Long-term efficacy of nusinersen and its evaluation in adolescent and adult patients with spinal muscular atrophy types 1 and 2.

BACKGROUNDS: The efficacy of nusinersen and its evaluation in patients with spinal muscular atrophy (SMA) has been established in clinical trials only for pediatric patients, not for adolescent and adult patients who developed SMA in infancy or early childhood. We report a long-term follow-up in adolescent and adult patients with SMA types 1 and 2. METHODS: Nusinersen-treated patients with SMA types 1 and 2 between 2017 and 2022 were retrospectively reviewed. We compared baseline motor function tests with those after the final treatment. Physical and occupational therapists performed Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND), Hammersmith Functional Motor Scale-Expanded (HFMSE), and Revised Upper Limb Module (RULM). The Landau and Galant reflexes were not performed in CHOP-INTEND. Meaningful improvement was defined as CHOP-INTEND, 4; HFSME, 3; and RULM, 2. RESULTS: Seven patients with SMA (type 1, 1; type 2, 6) with a median age of 23 (range, 12-40)years were treated with nusinersen for 3.55 (1.78-4.53)years. Improvement was detected in CHOP-INTEND (pre, 5 [0-31]; post, 21 [0-39]; difference, 5 [0-26]; p = 0.100) without significance, although not in HFMSE (pre, 0 [0-3]; post, 0 [0-5]; difference, 0 [0-2]; p = 0.346) and RULM (pre, 1 [0-20]; post, 3 [0-21]; difference, 1 [0-2]; p = 0.089). Owing to prolonged treatment intervals with the COVID-19 pandemic, RULM worsened in two patients. CONCLUSION: Nusinersen was effective in long-term follow-up. Only CHOP-INTEND showed meaningful improvement. The interval between doses of nusinersen should not be prolonged even with the COVID-19 pandemic.

Early immunological responses to the mRNA SARS-CoV-2 vaccine in patients with neuromuscular disorders.

Backgrounds: Intramuscular injection of the SARS-CoV-2 vaccine has raised concerns about its use in patients with neuromuscular disorders (NMDs). We evaluated the response of patients with NMDs to the BNT162b2 vaccine. Methods: Healthy subjects, patients with spinal muscular atrophy (SMA), and patients with Duchenne muscular dystrophy (DMD) were included. All participants received two BNT162b2 doses. SARS-CoV-2 antibody titers at baseline and 2 weeks after each vaccination were compared between groups. Residual muscle volume was evaluated in NMDs group. A questionnaire documented adverse reactions. Results: Eleven patients with NMDs (9 with SMA, 2 with DMD; 7 males; aged 32.7 ± 19.3 years) and 346 healthy subjects (60 males, aged 40.0 ± 12.4 years) were included. Antibody titers (U/mL) were similar between groups (baseline: <0.40 vs. <0.40, first vaccination, 145 ± 258 vs. 103 ± 1192, and second vaccination, 1528 ± 1265 vs. 1429 ± 944; p = 1.000, 0.909, and 0.736, respectively). A negative correlation was found between antibody titers and residual muscle volume but was not significant (Mercuri scale, r = -0.429, p = 0.249; fat infiltration rate, r = -0.194, p = 0.618). The adverse reactions were comparable between groups. Conclusion: The BNT162b2 vaccine is safe and effective in patients with NMDs.

Administration of nusinersen via paramedian approach for spinal muscular atrophy.

