Proteomics profiling and machine learning in nusinersen-treated patients with spinal muscular atrophy.

AIM: The availability of disease-modifying therapies and newborn screening programs for spinal muscular atrophy (SMA) has generated an urgent need for reliable prognostic biomarkers to classify patients according to disease severity. We aim to identify cerebrospinal fluid (CSF) prognostic protein biomarkers in CSF samples of SMA patients collected at baseline (T0), and to describe proteomic profile changes and biological pathways influenced by nusinersen before the sixth nusinersen infusion (T302). METHODS: In this multicenter retrospective longitudinal study, we employed an untargeted liquid chromatography mass spectrometry (LC-MS)-based proteomic approach on CSF samples collected from 61 SMA patients treated with nusinersen (SMA1 n=19, SMA2 n=19, SMA3 n=23) at T0 at T302. The Random Forest (RF) machine learning algorithm and pathway enrichment analysis were applied for analysis. RESULTS: The RF algorithm, applied to the protein expression profile of naïve patients, revealed several proteins that could classify the different types of SMA according to their differential abundance at T0. Analysis of changes in proteomic profiles identified a total of 147 differentially expressed proteins after nusinersen treatment in SMA1, 135 in SMA2, and 289 in SMA3. Overall, nusinersen-induced changes on proteomic profile were consistent with i) common effects observed in allSMA types (i.e. regulation of axonogenesis), and ii) disease severity-specific changes, namely regulation of glucose metabolism in SMA1, of coagulation processes in SMA2, and of complement cascade in SMA3. CONCLUSIONS: This untargeted LC-MS proteomic profiling in the CSF of SMA patients revealed differences in protein expression in naïve patients and showed nusinersen-related modulation in several biological processes after 10 months of treatment. Further confirmatory studies are needed to validate these results in larger number of patients and over abroader timeframe.

Titin copy number variations associated with dominant inherited phenotypes.

BACKGROUND: Titinopathies are caused by mutations in the titin gene (TTN). Titin is the largest known human protein; its gene has the longest coding phase with 364 exons. Titinopathies are very complex neuromuscular pathologies due to the variable age of onset of symptoms, the great diversity of pathological and muscular impairment patterns (cardiac, skeletal muscle or mixed) and both autosomal dominant and recessive modes of transmission. Until now, only few CNVs in TTN have been reported without clear genotype-phenotype associations. METHODS: Our study includes eight families with dominant titinopathies. We performed next-generation sequencing or comparative genomic hybridisation array analyses and found CNVs in the TTN gene. We characterised these CNVs by RNA sequencing (RNAseq) analyses in six patients' muscles and performed genotype-phenotype inheritance association study by combining the clinical and biological data of these eight families. RESULTS: Seven deletion-type CNVs in the TTN gene were identified among these families. Genotype and RNAseq results showed that five deletions do not alter the reading frame and one is out-of-reading frame. The main phenotype identified was distal myopathy associated with contractures. The analysis of morphological, clinical and genetic data and imaging let us draw new genotype-phenotype associations of titinopathies. CONCLUSION: Identifying TTN CNVs will further increase diagnostic sensitivity in these complex neuromuscular pathologies. Our cohort of patients enabled us to identify new deletion-type CNVs in the TTN gene, with unexpected autosomal dominant transmission. This is valuable in establishing new genotype-phenotype associations of titinopathies, mainly distal myopathy in most of the patients.

Clinical Phenotype of Pediatric and Adult Patients With Spinal Muscular Atrophy With Four SMN2 Copies: Are They Really All Stable?

