Kenzo Hamano

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 (micro-polygria) 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 (refs 2–4). 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.

A nonsense mutation of the MASS1 gene in a family with febrile and afebrile seizures

A naturally occurring mutation of the mass1 (monogenic audiogenic seizure‐susceptible) gene recently has been reported in the Frings mouse strain, which is prone to audiogenic seizures. The human orthologous gene, MASS1, was mapped to chromosome 5q14, for which we previously have reported significant evidence of linkage to febrile seizures (FEB4). We screened for MASS1 mutations in individuals from 48 families with familial febrile seizures and found 25 DNA alterations. None of nine missense polymorphic alleles was significantly associated with febrile seizures; however, a nonsense mutation (S2652X) causing a deletion of the C‐terminal 126 amino acid residues was identified in one family with febrile and afebrile seizures. Our results suggest that a loss‐of‐function mutation in MASS1 might be responsible for the seizure phenotypes, though it is not likely that MASS1 contributed to the cause of febrile seizures in most of our families.

A quantitative analysis of rat central nervous system myelination using the immunohistochemical method for MBP

The temporal order of the myelination of the nervous pathways in 0-42-day-old Wistar rats was quantitatively analyzed using immunohistochemistry with anti-myelin basic protein (MBP) antibody. Immunohistochemistry was performed on paraffin-embedded tissue according to the standard ABC technique. For the objective evaluation of myelination, we converted the level stained with the immunohistochemical method into continuous numbers of 0-256 giving the intensity of myelination, using an image analyzing system. We analyzed nine nervous pathways: corpus callosum, optic tract, internal capsule, spinal tract of trigeminal nerve, inferior cerebellar peduncle, cerebellar white matter, pyramidal tract, medial longitudinal fasciculus, and cuneate fasciculus. The onset of the myelination of the spinal tract of the trigeminal nerve, inferior cerebellar peduncle, medial longitudinal fasciculus and cuneate fasciculus was day 7 (postnatal). That of the corpus callosum, optic tract, internal capsule and cerebellar white matter was day 14, and that of the pyramidal tract was day 21. The time required to reach the level of myelination of day 42 was day 21 for the spinal tract of the trigeminal nerve and the inferior cerebellar peduncle, day 28 for the internal capsule, day 35 for the corpus callosum, optic tract, cerebellar white matter and pyramidal tract, and day 42 for the medial longitudinal fasciculus. Our method using immunohistochemistry with anti-MBP antibody provided a highly sensitive and objective criterion for judging precisely the time and the progress of myelination in each nervous pathway and compare one nervous pathway with another.

A quantitative study of the progress of myelination in the rat central nervous system, using the immunohistochemical method for proteolipid protein

The temporal changes in intensity of myelination of the nervous pathways in 0 to 42-day-old Wistar rats were quantitatively analyzed by immunohistochemistry with anti-proteolipid protein and compared with that obtained by immunohistochemistry with anti-myelin basic protein. Immunohistochemistry was performed on paraffin-embedded tissue according to the standard ABC technique. Intensity of myelination was examined by an image analyzing system. We analyzed nine nervous pathways: corpus callosum, optic tract, internal capsule, spinal tract of the trigeminal nerve, inferior cerebellar peduncle, cerebellar white matter, pyramidal tract, medial longitudinal fasciculus, and cuneate fasciculus. The presence of immunoreactive fibers for proteolipid protein (PLP) in the spinal tract of the trigeminal nerve, medial longitudinal fasciculus and cuneate fasciculus was noted on postnatal day 0. Those of the corpus callosum, inferior cerebellar peduncle, cerebellar white matter, pyramidal tract and internal capsule were noted on day 7, and that of optic tract on day 14. The time required to reach the intensity of myelination of day 42 was day 14 for the cuneate fasciculus, day 21 for the spinal tract of the trigeminal nerve, inferior cerebellar peduncle and medial longitudinal fasciculus, day 28 for the optic and pyramidal tracts, day 35 for the corpus callosum and day 42 for the internal capsule and cerebellar white matter. The appearance of immunoreactive fibers for PLP was usually earlier than that for myelin basic protein (MBP) and the pattern of difference between PLP and MBP can be classified into three groups: (1) their time of appearance and progress are almost the same, as in the optic tract; (2) the appearance and progress of PLP occurs earlier than those of MBP, as in the pyramidal tract; (3) the appearance of PLP occurs earlier than that of MBP, but their progress is the same. Our findings revealed that the time of appearance and progress of myelination as measured by PLP are different among the nervous pathways, and that there is also a difference between PLP and MBP. This difference between PLP and MBP may indicate a functional difference between them.

Volumetric quantification of brain development using MRI

Abstract We devised a three-dimensional method for estimation of cerebral development and myelination which measures cerebral volume using MRI. Accuracy of the system was estimated using cadaver brains. The mean percentage error in the calculated volumes compared with the real volumes was 2.33 %, range 0.00–5.33 %. We applied the method to the volume of both cerebral hemispheres (CH), basal ganglia, thalamus and internal capsule (BT), and myelinated white matter (WM) in 44 neurologically normal individuals (4 months to 28 years of age), 13 patients with spastic motor disturbances (2–25 years of age), and 9 patients with athetotic motor disturbances (2–23 years of age). In the neurologically normal cases, the volumes of CH, BT and WM increased with age; the volume of MW more slowly than that of CH. In cases with spastic motor disturbances, the volumes of CH, BT and WM were between –1.4 and 3.5 SD, –1.0 and –3.5 SD, and 0.0 and –5.2 SD respectively, of those of neurologically-normal cases. On the other hand, 7 of the 9 cases with athetotic motor disturbances were within 2 SD of the volume of CH in neurologically normal cases. Our method for direct measurement of cerebral volume based on serial MRI should be useful for the accurate assessment of brain development and quantitative analysis of delayed myelination.

