OBJECTIVE: To carry out carrier screening for Spinal muscular atrophy (SMA) in reproductive-aged individuals from Dongguan region and determine the carrier frequency of SMN1 gene mutations. METHODS: Reproductive-aged individuals who underwent SMN1 genetic screening at the Dongguan Maternal and Child Health Care Hospital from March 2020 to August 2022 were selected as the study subjects. Deletions of exon 7 and 8 (E7/E8) of the SMN1 gene were detected by real-time fluorescence quantitative PCR (qPCR), and prenatal diagnosis was provided for carrier couples by multiple ligation-dependent probe amplification (MLPA). RESULTS: Among the 35 145 subjects, 635 were found to be carriers of SMN1 E7 deletion (586 with heterozygous E7/E8 deletion, 2 with heterozygous E7 deletion and homozygous E8 deletion, and 47 with sole heterozygous E7 deletion). The carrier frequency was 1.81% (635/35 145), with 1.59% (29/1 821) in males and 1.82% (606/33 324) in females. There was no significant difference between the two genders (χ² = 0.497, P = 0.481). A 29-year-old woman was found to harbor homozygous deletion of SMN1 E7/E8, and was verified to have a SMN1∶SMN2 ratio of [0∶4], none of her three family members with a [0∶4] genotype had clinical symptoms. Eleven carrier couples had accepted prenatal diagnosis, and one fetus was found to have a [0∶4] genotype, and the pregnancy was terminated. CONCLUSION This study has determined the SMA carrier frequency in Dongguan region for the first time and provided prenatal diagnosis for carrier couples. The data can provide a reference for genetic counseling and prenatal diagnosis, which has important clinical implications for the prevention and control of birth defects associated with SMA.
Humans ; Child ; Pregnancy ; Male ; Female ; Adult ; Homozygote ; Sequence Deletion ; Prenatal Diagnosis ; Genetic Testing ; Muscular Atrophy, Spinal/genetics* ; Survival of Motor Neuron 1 Protein/genetics* ; Genetic Carrier Screening

Objective: To analyze the clinical and genetic characteristics of pediatric patients with dual genetic diagnoses (DGD). Methods: Clinical and genetic data of pediatric patients with DGD from January 2021 to February 2022 in Peking University First Hospital were collected and analyzed retrospectively. Results: Among the 9 children, 6 were boys and 3 were girls. The age of last visit or follow-up was 5.0 (2.7,6.8) years. The main clinical manifestations included motor retardation, mental retardation, multiple malformations, and skeletal deformity. Cases 1-4 were all all boys, showed myopathic gait, poor running and jumping, and significantly increased level of serum creatine kinase. Disease-causing variations in Duchenne muscular dystrophy (DMD) gene were confirmed by genetic testing. The 4 children were diagnosed with DMD or Becker muscular dystrophy combined with a second genetic disease, including hypertrophic osteoarthropathy, spinal muscular atrophy, fragile X syndrome, and cerebral cavernous malformations type 3, respectively. Cases 5-9 were clinically and genetically diagnosed as COL9A1 gene-related multiple epiphyseal dysplasia type 6 combined with NF1 gene-related neurofibromatosis type 1, COL6A3 gene-related Bethlem myopathy with WNT1 gene-related osteogenesis imperfecta type XV, Turner syndrome (45, X0/46, XX chimera) with TH gene-related Segawa syndrome, Chromosome 22q11.2 microduplication syndrome with DYNC1H1 gene-related autosomal dominant lower extremity-predominant spinal muscular atrophy-1, and ANKRD11 gene-related KBG syndrome combined with IRF2BPL gene-related neurodevelopmental disorder with regression, abnormal movement, language loss and epilepsy. DMD was the most common, and there were 6 autosomal dominant diseases caused by de novo heterozygous pathogenic variations. Conclusions: Pediatric patients with coexistence of double genetic diagnoses show complex phenotypes. When the clinical manifestations and progression are not fully consistent with the diagnosed rare genetic disease, a second rare genetic disease should be considered, and autosomal dominant diseases caused by de novo heterozygous pathogenic variation should be paid attention to. Trio-based whole-exome sequencing combining a variety of molecular genetic tests would be helpful for precise diagnosis.
Humans ; Abnormalities, Multiple ; Retrospective Studies ; Intellectual Disability/genetics* ; Bone Diseases, Developmental/complications* ; Tooth Abnormalities/complications* ; Facies ; Muscular Dystrophy, Duchenne/complications* ; Muscular Atrophy, Spinal/complications* ; Carrier Proteins ; Nuclear Proteins

