Structural organization, complete genomic sequences and mutational analyses of the Fukuyama-type congenital muscular dystrophy gene, fukutin.

Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive severe muscular dystrophy in combination with cerebral cortical dysplasia. Previously, we identified the gene responsible for FCMD, termed fukutin, through positional cloning. In this study, we have sequenced 131892 bp of genomic DNA in the region of the fukutin gene on chromosome 9q31 and obtained its complete genomic structure. The fukutin genomic sequence spans approximately 100 kb and is organized into 10 exons (41-6067 bp) and nine introns (1841-21460 bp). Using these sequence data, we have identified three novel fukutin mutations in FCMD patients. We have also located a putative TATA box in the flanking 5' region and identified numerous alternatively spliced fukutin mRNA transcripts. Analysis of expressed sequence tag clusters within the region revealed two novel genes upstream of the fukutin gene. These data provide fundamental information to support detailed genetic and functional analyses of the fukutin gene.

A case of Walker-Warburg syndrome.

Walker-Warburg syndrome (WWS) is an autosomal recessive disorder characterized by type II lissencephaly, cerebellar and retinal anomalies, and congenital muscular dystrophy. We report a female diagnosed with WWS based on clinical criteria. This patient was found to have fetal hydrocephalus on ultrasonography at 29 weeks of gestation, and exhibited severe hypotonia, ocular malformations, and hydrocephalus at birth. MRI revealed type II lissencephaly, hydrocephalus, and other severe brain malformations. Genetic analysis was performed to distinguish WWS from severe Fukuyama-type congenital muscular dystrophy (FCMD), which has numerous findings in common. This revealed no expression of the founder haplotype or single-stranded conformation polymorphism (SSCP) abnormalities. Since the life expectancy of patients with FCMD is longer, differential diagnosis should be performed precisely.

Neuronal expression of the fukutin gene.

Fukuyama-type congenital muscular dystrophy (FCMD), a relatively common autosomal recessive disorder in Japan, is characterized by severe congenital muscular dystrophy in combination with cortical dysgenesis (polymicrogyria). The gene responsible for FCMD encodes a novel protein, fukutin, which is likely to be an extracellular protein. Pathological study of brain tissue from FCMD fetuses revealed frequent breaks in the glia limitans and basement membrane complex. Disruption of the basal lamina in FCMD muscle was also seen. Thus, structural alteration of the basal lamina appears to play a key role in the pathophysiology of FCMD. To investigate the role of fukutin in brain anomalies, we examined fukutin mRNA expression in the human brain. Northern blot and RT-PCR analysis revealed that the fukutin gene is expressed at similar levels in fetal and adult brain, whereas its expression is much reduced in FCMD brains. Tissue in situ hybridization analysis revealed fukutin mRNA expression in the migrating neurons, including Cajar-Retzius cells and adult cortical neurons, as well as in hippocampal pyramidal cells and cerebellar Purkinje cells. However, we observed no expression in the glia limitans, the subpial astrocytes (which contribute to basement membrane formation) or other glial cells. In the FCMD brain, neurons in regions with no dysplasia showed fair expression, whereas transcripts were nearly undetectable in the overmigrated dysplastic region. These observations suggest that fukutin function may influence neuronal migration itself rather than formation of the basement membrane. Furthermore, differences in mRNA levels among neurons in early developmental stages may partially differentiate normal and abnormal regions.

Walker-Warburg syndrome is genetically distinct from Fukuyama type congenital muscular dystrophy.

A female patient who fulfilled the diagnostic criteria of Walker-Warburg syndrome had muscle biopsy finding of muscular dystrophy. There was normal expression of merosin (laminin alpha2 chain) and dystrophin and only slightly reduced dystrophin-associated glycoprotein expression. On genetic analysis, she had no specific haplotype, the common mutation of 3kb insertion, or point mutations in the Fukuyama-type congenital muscular dystrophy gene, suggesting that the two diseases are not genetically identical.

Haplotype-phenotype correlation in Fukuyama congenital muscular dystrophy.