OBJECTIVE: To assess the success rate, procedure time, and adverse events of intrathecal administration of nusinersen via the paramedian approach in adolescents and adults with spinal muscular atrophy (SMA) associated with scoliosis. METHODS: Seven patients with genetically confirmed SMA (age, 12-40 years) were included. Intrathecal administration of nusinersen was performed via paramedian approach using fluoroscopy after determination of the largest interlaminal foramen among L2-L3, L3-L4, or L4-L5 by three-dimensional computed tomography. We measured the times for preparation, positioning, and puncture, and the total time of stay. Adverse effects of intrathecal administration were noted. RESULTS: Intrathecal administration via paramedian approach was successful for all 38 opportunities. The median total time of stay was 44.0 min (interquartile range, 37.3-50.0 min). The total time of stay was significantly longer in patients with SMA type 1 than in those with SMA type 2, but was not different according to the severity of scoliosis. Adverse effects included oxygen supplementation, headache, and back pain. Sedation was correlated with oxygen supplementation and headache. CONCLUSIONS: Intrathecal administration of nusinersen via the paramedian approach had the advantages of a high success rate and short procedure time with fewer adverse events in SMA patients associated with scoliosis.

Mutations in HADHB, which encodes the ß-subunit of mitochondrial trifunctional protein, cause infantile onset hypoparathyroidism and peripheral polyneuropathy.

Mitochondrial trifunctional protein (MTP) is a hetero-octamer composed of four α- and four ß-subunits that catalyzes the final three steps of mitochondrial ß-oxidation of long chain fatty acids. HADHA and HADHB encode the α-subunit and the ß-subunit of MTP, respectively. To date, only two cases with MTP deficiency have been reported to be associated with hypoparathyroidism and peripheral polyneuropathy. Here, we report on two siblings with autosomal recessive infantile onset hypoparathyroidism, peripheral polyneuropathy, and rhabdomyolysis. Sequence analysis of HADHA and HADHB in both siblings shows that they were homozygous for a mutation in exon 14 of HADHB (c.1175C>T, [p.A392V]) and the parents were heterozygous for the mutation. Biochemical analysis revealed that the patients had MTP deficiency. Structural analysis indicated that the A392V mutation identified in this study and the N389D mutation previously reported to be associated with hypoparathyroidism are both located near the active site of MTP and affect the conformation of the ß-subunit. Thus, the present patients are the second and third cases of MTP deficiency associated with missense HADHB mutation and infantile onset hypoparathyroidism. Since MTP deficiency is a treatable disease, MTP deficiency should be considered when patients have hypoparathyroidism as the initial presenting feature in infancy.

Clinical characterization and identification of duplication breakpoints in a Japanese family with Xq28 duplication syndrome including MECP2.

Xq28 duplication syndrome including MECP2 is a neurodevelopmental disorder characterized by axial hypotonia at infancy, severe intellectual disability, developmental delay, mild characteristic facial appearance, epilepsy, regression, and recurrent infections in males. We identified a Japanese family of Xq28 duplications, in which the patients presented with cerebellar ataxia, severe constipation, and small feet, in addition to the common clinical features. The 488-kb duplication spanned from L1CAM to EMD and contained 17 genes, two pseudo genes, and three microRNA-coding genes. FISH and nucleotide sequence analyses demonstrated that the duplication was tandem and in a forward orientation, and the duplication breakpoints were located in AluSc at the EMD side, with a 32-bp deletion, and LTR50 at the L1CAM side, with "tc" and "gc" microhomologies at the duplication breakpoints, respectively. The duplicated segment was completely segregated from the grandmother to the patients. These results suggest that the duplication was generated by fork-stalling and template-switching at the AluSc and LTR50 sites. This is the first report to determine the size and nucleotide sequences of the duplicated segments at Xq28 of three generations of a family and provides the genotype-phenotype correlation of the patients harboring the specific duplicated segment.

SIL1, a causative cochaperone gene of Marinesco-Söjgren syndrome, plays an essential role in establishing the architecture of the developing cerebral cortex.

