Pseudoexon activation by deep intronic variation in GNE myopathy with thrombocytopenia.

INTRODUCTION/AIMS: GNE myopathy is a rare autosomal recessive disorder caused by pathogenic variants in the GNE gene, which is essential for the sialic acid biosynthesis pathway. Although over 300 GNE variants have been reported, some patients remain undiagnosed with monoallelic pathogenic variants. This study aims to analyze the entire GNE genomic region to identify novel pathogenic variants. METHODS: Patients with clinically compatible GNE myopathy and monoallelic pathogenic variants in the GNE gene were enrolled. The other GNE pathogenic variant was verified using comprehensive methods including exon 2 quantitative polymerase chain reaction and nanopore long-read single-molecule sequencing (LRS). RESULTS: A deep intronic GNE variant, c.862+870C>T, was identified in nine patients from eight unrelated families. This variant generates a cryptic splice site, resulting in the activation of a novel pseudoexon between exons 5 and 6. It results in the insertion of an extra 146 nucleotides into the messengerRNA (mRNA), which is predicted to result in a truncated humanGNE1(hGNE1) protein. Peanut agglutinin（PNA） lectin staining of muscle tissues showed reduced sialylation of mucin O-glycans on sarcolemmal glycoproteins. Notably, a third of patients with the c.862+870C>T variant exhibited thrombocytopenia. A common core haplotype harboring the deep intronic GNE variant was found in all these patients. DISCUSSION: The transcript with pseudoexon activation potentially affects sialic acid biosynthesis via nonsense-mediated mRNA decay, or resulting in a truncated hGNE1 protein, which interferes with normal enzyme function. LRS is expected to be more frequently incorporated in genetic analysis given its efficacy in detecting hard-to-find pathogenic variants.

Diagnosis of Challenging Spinal Muscular Atrophy Cases with Long-Read Sequencing.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder primarily caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene. This study assesses the diagnostic potential of long-read sequencing (LRS) in three patients with SMA. For Patient 1, who has a heterozygous SMN1 deletion, LRS unveiled a missense mutation in SMN1 exon 5. In Patient 2, an Alu/Alu-mediated rearrangement covering the SMN1 promoter and exon 1 was identified through a blend of multiplex ligation-dependent probe amplification, LRS, and PCR across the breakpoint. The third patient, born to a consanguineous family, bore four copies of hybrid SMN genes. LRS determined the genomic structures, indicating two distinct hybrids of SMN2 exon 7 and SMN1 exon 8. However, a discrepancy was found between the SMN1/SMN2 ratio interpretations by LRS (0:2) and multiplex ligation-dependent probe amplification (0:4), which suggested a limitation of LRS in SMA diagnosis. In conclusion, this newly adapted long PCR-based third-generation sequencing introduces an additional avenue for SMA diagnosis.

Long-term impact of nusinersen on motor and electrophysiological outcomes in adolescent and adult spinal muscular atrophy: insights from a multicenter retrospective study.

