedullary kink, and pontine midline cleft (30).

Severe deficiency of alpha-dystroglycan in biopsied muscle offers good evidence for an O-glycosylation defect and warrants a search for mutations in POMT1, POMT2, POMGnT1, FKTN, FKRP, LARGE, and ISPD. Recessive mutations in LAMB1 may cause cobblestone lissencephaly with minimal involvement of eye and muscle. Two genes not related to alpha-dystroglycan glycosylation, GPR56 and COL4A1, should be added in the case of negative results. COL4A1 mutations causing Walker-Warburg syndrome are dominant mutations and unlikely to be found in consanguineous families. Recessive GPR56 mutations cause frontoparietal polymicrogyria and cerebellar hypoplasia, symptoms that overlap with Walker-Warburg syndrome (06).

Mutations in B3GALNT2 cause muscular dystrophy and structural brain defects similar to muscle-eye-brain disease (59).

Management

No specific therapy is available. Seizures may be treated with standard antiepileptic drugs. Hydrocephalus requires surgical treatment by ventriculoperitoneal shunt, and encephaloceles may require excision of extracranial neural and meningeal tissue and closure of the defect, at least the cutaneous surface.

Special considerations

Pregnancy

Prenatal detection has been accomplished before 20 weeks’ gestation. Given the variability of expression, it is not certain that detection is possible in all cases.

Prenatal diagnosis by gene analysis is possible.