Marinesco-Sjögren syndrome (MSS) is a rare autosomal recessively inherited disorder with mental retardation (MR). Recently, mutations in the SIL1 gene, encoding a co-chaperone which regulates the chaperone HSPA5, were identified as a major cause of MSS. We here examined the pathophysiological significance of SIL1 mutations in abnormal corticogenesis of MSS. SIL1-silencing caused neuronal migration delay during corticogenesis ex vivo. While RNAi-resistant SIL1 rescued the defects, three MSS-causing SIL1 mutants tested did not. These mutants had lower affinities to HSPA5 in vitro, and SIL1-HSPA5 interaction as well as HSPA5 function was found to be crucial for neuronal migration ex vivo. Furthermore time-lapse imaging revealed morphological disorganization associated with abnormal migration of SIL1-deficient neurons. These results suggest that the mutations prevent SIL1 from interacting with and regulating HSPA5, leading to abnormal neuronal morphology and migration. Consistent with this, when SIL1 was silenced in cortical neurons in one hemisphere, axonal growth in the contralateral hemisphere was delayed. Taken together, abnormal neuronal migration and interhemispheric axon development may contribute to MR in MSS.

Phenotypic spectrum of COL4A1 mutations: porencephaly to schizencephaly.

OBJECTIVE: Recently, COL4A1 mutations have been reported in porencephaly and other cerebral vascular diseases, often associated with ocular, renal, and muscular features. In this study, we aimed to clarify the phenotypic spectrum and incidence of COL4A1 mutations. METHODS: We screened for COL4A1 mutations in 61 patients with porencephaly and 10 patients with schizencephaly, which may be similarly caused by disturbed vascular supply leading to cerebral degeneration, but can be distinguished depending on time of insult. RESULTS: COL4A1 mutations were identified in 15 patients (21%, 10 mutations in porencephaly and 5 mutations in schizencephaly), who showed a variety of associated findings, including intracranial calcification, focal cortical dysplasia, pontocerebellar atrophy, ocular abnormalities, myopathy, elevated serum creatine kinase levels, and hemolytic anemia. Mutations include 10 missense, a nonsense, a frameshift, and 3 splice site mutations. Five mutations were confirmed as de novo events. One mutation was cosegregated with familial porencephaly, and 2 mutations were inherited from asymptomatic parents. Aberrant splicing was demonstrated by reverse transcriptase polymerase chain reaction analyses in 2 patients with splice site mutations. INTERPRETATION: Our study first confirmed that COL4A1 mutations are associated with schizencephaly and hemolytic anemia. Based on the finding that COL4A1 mutations were frequent in patients with porencephaly and schizencephaly, genetic testing for COL4A1 should be considered for children with these conditions.

[Survival analysis for patients with severe motor and intellectual disabilities following tracheotomy].

To investigate the survival rate and causes of death in patients with severe motor and intellectual disabilities (SMIDs) that necessitated tracheotomy, we retrospectively analyzed 90 patients who underwent tracheotomy between 1990 and 2009. Indications for tracheotomy in these patients were upper airway obstruction (44 patients), recurrent aspiration pneumonia (28 patients), retained secretions (23 patients), prolonged mechanical ventilation (18 patients), chronic respiratory failure (9 patients), central respiratory failure (5 patients), and gastroesophageal reflux (8 patients). Most of the patients underwent tracheotomy at the age of 0-5 years or 10-19 years. As of April 1, 2010, 28 patients had died. The survival rate was 0.91 at 1 year, 0.74 at 5 years, 0.59 at 10 years, 0.54 at 15 years, and 0.40 at 19 years after tracheotomy. Massive tracheal bleeding due to development of tracheo-innominate artery fistulas occurred in 5 patients, and 4 of them died. They were thirteen years of age or older when they underwent tracheotomy, and developed fistulas after 2 weeks or later. In contrast, 7 patients at high risk for fistula formation, including those that had developed severe tracheomalacia associated with granulation or warning hemorrhages, underwent preventive resection of the innominate artery, and all of them had survived. It is important to regularly evaluate patients with SMIDs who have undergone tracheotomy by using bronchofiberscopy to identify risk factors for tracheoinnominate artery fistulas, a preventable cause of death.

Clinical and genomic characterization of siblings with a distal duplication of chromosome 9q (9q34.1-qter).