OBJECTIVE: The aim of this study was to provide an overview of the clinical phenotypes associated with 4 SMN2 copies. METHODS: Clinical phenotypes were analyzed in all the patients with 4 SMN2 copies as part of a nationwide effort including all the Italian pediatric and adult reference centers for spinal muscular atrophy (SMA). RESULTS: The cohort includes 169 patients (102 men and 67 women) with confirmed 4 SMN2 copies (mean age at last follow-up = 36.9 ± 19 years). Six of the 169 patients were presymptomatic, 8 were classified as type II, 145 as type III (38 type IIIA and 107 type IIIB), and 8 as type IV. The remaining 2 patients were asymptomatic adults identified because of a familial case. The cross-sectional functional data showed a reduction of scores with increasing age. Over 35% of the type III and 25% of the type IV lost ambulation (mean age = 26.8 years ± 16.3 SD). The risk of loss of ambulation was significantly associated with SMA type (p < 0.0001), with patients with IIIB and IV less likely to lose ambulation compared to type IIIA. There was an overall gender effect with a smaller number of women and a lower risk for women to lose ambulation. This was significant in the adult (p = 0.009) but not in the pediatric cohort (p = 0.43). INTERPRETATION: Our results expand the existing literature on natural history of 4 SMN2 copies confirming the variability of phenotypes in untreated patients, ranging from type II to type IV and an overall reduction of functional scores with increasing age. ANN NEUROL 2023;94:1126-1135.

Early treatment of type II SMA slows rate of progression of scoliosis.

BACKGROUND: Type II spinal muscular atrophy (SMA) often leads to scoliosis in up to 90% of cases. While pharmacological treatments have shown improvements in motor function, their impact on scoliosis progression remains unclear. This study aims to evaluate potential differences in scoliosis progression between treated and untreated SMA II patients. METHODS: Treatment effect on Cobb's angle annual changes and on reaching a 50° Cobb angle was analysed in treated and untreated type II SMA patients with a minimum 1.5-year follow-up. A sliding cut-off approach identified the optimal treatment subpopulation based on age, Cobb angle and Hammersmith Functional Motor Scale Expanded at the initial visit. Mann-Whitney U-test assessed statistical significance. RESULTS: There were no significant differences in baseline characteristics between the untreated (n=46) and treated (n=39) populations. The mean Cobb angle variation did not significantly differ between the two groups (p=0.4). Optimal cut-off values for a better outcome were found to be having a Cobb angle <26° or an age <4.5 years. When using optimal cut-off, the treated group showed a lower mean Cobb variation compared with the untreated group (5.61 (SD 4.72) degrees/year vs 10.05 (SD 6.38) degrees/year; p=0.01). Cox-regression analysis indicated a protective treatment effect in reaching a 50° Cobb angle, significant in patients <4.5 years old (p=0.016). CONCLUSION: This study highlights that pharmacological treatment, if initiated early, may slow down the progression of scoliosis in type II SMA patients. Larger studies are warranted to further investigate the effectiveness of individual pharmacological treatment on scoliosis progression in this patient population.

De Novo GRID2 Variant as a Cause of Ataxia with Oculomotor Apraxia and Alpha-Fetoprotein Elevation.

Bi-allelic pathogenic variants in GRID2 have been initially associated to an autosomal recessive form of spinocerebellar ataxia, namely SCAR18. Subsequently, few monoallelic cases have been described. Here we present a new subject harboring a novel de novo heterozygous GRID2 missense variant presenting with progressive ataxia together with cerebellar atrophy and, for the first time, alpha-fetoprotein (AFP) elevation. We retrospectively collected data of the patient followed at our clinic. Genetic analysis was performed through clinical exome sequencing with an in-house in-silico ataxia-related genes panel. Variant effect prediction was performed through in silico modeling. The patient had normal psychomotor development except for mild fine and gross motor impairment. In adolescence, he started presenting dysarthria and progressive ataxia. Blood tests showed significant AFP elevation. Brain MRI showed cerebellar atrophy mainly involving the vermis. The novel de novo heterozygous GRID2 (c.1954C>A; p.Leu652Ile) missense variant was disclosed. This variant is located within a highly conserved site with low tolerance to variation and it is predicted to cause protein structure destabilization. GRID2 expression appears to be influenced by other genes related with ataxia and AFP elevation, like ATM and APTX, suggesting a possible shared mechanism. This additional patient increases the scarce literature and genotypic spectrum of the GRID2-related ataxia and evidences a fairly homogeneous phenotype of ataxia with oculomotor abnormalities for the autosomal-dominant form. Alfa-fetoprotein elevation is a novel finding in this condition and this data must be confirmed in larger case-series to definitively state that GRID2-related ataxia can be included among ataxias with AFP increase.