Molecular Genetics of Febrile Seizures

Summary: Febrile seizures are the most common form of convulsion, occurring in 2–5% of infants in Europe and North America and in 6–9% in Japan. In large families, the febrile seizure (FS) susceptibility trait is inherited by the autosomal dominant pattern with reduced penetrance. Two putative FS loci, FEB1 (chromosome 8q13‐q21) and FEB2 (chromosome 19p13.3) have been mapped. A clinical subset of FS, termed generalized epilepsy with febrile seizures plus (GEFS+), was reported. In GEFS+ families, a mutation in the voltage‐gated sodium channel β1 subunit gene (SCN1B) at chromosome 19q13.1 and two mutations of the same α1 subunit gene (SCN1A) at chromosome 2q24 were identified. These loci are linked to febrile convulsions in large families. We conducted a genome‐wide linkage search for FS in one large family with subsequent linkage confirmation in 39 nuclear families using nonparametric allele‐sharing methods, and found a new FS susceptibility locus, FEB4 (chromosome 5q14‐q15). In contrast to the FEB1, FEB2, and GEFS+ genetic loci, linkage to FEB4 was suggested in nuclear FS families, indicating that FEB4 may be the most common linkage locus in FS families.

Failure to find causal mutations in the GABAA-receptor γ2 subunit (GABRG2) gene in Japanese febrile seizure patients

Recently, mutations in the GABA(A)-receptor gamma2 subunit (GABRG2) gene were identified in two families with generalized epilepsy with febrile seizures plus (GEFS+) and two families with childhood absence epilepsy (CAE) and febrile seizures (FS). We tested the hypothesis that genetic variations in the GABRG2 gene confer susceptibility to FS in the Japanese population. We performed a systematic search for mutations in 94 unrelated Japanese patients with FS and detected six variants (-158C>T, 315C>T, 588T>C, IVS5-55C>T, IVS7+20G>A, and IVS7-141T>A). No non-synonymous mutation was detected. We genotyped three exonic polymorphisms and performed a case control study and a transmission disequilibrium test using 55 independent complete trios with FS and 106 control subjects. None of these polymorphic alleles were significantly associated with FS. Our results indicate that genomic variations of GABRG2 are not likely to be substantially involved in the etiology of FS in the Japanese population.

Aicardi syndrome accompanied by auditory disturbance and multiple brain tumors

We described a 5-month-old girl with Aicardi syndrome accompanied by auditory disturbance and multiple brain tumors. She was admitted to our hospital because she suffered from intractable flexor spasms. Physical examination revealed craniofacial asymmetry, left auricular deformity, scoliosis, and remarkable hypotonia with psychomotor retardation. Abnormal ophthalmological findings included chorioretinopathy with pale and round-shaped peripapillary lacunae, and there was modified hypsarrhythmia in her EEG. MRI revealed multiple brain tumors in the 3rd and the lateral ventricles which are considered to be choroid plexus papilloma with agenesis of the corpus callosum. ACTH therapy was administered because of the intractable seizures. After ACTH therapy, the thresholds of waves I and V were much improved. The interpeak latency of waves I-V of the left ear and the peak latency of wave I of the right ear had been lengthened. Acoustic reflex with contralateral stimulation showed no response in the left ear. These findings indicate that the auditory system is also involved in the Aicardi syndrome and that ACTH is effective for its dysfunction.

Failure to find evidence for association between voltage-gated sodium channel gene SCN2A variants and febrile seizures in humans

The voltage-gated sodium channel type II alpha polypeptide gene (SCN2A) R188W mutation with channel dysfunction was recently identified in a patient with febrile and afebrile seizures. A possible association between SCN2A R19K polymorphism and febrile seizures (FS) associated with afebrile seizures including generalized epilepsy with febrile seizures plus (GEFS+) was also noted. We attempted to identify the R188W mutation and confirm association of the R19K polymorphism in 93 Japanese patients with FS, 35 Japanese patients with FS associated with afebrile seizures including GEFS+, and 100 control subjects. The R188W mutation was not found. There were no significant differences in genotype or allele frequencies of the R19K polymorphism between groups. Our study failed to provide evidence supporting a causal relation between the SCN2A mutation/polymorphism and FS or FS associated with afebrile seizures including GEFS+ in the Japanese population.

Volumetric quantification of brain volume in children using sequential CT scans

SummaryWe devised a three dimensional method for the accurate measurement of brain volume and applied it to 32 neurologically normal children, 7 children with only mental retardation and 15 children with both mental retardation and motor disturbance. In the group of neurologically normal children, the total brain volume increased from 723 cm3 to 1407 cm3 in order of age. The correlation ratio between the total brain volume and age was significant (P<0.0001). The values of the total brain volume and the developmental curve were similar to those of the total brain weight of normal children previously reported. The combined volume of the cerebellum, the midbrain, the pons and the medulla also increased from 76 cm3 to 200 cm3 in a manner similar to that of the total brain. The correlation between total brain volume and head circumference was significant (P<0.0001). In the group of children with mental retardation, the total brain volume was relatively smaller than that of neurologically normal children. In the group of the children with mental retardation and motor disturbance, 10 out of 15 cases showed values below — 2 SD of those of neurologically normal children. The values of the total brain volume were each less than — 3 SD in 3 cases whose head circumferences were each more than — 3 SD. Our method for the direct measurement of brain volume based on serial CT scans may be useful for the accurate examination of brain development.