We report herein on two female siblings exhibiting mild intellectual disability, hypotonia in infancy, postnatal growth retardation, characteristic appearance of the face, fingers, and toes. Their healthy mother had a translocation between 9q34.1 and the 13pter. FISH and array CGH analysis demonstrated that the two children had an additional 8.5 Mb segment of the 9q34.1-qter at 13pter. The clinical features of the present cases were similar to those of previously reported 9q34 duplication cases; however, the present cases did not exhibit other abnormal behaviors, such as autistic features or attention deficit disorders, those are reportedly associated with 9q34 duplications. A 3.0 Mb region (9q34.1-q34.3) within 9q34 duplication in our patients are overlapped with duplication region of previously reported cases and is proposed to be critical for the presentation of several phenotypes associated with 9q34 duplications.

A wide spectrum of clinical and brain MRI findings in patients with SLC19A3 mutations.

BACKGROUND: SLC19A3 (solute carrier family 19, member 3) is a thiamin transporter with 12 transmembrane domains. Homozygous or compound heterozygous mutations in SLC19A3 cause two distinct clinical phenotypes, biotin-responsive basal ganglia disease and Wernicke's-like encephalopathy. Biotin and/or thiamin are effective therapies for both diseases. METHODS: We conducted on the detailed clinical, brain MRI and molecular genetic analysis of four Japanese patients in a Japanese pedigree who presented with epileptic spasms in early infancy, severe psychomotor retardation, and characteristic brain MRI findings of progressive brain atrophy and bilateral thalami and basal ganglia lesions. RESULTS: Genome-wide linkage analysis revealed a disease locus at chromosome 2q35-37, which enabled identification of the causative mutation in the gene SLC19A3. A pathogenic homozygous mutation (c.958G > C, [p.E320Q]) in SLC19A3 was identified in all four patients and their parents were heterozygous for the mutation. Administration of a high dose of biotin for one year improved neither the neurological symptoms nor the brain MRI findings in one patient. CONCLUSION: Our cases broaden the phenotypic spectrum of disorders associated with SLC19A3 mutations and highlight the potential benefit of biotin and/or thiamin treatments and the need to assess the clinical efficacy of these treatments.

[Intervention to reduce the difficulty in kanji copying related to the visuo-spatial dysfunction in patients with Williams syndrome].

Williams syndrome (WS) is known for its uneven cognitive abilities. Visuo-spatial cognition is more disturbed, whereas cognition of colors or shapes of objects is relatively preserved. This tendency is attributed to the greater deficits of functions in the dorsalpathway compared to those in the ventral pathway in the visual system. When patients with WS are asked to copy two dimensional figures, they often show difficulty in locating components of the figures to make global shapes. Similar findings are observed in copying kanji, Japanese semantic characters. Patients with WS often can copy only the components of a kanji character but can not locate the components properly and fail to make appropriate global shapes of the character even though they can read it. In order to ameliorate this difficulty, we rely on the preserved cognitive function, that is, the cognition of colors. Four participants with WS, who have difficulty copying two dimensional figures, joined the study. A kanji written on a square which is divided into four sections was used as a copy model. Each divided part of the square was colored red, green, yellow and blue, respectively. A similarly colored square was given to copy the kanji. This method was successful and made it easier for the participants with WS to copy a given kanji as it became easier to realize where to locate each component. Intervention using similar squares without colors or only with gray scaled back ground did not work. In addition, kanji with differently colored components was also presented to one of the participants as a model but it was not copied successfully. The similar difficulty in copying two dimensional objects and kanji was observed in a patient with Kabuki syndrome and the same interventional method with colored squares was effective for copying kanji.

Low incidence of limb-girdle muscular dystrophy type 2C revealed by a mutation study in Japanese patients clinically diagnosed with DMD.