Determining minimal clinically important differences in the Hammersmith Functional Motor Scale Expanded for untreated spinal muscular atrophy patients: An international study.

BACKGROUND AND PURPOSE: Spinal muscular atrophy (SMA) is a rare and progressive neuromuscular disorder with varying severity levels. The aim of the study was to calculate minimal clinically important difference (MCID), minimal detectable change (MDC), and values for the Hammersmith Functional Motor Scale Expanded (HFMSE) in an untreated international SMA cohort. METHODS: The study employed two distinct methods. MDC was calculated using distribution-based approaches to consider standard error of measurement and effect size change in a population of 321 patients (176 SMA II and 145 SMA III), allowing for stratification based on age and function. MCID was assessed using anchor-based methods (receiver operating characteristic [ROC] curve analysis and standard error) on 76 patients (52 SMA II and 24 SMA III) for whom the 12-month HFMSE could be anchored to a caregiver-reported clinical perception questionnaire. RESULTS: With both approaches, SMA type II and type III patients had different profiles. The MCID, using ROC analysis, identified optimal cutoff points of -2 for type II and -4 for type III patients, whereas using the standard error we found the optimal cutoff points to be 1.5 for improvement and -3.2 for deterioration. Furthermore, distribution-based methods uncovered varying values across age and functional status subgroups within each SMA type. CONCLUSIONS: These results emphasize that the interpretation of a single MCID or MDC value obtained in large cohorts with different functional status needs to be made with caution, especially when these may be used to assess possible responses to new therapies.

Parental diagnostic delay and developmental outcomes in congenital and childhood-onset myotonic dystrophy type 1.

AIM: To investigate the timing of type 1 myotonic dystrophy (DM1) diagnosis in parents of affected children and describe children's perinatal characteristics and developmental outcomes. METHOD: This was a descriptive case series of children with congenital myotonic dystrophy (CDM) and childhood-onset myotonic dystrophy (ChDM). Parental timing of DM1 diagnosis and the perinatal, motor, and cognitive outcomes of paediatric patients were recorded. RESULTS: A total of 139 children followed by 12 highly specialized tertiary care neuromuscular centres in Italy and one tertiary neuromuscular centre in the USA were included: 105 children with CDM and 34 children with ChDM (mean age 8 years 8 months and 12 years 2 months respectively; 49 males and 17 males respectively). Seventy (50%) parents were diagnosed with adult-onset DM1 after the affected child was diagnosed. Only 12 (17%) of the 69 parents known to be affected had prenatal testing. Of the 105 children with CDM, 98% had maternally inherited CDM, 36% were born preterm, 83% required a stay in the neonatal intensive care unit for more than 48 hours, 84% and 79% had ambulation and speech delay, and 84% had an IQ lower than 70. Of the 34 children with ChDM, 59% had paternally inherited ChDM, 91% were born at term, and 36% had an IQ lower than 70. INTERPRETATION: Delay in diagnosing DM1 affects family planning. The prenatal and perinatal outcomes of the affected offspring emphasize the need for proactive counselling as parents may be reluctant to conduct prenatal testing.

Prognostic factors for tube feeding in type I SMA patients treated with disease-modifying therapies: a cohort study.