BACKGROUND: 5q spinal muscular atrophy (SMA) is a progressive autosomal recessive motor neuron disease. OBJECTIVE: We aimed to assess the effects of nusinersen on motor function and electrophysiological parameters in adolescent and adult patients with 5q SMA. METHODS: Patients with genetically confirmed 5q SMA were eligible for inclusion, and clinical data were collected at baseline (V1), 63 days (V4), 180 days (V5), and 300 days (V6). The efficacy of nusinersen was monitored by encompassing clinical assessments, including the Revised Upper Limb Module (RULM), Hammersmith Functional Motor Scale Expanded (HFMSE), 6-Minute Walk Test (6MWT), and percent-predicted Forced Vital Capacity in sitting position (FVC%) and Compound Muscle Action Potential (CMAP) amplitude. The patients were divided into "sitter" and "walker" subgroups according to motor function status. RESULTS: 54 patients were screened, divided into "sitter" (N = 22) and "walker" (N = 32), with the mean age at baseline of 27.03 years (range 13-53 years). The HFMSE in the walker subgroup increased significantly from baseline to V4 (mean change +2.32-point, P = 0.004), V5 (+3.09, P = 0.004) and V6 (+4.21, P = 0.005). The patients in both the sitter and walker subgroup had no significant changes in mean RULM between V1 and the following time points. Significant increases in CMAP amplitudes were observed in both upper and lower limbs after treatment. Also, patients with RULM ≥ 36 points showed significant CMAP improvements. Our analysis predicted that patients with CMAP amplitudes of trapezius ≥ 1.76 mV were more likely to achieve significant motor function improvements. CONCLUSIONS: Nusinersen effectively improves motor function and electrophysiological data in adolescent and adult patients with SMA. This is the first report on the CMAP amplitude changes in the trapezius after treatment in patients with SMA. The CMAP values effectively compensate for the ceiling effect observed in the RULM, suggesting that CMAP could serve as an additional biomarker for evaluating treatment efficacy.

Diagnostic delay in late-onset Pompe disease among Chinese patients: A retrospective study.

Surveys and retrospective studies have revealed considerable delays in diagnosing late-onset Pompe disease (LOPD) in China, where the contributing factors remain poorly represented. Our study analyzed the diagnostic journey of 34 LOPD patients seen at our neuromuscular clinic from 2005 to 2022. We defined diagnostic delay as the time from the onset of the first relevant symptoms and laboratory findings suggestive of LOPD to the eventual diagnosis, and we constructed a correlation matrix to assess relationships among these variables. The cohort consisted of 34 patients with an equal male-to-female ratio, and the mean age at diagnosis was 27.68 ± 10.03 years. We found the median diagnostic delay to be 5 years, with a range of 0.3 to 20 years, with 97.1% having been misdiagnosed previously, most commonly with "Type II Respiratory insufficiency" (36.7%). Notably, patients at earlier onset (mean age, 18.19 years vs. 31 years; p < 0.005) tended to have higher creatine kinase (CK) levels. Furthermore, 92.6% reported difficulty in sitting up from a supine position since childhood. Our research emphasizes the role of early indicators like dyspnea and difficulty performing sit-ups in adolescents for timely LOPD diagnosis and treatment initiation. The importance of early high-risk screening using dried blood spot testing cannot be overstated.

Early respiratory muscle involvement in LGMDR25: a case report.

Limb girdle muscular dystrophy type R25 (LGMDR25) is a rare genetic disorder due to loss-of-function mutations in BVES, characterized by progressive proximal lower limb weakness and atrioventricular block. Here we report a young Chinese man with LGMDR25 who presented with asymmetrical lower limb weakness, myalgia, palpitations and dyspnea on exertion. Muscle imaging demonstrated fatty infiltration of the long head of biceps femoris, adductor magnus, gastrocnemius and soleus, and myoedema of semitendinosus and quadriceps, sparing rectus femoris. ECG showed only mild sinus tachycardia but pulmonary function test suggested prominent respiratory muscle weakness. Our report expands the phenotypical spectrum and indicates the importance of monitoring respiratory function in LGMDR25 patients.

Human-induced pluripotent stem cell line (FDHSi001-A) derived from a patient with a CGG repeat expansion in the 5'UTR of GIPC1.

Oculopharyngodistal myopathy (OPDM) is a late-onset degenerative muscle disorder characterized by ptosis and weakening of the facial, pharyngeal, and distal limb muscles. Our study reprogrammed human-induced pluripotent stem cells (iPSC) from the peripheral blood mononuclear cells (PBMCs) of a patient with a CGG repeat expansion in the 5'UTR of GIPC1 gene that co-segregated with the disease. The generated iPSCs express the pluripotent cell markers with no mycoplasma contamination. Besides, it showed normal karyotypes and the capacity to differentiate into three germ layers. We also identified that it had the same specific mutation as the patient did.