Humans ; Amyotrophic Lateral Sclerosis/drug therapy* ; Uric Acid

OBJECTIVE: To explore the molecular pathological mechanism of liver metabolic disorder in severe spinal muscular atrophy (SMA). METHODS: The transgenic mice with type Ⅰ SMA (Smn^-/- SMN2^0tg/2tg) and littermate control mice (Smn^+/- SMN2^0tg/2tg) were observed for milk suckling behavior and body weight changes after birth. The mice with type Ⅰ SMA mice were given an intraperitoneal injection of 20% glucose solution or saline (15 μL/12 h), and their survival time was recorded. GO enrichment analysis was performed using the RNA-Seq data of the liver of type Ⅰ SMA and littermate control mice, and the results were verified using quantitative real-time PCR. Bisulfite sequencing was performed to examine CpG island methylation level in Fasn gene promoter region in the liver of the neonatal mice. RESULTS: The neonatal mice with type Ⅰ SMA showed normal milk suckling behavior but had lower body weight than the littermate control mice on the second day after birth. Intraperitoneal injection of glucose solution every 12 h significantly improved the median survival time of type Ⅰ SMA mice from 9±1.3 to 11± 1.5 days (P < 0.05). Analysis of the RNA-Seq data of the liver showed that the expression of the target genes of PPARα related to lipid metabolism and mitochondrial β oxidation were down-regulated in the liver of type Ⅰ SMA mice. Type Ⅰ SMA mice had higher methylation level of the Fasn promoter region in the liver than the littermate control mice (76.44% vs 58.67%). In primary cultures of hepatocytes from type Ⅰ SMA mice, treatment with 5-AzaC significantly up-regulated the expressions of the genes related to lipid metabolism by over 1 fold (P < 0.01). CONCLUSION Type Ⅰ SMA mice have liver metabolic disorder, and the down-regulation of the target genes of PPARα related to lipid and glucose metabolism due to persistent DNA methylation contributes to the progression of SMA.
Mice ; Animals ; PPAR alpha ; Liver Diseases ; Muscular Atrophy, Spinal/genetics* ; Mice, Transgenic ; Body Weight ; Glucose

Background: Amyotrophic lateral sclerosis is one of the neurodegenerative disorders with very limited treatment options owing to its progressive course and diverse pathophysiology. Majority of patients succumb to death within three to five years after the onset of symptoms, mostly due to respiratory failure. This study aimed to determine the efficacy and safety of ultra-high dose methylcobalamin versus placebo among patients with early-stage amyotrophic lateral sclerosis in terms of slowing down functional decline. Methods: MEDLINE, CENTRAL, and Google Scholar databases were searched from inception up to September 23, 2023. The impact of treatments was measured by risk ratios with 95% confidence interval. The overall certainty of the evidence was evaluated using GRADE. Results: No significant difference was detected for the outcome median change in the ALSFRS-R score for the whole cohort. Post-hoc analyses showed that ultra-high-dose methylcobalamin decreased ALSFRS-R scores (p=0.003 for 50 mg and p=0.01 for all methylcobalamin groups) in a dose-responsive manner. Mean difference was 1.97 in favor of methylcobalamin (95% CI, 0.44- 3.50; P = .01). Conclusion Ultra-high dose methylcobalamin can reduce ALSFRS-R scores of patients in its early stage but the scarcity of clinical trials makes it difficult to support a robust conclusion. Ultra-high dose methylcobalamin therapy remains to be investigational.
Amyotrophic Lateral Sclerosis ; Systematic Review

OBJECTIVE: To assess the value of single sperm sequencing in preimplantation genetic testing for monogenic disease (PGT-M). METHODS: A Chinese couple with two children whom had died of Spinal muscular atrophy (SMA) and attended the Jiangxi Provincial Maternal and Child Health Care Hospital in June 2020 was selected as the subject. Eleven single sperm samples were isolated by mechanical immobilization and subjected to whole genome amplification. Real-time PCR and Sanger sequencing were used to detect the SMN1 variants in the single sperm samples. Genomic DNA of the wife, her parents and the husband, as well as one single sperm sample harboring the SMN1 variant and two single sperm samples without the variant were used for the linkage analysis. Targeted capture and high-throughput sequencing were carried out to test 100 single nucleotide polymorphisms distributed within 2 Mb up- and downstream the variant site. The haplotypes linked with the SMN1 variants were determined by linkage analysis. Blastocyst embryos were harvested after fertilizing by intracytoplasmic sperm injection. Cells from the trophoblasts of each embryo were biopsied and subjected to whole genome amplification and targeted capture and high-throughput sequencing to determine their carrier status. Chromosomal aneuploidy of wild-type embryos was excluded. An euploid embryo of high quality was transferred. Amniotic fluid sample was taken at 18 weeks of gestation to confirm the status of the fetus. RESULTS: Genetic testing showed that the couple both had deletion of exons 7 ~ 8 of the SMN1 gene. The wife has inherited the deletion from her father, while the husband was de novo. The haplotypes of the husband were successfully constructed by single sperm sequencing. Preimplantation genetic testing has indicated that 5 embryos had harbored the heterozygous variant, 4 embryos were of the wild type, among which 3 were euploid. Prenatal diagnosis during the second trimester of pregnancy has confirmed that the fetus did not carry the deletion. CONCLUSION By single sperm sequencing and PGT-M, the birth of further affected child has been successfully avoided.
Humans ; Pregnancy ; Female ; Child ; Male ; Preimplantation Diagnosis ; East Asian People ; Semen ; Genetic Testing ; Muscular Atrophy, Spinal/genetics* ; Aneuploidy ; Blastocyst/pathology* ; High-Throughput Nucleotide Sequencing ; Spermatozoa