BACKGROUND: Limb-girdle muscular dystrophy type 2C (LGMD2C) is an autosomal recessive muscle dystrophy that resembles Duchenne muscular dystrophy (DMD). Although DMD is known to affect one in every 3500 males regardless of race, a widespread founder mutation causing LGMD2C has been described in North Africa. However, the incidence of LGMD2C in Japanese has been unknown because the genetic background remains uncharacterized in many patients clinically diagnosed with DMD. METHODS: We enrolled 324 patients referred to the Kobe University Hospital with suspected DMD. Mutations in the dystrophin or the SGCG genes were analyzed using not only genomic DNA but also cDNA. RESULTS: In 322 of the 324 patients, responsible mutations in the dystrophin were successfully revealed, confirming DMD diagnosis. The remaining two patients had normal dystrophin expression but absence of gamma-sarcoglycan in skeletal muscle. Mutation analysis of the SGCG gene revealed homozygous deletion of exon 6 in one patient, while the other had a novel single nucleotide insertion in exon 7 in one allele and deletion of exon 6 in the other allele. These mutations created a stop codon that led to a gamma-sarcoglycan deficiency, and we therefore diagnosed these two patients as having LGMD2C. Thus, the relative incidence of LGMD2C among Japanese DMD-like patients can be calculated as 1 in 161 patients suspected to have DMD (2 of 324 patients = 0.6%). Taking into consideration the DMD incidence for the overall population (1/3,500 males), the incidence of LGMD2C can be estimated as 1 per 560,000 or 1.8 per million. CONCLUSIONS: To the best of our knowledge, this is the first study to demonstrate a low incidence of LGMD2C in the Japanese population.

Development of visuospatial ability and kanji copying in Williams Syndrome.

Williams syndrome is known for uneven cognitive abilities. Visuospatial difficulties such as a failure in constructing objects are considered to be characteristic and may influence the copying of Japanese semantic characters, kanji. In contrast to previous investigations, which were done mainly cross-sectionally, this study focused on the developmental aspects of the symptoms, to get a better view of the mechanism. Developmental changes in visuospatial abilities (including copying two-dimensional figures, three-dimensional figures, and kanji) in four boys with Williams syndrome, ages 4 to 11 years, were longitudinally observed for 6-9 years. The Benton's three-dimensional block construction tests and the Yerkes test were also performed. Some of the results were compared with those of mental age-matched children. The observation revealed improvements in performance for copying two-dimensional figures, as well as for copying kanji, in the Williams syndrome participants; however, copying three-dimensional figures tended to remain difficult, especially if in a transparent view. Obtaining three-dimensional information using pictorial cues seemed to remain difficult for the Williams syndrome participants even at the later stage of study monitoring. This difficulty might be correlated with the core dysfunction of Williams syndrome.

[Various central nervous system involvements in dystrophinopathy: clinical and genetic considerations].

Duchenne and Becker muscular dystrophies, generically called dystrophinopathy, are caused by mutations of the dystrophin gene. It is not surprising that mutations of the dystrophin gene cause various neurological symptoms, since dystrophin protein is found in the brain tissue as well as in the muscle fiber cell membrane. However, few studies have reported on the frequency of central nervous complications other than mental retardation. Also, the relationship between the types of abnormal dystrophin gene and central nervous symptoms remains to be revealed. Medical records of 200 patients with dystrophinopathy from 167 extended families who had visited our institution during the past 15 years were reviewed to elucidate the frequency of central nervous complications. Fifty-four (27%) had mental retardation (an intelligence quotient less than 70), 15 (7.5%) had autism, 12 (6%) had epilepsy. 8 (4%) had febrile convulsion. 131 of these patients also underwent genetic testing. All patients with central nervous symptoms except one pair of siblings had some form of genetic deficiency or duplication distal to exon 44. Central nervous symptoms other than mental retardation are also common in patients with dystrophinopathy. These central nervous complications may be associated with mutations in the isoforms derived from exon 44 to 79.

Age-related changes in BDNF protein levels in human serum: differences between autism cases and normal controls.