The aim of this study was to assess the need for tube feeding in a cohort of treated infants with type I SMA and to identify predictive factors. All patients were classified at baseline, when treatment started, and at follow-up according to their functional level and the need for tube feeding. Fisher's exact test was used to examine the associations between the outcome at the last follow-up and SMA type, SMN2 copy number, and baseline nutritional status. ANOVA was performed to compare CHOP INTEND scores and age at treatment initiation with outcomes. The cohort includes 75 type I SMA infants treated between 0.1 and 5 years of age. At the last follow-up, 34 had no need for tube feeding, 9 had tube feeding but were also able to be fed by mouth, and 32 had tube feeding and were unable to be fed by mouth. Thirty of the 41 infants with tube feeding at follow-up already had feeding difficulties when treatment was started. The need for tube feeding at follow-up was associated with the level of feeding involvement at baseline and with CHOP INTEND scores [p < 0.001] but not with SMN2 copy number, SMA type 1 subtypes or age at treatment. The results of this study suggest that the need for tube feeding is not frequent in treated infants with type I SMA and, when occurring, can be predicted by the level of feeding involvement and low CHOP INTEND scores at baseline. What is Known: • The advent of disease-modifying therapies is increasingly changing the approach to swallowing and nutritional management in type I SMA. • Clinical trials and real-world data using all three disease-modifying therapies report a rather wide variability of feeding outcome and need for tube feeding that is often related to different cohorts that makes comparison between studies very difficult. What is New: • The real-world findings of this study, including all the children treated since treatments became available, confirmed that the need for tube feeding is not an invariable finding. • The level of feeding involvement at baseline appears to be a reliable prognostic indicator of bulbar outcome. • The results highlight the need for interventional studies with structured Speech and Language Therapist protocols that will help to better understand the extent to which bulbar function can be maintained or regained even in children requiring tube feeding.

Experience of a 2-year spinal muscular atrophy NBS pilot study in Italy: towards specific guidelines and standard operating procedures for the molecular diagnosis.

BACKGROUND: Spinal muscular atrophy (SMA) is due to the homozygous absence of SMN1 in around 97% of patients, independent of the severity (classically ranked into types I-III). The high genetic homogeneity, coupled with the excellent results of presymptomatic treatments of patients with each of the three disease-modifying therapies available, makes SMA one of the golden candidates to genetic newborn screening (NBS) (SMA-NBS). The implementation of SMA in NBS national programmes occurring in some countries is an arising new issue that the scientific community has to address. We report here the results of the first Italian SMA-NBS project and provide some proposals for updating the current molecular diagnostic scenario. METHODS: The screening test was performed by an in-house-developed qPCR assay, amplifying SMN1 and SMN2. Molecular prognosis was assessed on fresh blood samples. RESULTS: We found 15 patients/90885 newborns (incidence 1:6059) having the following SMN2 genotypes: 1 (one patient), 2 (eight patients), 2+c.859G>C variant (one patient), 3 (three patients), 4 (one patient) or 6 copies (one patient). Six patients (40%) showed signs suggestive of SMA at birth. We also discuss some unusual cases we found. CONCLUSION: The molecular diagnosis of SMA needs to adapt to the new era of the disease with specific guidelines and standard operating procedures. In detail, SMA diagnosis should be felt as a true medical urgency due to therapeutic implications; SMN2 copy assessment needs to be standardised; commercially available tests need to be improved for higher SMN2 copies determination; and the SMN2 splicing-modifier variants should be routinely tested in SMA-NBS.

Type I spinal muscular atrophy patients treated with nusinersen: 4-year follow-up of motor, respiratory and bulbar function.

BACKGROUND: We report the 4-year follow-up in type I patients treated with nusinersen and the changes in motor, respiratory and bulbar function in relation to subtype, age and SMN2 copy number. METHODS: The study included SMA 1 patients with at least one assessment after 12, 24 and 48 months from the first dose of nusinersen. The assessments used were Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) and the Hammersmith Infant Neurological Examination (HINE-II). RESULTS: Forty-eight patients, with ages ranging from 7 days to 12 years (mean 3.3 years, SD 3.6 years) were included in the study. The CHOP INTEND and HINE-II scores significantly increased between baseline and 48 months (p < 0.001). When age at starting treatment subgroups (<210 days, <2 years, 2-4 years, 5-11 years, ≥12 years) were considered, the CHOP INTEND increased significantly in patients younger than 4 years at treatment, while the HINE-2 increased significantly in patients younger than 2 years at treatment. In a mixed-model analysis, age, nutritional and respiratory status were predictive of changes on both scales while SMN2 copy number and decimal classification were not. CONCLUSIONS: Our results confirm the safety profile previously reported and support the durability of the efficacy of nusinersen at 4 years with an overall stability or mild improvement and no evidence of deterioration over a long period of time.