BACKGROUND: Juvenile amyotrophic lateral sclerosis (JALS) is an uncommon form of amyotrophic lateral sclerosis whose age at onset (AAO) is defined as prior to 25 years. FUS mutations are the most common cause of JALS. SPTLC1 was recently identified as a disease-causative gene for JALS, which has rarely been reported in Asian populations. Little is known regarding the difference in clinical features between JALS patients carrying FUS and SPTLC1 mutations. This study aimed to screen mutations in JALS patients and to compare the clinical features between JALS patients with FUS and SPTLC1 mutations. METHODS: Sixteen JALS patients were enrolled, including three newly recruited patients between July 2015 and August 2018 from the Second Affiliated Hospital, Zhejiang University School of Medicine. Mutations were screened by whole-exome sequencing. In addition, clinical features such as AAO, onset site and disease duration were extracted and compared between JALS patients carrying FUS and SPTLC1 mutations through a literature review. RESULTS: A novel and de novo SPTLC1 mutation (c.58G>A, p.A20T) was identified in a sporadic patient. Among 16 JALS patients, 7/16 carried FUS mutations and 5/16 carried respective SPTLC1 , SETX , NEFH , DCTN1 , and TARDBP mutations. Compared with FUS mutation patients, those with SPTLC1 mutations had an earlier AAO (7.9 ± 4.6 years vs. 18.1 ± 3.9 years, P  < 0.01), much longer disease duration (512.0 [416.7-607.3] months vs. 33.4 [21.6-45.1] months, P  < 0.01), and no onset of bulbar. CONCLUSION Our findings expand the genetic and phenotypic spectrum of JALS and help to better understand the genotype-phenotype correlation of JALS.
Humans ; Amyotrophic Lateral Sclerosis/genetics* ; DNA Helicases/genetics* ; Genetic Association Studies ; Multifunctional Enzymes/genetics* ; Mutation/genetics* ; RNA Helicases/genetics* ; RNA-Binding Protein FUS/genetics* ; Serine C-Palmitoyltransferase/genetics* ; Child, Preschool ; Child ; Adolescent ; Young Adult

Humans ; Amyotrophic Lateral Sclerosis/drug therapy* ; Benzofurans/therapeutic use* ; Double-Blind Method ; Treatment Outcome

Spinal muscular atrophy (SMA) is the most common inherited lethal disease in children. Confirmatory diagnosis is based on molecular genetic testing of survival motor neuron (SMN) genes. We aimed to describe the phenotypic presentation of Filipino infants and children with SMA based on the copy number analysis of SMN genes. Medical records of 17 Filipino children were reviewed from January 2017 to December 2019. De-identified clinical data fulfilled the diagnostic criteria defined by the International SMA Consortium. Among Filipino children, the predominant SMA type by copy number was type I having two copies of SMN2 gene. The clinical severity based on symptom onset and highest functional motor capacity attained correlated with SMN2 copy number congruent with existing data. A significant time lag between symptom onset to confirmation of genetic diagnosis was noted. Nine out of the 17 (52%) children did not have a family history of the disease, raising the possibility of mutation carriers in these families since the incidence of de novo mutations in literature is about 2%. These data offered the first epidemiological pattern of genetically confirmed SMA among Filipino children; provided additional information for genetic counselling; and an avenue to consider pre-symptomatic newborn screening and carrier testing that would change proactive measures and opportunities for therapy. These measures unavoidably will decrease the incidence and prevalence of disease in the future.
Muscular Atrophy, Spinal

A decline in the activities of oxidative phosphorylation (OXPHOS) complexes has been consistently reported in amyotrophic lateral sclerosis (ALS) patients and animal models of ALS, although the underlying molecular mechanisms are still elusive. Here, we report that receptor expression enhancing protein 1 (REEP1) acts as an important regulator of complex IV assembly, which is pivotal to preserving motor neurons in SOD1^G93A mice. We found the expression of REEP1 was greatly reduced in transgenic SOD1^G93A mice with ALS. Moreover, forced expression of REEP1 in the spinal cord extended the lifespan, decelerated symptom progression, and improved the motor performance of SOD1^G93A mice. The neuromuscular synaptic loss, gliosis, and even motor neuron loss in SOD1^G93A mice were alleviated by increased REEP1 through augmentation of mitochondrial function. Mechanistically, REEP1 associates with NDUFA4, and plays an important role in preserving the integrity of mitochondrial complex IV. Our findings offer insights into the pathogenic mechanism of REEP1 deficiency in neurodegenerative diseases and suggest a new therapeutic target for ALS.
Mice ; Animals ; Amyotrophic Lateral Sclerosis/metabolism* ; Superoxide Dismutase-1/metabolism* ; Superoxide Dismutase/metabolism* ; Mice, Transgenic ; Spinal Cord/pathology* ; Mitochondria/physiology* ; Disease Models, Animal