Accumulating evidence suggests the possible association between the concentrations of serum brain-derived neurotrophic factor (BDNF) and psychiatric disease with impaired brain development. Yet the reasons remain unclear. We therefore investigated the characteristics of serum BDNF as well as its age-related changes in healthy controls in comparison to autism cases. BDNF was gradually released from platelets at 4 degrees C, reached a maximal concentration after around 24 h, and remained stable until 42 h. At room temperature, BDNF was found to be immediately degraded. Circadian changes, but not seasonal changes, were found in serum levels of BDNF existing as the mature form with a molecular mass of 14 kDa. In healthy controls, the serum BDNF concentration increased over the first several years, then slightly decreased after reaching the adult level. There were no sex differences between males and females. In the autism cases, mean levels were significantly lower in children 0-9 years old compared to teenagers or adults, or to age-matched healthy controls, indicating a delayed BDNF increase with development. In a separate study of adult rats, a circadian change in serum BDNF was found to be similar to that in the cortex, indicating a possible association with cortical functions.

[Tracheostomy for home-care patients with severe motor and intellectual disabilities].

We discuss here the indication and complications of tracheostomy performed in 57 home-care pateints with severe motor and intellectual disabilities (SMID) during the past 13 years at our hospital. Thirty-five cases underwent tracheostomy following emergency endotracheal intubation for acute respiratory failure. Recently, the number of cases without preceding endotracheal intubation have increased. Many patients underwent tracheostomy at the age of 1 to 4 years and 10 to 14 years. The quality of life (QOL) of almost all the patients without preceding intubation markedly improved, as well as that of their families, and they were able to return to home. The most decisive reason for tracheostomy was secretions and recurrent aspiration pneumonia in 8 patients, gastroesophageal reflux in 4 and upper airway obstructions in 3. Several complications of tracheostomy were observed: tracheal granulations in 9 patients, tracheal malacia in 8, and tracheoinnominate artery fistula in 5. Among 8 patients with tracheal malacia, bleeding from the tracheoinnominate artery fistula occurred in 3. In 7 patients, self-made long tracheostomy tubes were necessary for the initial management of the tracheal malacia or tracheal granulations. Subsequently, made-to-order long tracheostomy tubes were used in three of these patients. In 12 patients, improved endotracheal T-tube with the tip sealed on the vocal cord side was used to prevent aspiration. Home-care SMID patients with respiratory disturbance require tracheostomy timely performed, followed by careful observation to prevent postoperative complications.

Evolution of seizures and electroencephalographical findings in 23 cases of deletion type Angelman syndrome.

Angelman syndrome (AS) is a genetic disorder with characteristic clinical and EEG findings. We report here the results of long-term follow-up studies on the epileptic seizures and EEG findings of 23 cases of deletion type AS confirmed by FISH analysis, including seven cases previously reported by Matsumoto et al. in 1992. The age at last follow-up in 23 patients was from 1 to 37 years of age (average: 18.0 years), with 10 patients (43.5%) in their 20s, and five over 30. Epileptic seizures were seen in all patients, and the age at seizure onset ranged from 3 to 50 months (average: 21.7 months). Status epilepticus was seen in 11 patients (47.8%). The percentages of cases seizure-free for more than 3 years were 25% (4/16) at 10 years of age, 70% (7/10) at 20, and 80% (4/5) at 30. The EEG findings were classified into six patterns according to the previous report: N (no spike, including focal slow waves), HVS (diffuse high-voltage slow bursts with or without spikes), F (focal spikes or multifocal spikes), S (diffuse spike and waves), C (continuous diffuse spike and waves), Hy (hypsarrythmia or hypsarrhythmia like waves). Hy was noted at ages 0-2 years in two cases. C was observed from the ages 2 to 15 years, being most frequently noted at 3-6 years of age, and it was never seen after 16 years of age. S was observed from ages 1 to 21 years. F was seen from 2 to 21 years of age, and most frequently during the ages of 2-7 years. HVS was seen from 0 years, and still remained after the age of 20. After 22 years of age, all patients showed N pattern including focal slow waves. One of the two patients who had bilateral frontal dominant delta slow waves in their 30s, had a recent seizure. Even if the spikes disappear with age, when bi-frontal focal slow waves remain, seizures may occur even in patients over 30.