In typical Fukuyama congenital muscular dystrophy (FCMD), peak motor function is usually only unassisted sitting or sliding on the buttocks, though a few patients are able to walk at some point. However, a few patients have a severe phenotype and never acquire head control. In addition, it is clinically difficult to differentiate this severe FCMD from Walker-Warburg syndrome (WWS) or from muscle-eye-brain disease (MEBD). In order to establish a genotype-phenotype correlation, we performed haplotype analysis using microsatellite markers closest to the FCMD gene (FCMD) in 56 Japanese FCMD families, including 35 families whose children were diagnosed as FCMD with the typical phenotype, 12 families with a mild phenotype, and 9 families with a severe phenotype. Of the 12 propositi with the mild phenotype, 8 could walk and the other 4 could stand with support; 10 cases were homozygous for the ancestral founder (A-F) haplotype whereas the other 2 were heterozygous for the haplotype. In the 9 severe cases, who had never acquired head control or the ability to sit without support, 3 had progressive hydrocephalus, 2 required a shunt operation, and 7 had ophthalmological abnormalities. Haplotype analysis showed that 8 of the 9 cases of the severe phenotype are heterozygous for the A-F haplotype, and the other one homozygous for the haplotype. We confirmed that at least one chromosome in each of the 56 FCMD patients has the A-F haplotype. The rate of heterozygosity for the A-F haplotypes was significantly higher in severe cases than in typical or mild cases (P < 0.005). Severe FCMD patients appeared to be compound heterozygotes for the founder mutation and another mutation. Thus, the present study yielded molecular genetic evidence of a broad clinical spectrum in FCMD.

The Fukuyama congenital muscular dystrophy story.

Fukuyama congenital muscular dystrophy is one of the most common autosomal recessive disorders in the Japanese population, characterized by congenital muscular dystrophy in combination with cortical dysgenesis (micropolygyria). Recently, we have identified the gene responsible for fukuyama congenital muscular dystrophy on 9q31, which encodes a novel 461-amino-acid protein termed fukutin. Most Fukuyama congenital muscular dystrophy-bearing chromosomes are derived from a single ancestral founder (87%), and a 3 kb-retrotransposal insertion into the 3' untranslated region of this gene was found to be a founder mutation. Two independent point mutations causing premature termination confirmed that that this gene is responsible for Fukuyama congenital muscular dystrophy. Fukuyama congenital muscular dystrophy is the first human disease to be caused by an ancient retrotransposal integration. Fukutin contains an amino-terminal signal sequence, which together with results from transfection experiments suggests that it is an extracellular protein. Discovery of the Fukuyama congenital muscular dystrophy gene represents an important step toward greater understanding of the pathogenesis of muscular dystrophies and also of normal brain development.

Novel mutations and genotype-phenotype relationships in 107 families with Fukuyama-type congenital muscular dystrophy (FCMD).

Fukuyama-type congenital muscular dystrophy (FCMD), one of the most common autosomal recessive disorders in the Japanese population, is characterized by congenital muscular dystrophy in combination with cortical dysgenesis (micropolygyria). Recently, we identified, on chromosome 9q31, the gene responsible for FCMD, which encodes a novel 461 amino acid protein which we have termed fukutin. Most FCMD-bearing chromosomes examined to date (87%) have been derived from a single ancestral founder, whose mutation consisted of a 3 kb retrotransposal insertion in the 3' non-coding region of the fukutin gene. FCMD is the first human disease known to be caused primarily by an ancient retrotransposal integration. We under-took a systematic analysis of the FCMD gene in 107 unrelated patients, and identified four novel non-founder mutations in five of them: one missense, one nonsense, one L1 insertion and a 1 bp insertion. The frequency of severe phenotypes, including Walker-Walberg syndrome-like manifestations such as hydrocephalus and microphthalmia, was significantly higher among probands who were compound heterozygotes carrying a point mutation on one allele and the founder mutation on the other, than it was among probands who were homozygous for the 3 kb retrotransposon. Remarkably, we detected no FCMD patients with non-founder (point) mutations on both alleles of the gene, and suggest that such cases might be embryonic-lethal. This could explain why few FCMD cases are reported in non-Japanese populations. Our results provided strong evidence that loss of function of fukutin is the major cause of FCMD, and appeared to shed some light on the mechanism responsible for the broad clinical spectrum seen in this disease.

Congenital muscular dystrophy with central and peripheral nervous system involvement in a Belgian patient.

We report a patient with congenital muscular dystrophy (CMD), developmental brain defects, and peripheral neuropathy. Marked hypotonia and plagiocephaly were noted at birth. Failure to thrive, generalized muscle weakness and wasting, absent deep tendon reflexes, partial seizures, and secondary microcephaly developed. Brain MRI showed a large area of cortical dysplasia, a thin but complete corpus callosum, and diffuse ventriculomegaly. Nerve conduction velocities were slow and creatine kinase levels only mildly elevated. Muscle biopsy showed dystrophic features with normal merosin, sarcoglycan, and dystrophin immunostaining. The Japanese Fukuyama CMD founder mutation was not detected. This is the first report of a patient with merosin-positive CMD, cobblestone lissencephaly, and demyelinating peripheral neuropathy.

An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy.

Fukuyama-type congenital muscular dystrophy (FCMD), one of the most common autosomal recessive disorders in Japan (incidence is 0.7-1.2 per 10,000 births), is characterized by congenital muscular dystrophy associated with brain malformation (micropolygria) due to a defect in the migration of neurons. We previously mapped the FCMD gene to a region of less than 100 kilobases which included the marker locus D9S2107 on chromosome 9q31. We have also described a haplotype that is shared by more than 80% of FCMD chromosomes, indicating that most chromosomes bearing the FCMD mutation could be derived from a single ancestor. Here we report that there is a retrotransposal insertion of tandemly repeated sequences within this candidate-gene interval in all FCMD chromosomes carrying the founder haplotype (87%). The inserted sequence is about 3 kilobases long and is located in the 3' untranslated region of a gene encoding a new 461-amino-acid protein. This gene is expressed in various tissues in normal individuals, but not in FCMD patients who carry the insertion. Two independent point mutations confirm that mutation of this gene is responsible for FCMD. The predicted protein, which we term fukutin, contains an amino-terminal signal sequence, which together with results from transfection experiments suggests that fukutin is a secreted protein. To our knowledge, FCMD is the first human disease to be caused by an ancient retrotransposal integration.

Prenatal diagnosis of Fukuyama type congenital muscular dystrophy in eight Japanese families by haplotype analysis using new markers closest to the gene.

We conducted prenatal diagnosis by haplotype analysis, using newly developed microsatellite markers, in eight Fukuyama type congenital muscular dystrophy (FCMD) families. In addition to six new families, two previously reported families were reexamined by haplotype analysis including detection of an ancestral founder haplotype (138-183-301) for 3 microsatellite markers closest to the FCMD gene, designated D9S2105-D9S2107-D9S172, the distances of which from the FCMD gene are presumed to be approximately 140, approximately 20, and approximately 280 kb, respectively. Five fetuses from five families were diagnosed as nonaffected, and were subsequently confirmed to be healthy. Three fetuses of the other three families were diagnosed as having a high probability of being affected by FCMD. In the prenatal diagnosis conducted for these eight families, the ancestral founder allele was observed in 13 of 16 (81%) FCMD-bearing chromosomes. Detection of the ancestral haplotype facilitated achieving accurate prenatal diagnosis of FCMD. The brains of all three fetuses prenatally diagnosed as FCMD-affected showed the initial stage of cortical dysplasia, strong evidence of FCMD.

Molecular genetic evidence of clinical heterogeneity in Fukuyama-type congenital muscular dystrophy.

Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive severe muscular dystrophy associated with brain malformation. The gene responsible for FCMD was mapped to chromosome 9q31, a region in which convincing evidence of strong linkage disequilibrium between FCMD and mfd220 (D9S306) was recently found. FCMD is also characterized clinically by a peak motor function which, at best, allows patients to sit unassisted or slide on the buttocks. However, a small fraction of patients acquire the capacity to walk unassisted. Whether such ambulant cases belong to the FCMD spectrum or to a different disease entity has been a topic of considerable debate. We performed linkage analysis for ten families with ambulant cases using DNA markers flanking the FCMD locus. The mfd220 locus yielded a significant lod score of 3.09 for ambulant FCMD. We also found evidence for linkage disequilibrium between ambulant FCMD and mfd220. We further conducted haplotype analysis in FCMD siblings with different phenotypes, one of whom was ambulant while the other was not. The results indicate that the FCMD siblings share exactly the same haplotype at nine marker loci spanning 23.3 cM surrounding the FCMD locus. On the basis of these results, we conclude that, genetically, ambulant cases are, in fact, part of the FCMD spectrum.

Polymorphism analysis of Fukuyama type congenital muscular dystrophy (FCMD) siblings with different phenotypes.

Peak motor function in Fukuyama type congenital muscular dystrophy (FCMD) is generally considered to be no better than sitting without help or sliding on the buttocks. There are a few patients who acquire the ability to stand and a small fraction of our total congenital muscular dystrophy (CMD) population are able to walk at some point. These ambulant cases may reflect a broad spectrum of motor disabilities in the category of FCMD, or may represent another CMD entity, which closely resembles but is distinct from FCMD. Since the localization of the FCMD gene to chromosome 9q3 1 by Toda et al. in 1993 and 1994, polymorphism analysis of this disease has become possible. We describe correlations between clinical features and genetic analysis using microsatellite markers flanking the FCMD locus in two FCMD families each having two affected children with distinctly different motor abilities. The results demonstrate that two sets of FCMD siblings share exactly the same haplotype at nine marker loci spanning 23.3 cM, surrounding the FCMD locus. Our results provide genetic confirmation that some FCMD cases may acquire the ability to walk.