[Clinical symptoms of the Rett syndrome patients with MECP2 gene abnormalities].

Mutations in a gene on the X-chromosome encoding methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome. We examined clinical symptoms of 27 patients with Rett syndrome (aged 2 to 37 years), diagnosed by the criteria of the Rett Syndrome Diagnostic Criteria Work Group. having MECP2 gene mutations. Two novel MECP2 mutations, 119 del AG resulting in amino acid frame-shift 40fs43X and C to G transversion resulting in amino acid change of F157L, were found. All patients had the most important symptoms of this syndrome, including loss of acquired purposeful hand skills followed by stereotyped hand movements. Two patients had mild perinatal abnormalities. Nine showed psychomotor delay or hypotonia before 6 months. Five patients over 4 years old did not have microcephaly. Speech was preserved in five patients. According to the criteria, 18 cases were diagnosed as Rett syndrome variants. Sixteen out of 26 patients over 3 years old were able to walk (61.5%), and 22 had epilepsy (84.6%). Mutations of the 5 patients without microcephaly were R133C, P225R, R255X, R306C and 376fs386X, whereas those of the 5 variants with preserved speech were 34fs123X, R133C, R255X and R270. Common T158M mutation was detected in 4 patients, R255X in 7 and R270X in 4. Patients with the same mutations showed different phenotypes. Patients with R133C and R306C presented a mild phenotype without microcephaly. Of the proposed diagnostic criteria, the following three may not be essential: apparently normal prenatal and perinatal period, apparently normal psychomotor development through the first 6 months, and deceleration of head growth between 5 months and 4 years.

Reduced aquaporin 4 expression in the muscle plasma membrane of patients with Duchenne muscular dystrophy.

BACKGROUND: In Duchenne muscular dystrophy (DMD), previous freeze-fracture electron microscopic studies demonstrated that muscle plasma membrane contained markedly decreased numbers of orthogonal arrays. Recent investigations showed that orthogonal arrays were composed of aquaporin 4 (AQP4) molecules, a member of the water channel protein family. OBJECTIVES: To study whether the immunostainability of anti-AQP4 antibody is reduced in muscles of patients with DMD and whether, if it is reduced, the problem is at the genomic DNA, messenger RNA (mRNA), or posttranscriptional level. PATIENTS AND METHODS: We analyzed the muscle and blood samples from 6 boys with DMD, 6 normal control subjects, and 12 patients with neuromuscular diseases at the protein, genomic DNA, and mRNA levels. At the protein level, immunohistochemical staining and immunoblot analysis were performed. At the genomic DNA and mRNA levels, the polymerase chain reaction and reverse transcription polymerase chain reaction, respectively, were used to screen for mutations in the AQP4 gene. RESULTS: At the protein level, immunohistochemical staining of our originally generated rabbit anti-AQP4 antibody in DMD muscles was markedly reduced. Most of the DMD myofibers showed negative staining with sporadic partially positive fibers at their myofiber surface, whereas the control muscles displayed continuous myofiber surface staining. Immunoblot analysis showed that the content of AQP4 in DMD muscles was remarkably decreased. Amplification of leukocyte genomic DNA by polymerase chain reaction showed that the patients with DMD had genomic DNA of the AQP4 molecule. Quantitative reverse transcription polymerase chain reaction demonstrated that DMD skeletal muscles contained markedly decreased AQP4 mRNA compared with controls. CONCLUSION: The reduction in AQP4 in DMD muscles results from decreased levels of AQP4 mRNA in DMD myofibers.