Full-Length SMN Transcript in Extracellular Vesicles as Biomarker in Individuals with Spinal Muscular Atrophy Type 2 Treated with Nusinersen.

BACKGROUND: Three therapeutic strategies have radically changed the therapeutic scenario for spinal muscular atrophy (SMA). However, therapeutic response differs between individuals. There is a need to identify biomarkers to further assess therapeutic response and to better understand which variables determine the extent of response. METHODS: We conducted a study using an optimized digital droplet PCR-based method for the ultra-sensitive detection of SMN transcript in serum EVs from SMA 2 individuals treated with nusinersen over 14 months. In parallel, we investigated levels of serum and CSF neurofilament heavy chain (pNF-H) in the same cohort. RESULTS: Expression of flSMN transcript in EVs of SMA 2 individuals prior to nusinersen was lower than in controls (0.40 vs 2.79 copies/ul; p < 0.05) and increased after 14 months of nusinersen (0.40 vs 1.11 copies/ul; p < 0.05). The increase in flSMN with nusinersen was significantly higher in younger individuals (p < 0.05). Serum pNF-h was higher in non-treated individuals with SMA 2 than in controls (230.72 vs 22.88 pg/ml; p < 0.05) and decreased with nusinersen (45.72 pg/ml at 6 months, 39.02 pg/ml at 14 months). CSF pNF-h in SMA 2 individuals also decreased with nusinersen (248.04 pg/ml prior to treatment, 197.10 pg/dl at 2 months, 104.43 pg/dl at 6 months, 131.03 pg/dl at 14 months). CONCLUSIONS: We identified an increase of flSMN transcript in serum EVs of SMA 2 individuals treated with nusinersen that was more pronounced in the younger individuals. Our results indicate that flSMN transcript expression in serum EVs is a possible biomarker in SMA to predict or monitor the response to treatment.

Case report: De novo pathogenic variant in WFS1 causes Wolfram-like syndrome debuting with congenital bilateral deafness.

Background: Congenital deafness could be the first manifestation of a syndrome such as in Usher, Pendred, and Wolfram syndromes. Therefore, a genetic study is crucial in this deficiency to significantly improve its diagnostic efficiency, to predict the prognosis, to select the most adequate treatment required, and to anticipate the development of other associated clinical manifestations. Case presentation: We describe a young girl with bilateral congenital profound deafness, who initially received a single cochlear implant. The genetic study of her DNA using a custom-designed next-generation sequencing (NGS) panel detected a de novo pathogenic heterozygous variant in the WFS1 gene related to Wolfram-like syndrome, which is characterized by the presence of other symptoms such as optic atrophy. Due to this diagnosis, a second implant was placed after the optic atrophy onset. The speech audiometric results obtained with both implants indicate that this work successfully allows the patient to develop normal speech. Deterioration of the auditory nerves has not been observed. Conclusion: The next-generation sequencing technique allows a precise molecular diagnosis of diseases with high genetic heterogeneity, such as hereditary deafness, while this was the only symptom presented by the patient at the time of analysis. The NGS panel, in which genes responsible for both syndromic and non-syndromic hereditary deafness were included, was essential to reach the diagnosis in such a young patient. Early detection of the pathogenic variant in the WFS1 gene allowed us to anticipate the natural evolution of the disease and offer the most appropriate management to the patient.

Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G>C Variant in SMN2

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype−phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype−phenotype correlations and improve prognostic outcomes.

High Mutational Heterogeneity, and New Mutations in the Human Coagulation Factor V Gene. Future Perspectives for Factor V Deficiency Using Recombinant and Advanced Therapies.

Factor V is an essential clotting factor that plays a key role in the blood coagulation cascade on account of its procoagulant and anticoagulant activity. Eighty percent of circulating factor V is produced in the liver and the remaining 20% originates in the α-granules of platelets. In humans, the factor V gene is about 80 kb in size; it is located on chromosome 1q24.2, and its cDNA is 6914 bp in length. Furthermore, nearly 190 mutations have been reported in the gene. Factor V deficiency is an autosomal recessive coagulation disorder associated with mutations in the factor V gene. This hereditary coagulation disorder is clinically characterized by a heterogeneous spectrum of hemorrhagic manifestations ranging from mucosal or soft-tissue bleeds to potentially fatal hemorrhages. Current treatment of this condition consists in the administration of fresh frozen plasma and platelet concentrates. This article describes the cases of two patients with severe factor V deficiency, and of their parents. A high level of mutational heterogeneity of factor V gene was identified, nonsense mutations, frameshift mutations, missense changes, synonymous sequence variants and intronic changes. These findings prompted the identification of a new mutation in the human factor V gene, designated as Jaén-1, which is capable of altering the procoagulant function of factor V. In addition, an update is provided on the prospects for the treatment of factor V deficiency on the basis of yet-to-be-developed recombinant products or advanced gene and cell therapies that could potentially correct this hereditary disorder.

Beyond copy number: A new, rapid, and versatile method for sequencing the entire SMN2 gene in SMA patients.

Spinal muscular atrophy (SMA) is caused by bi-allelic loss or pathogenic variants in the SMN1 gene. SMN2, the highly homologous copy of SMN1, is considered the major phenotypic modifier of the disease. Determination of SMN2 copy number is essential to establish robust genotype-phenotype correlations and predict disease evolution, to stratify patients for clinical trials, as well as to define those eligible for treatment. Discordant genotype-phenotype correlations are not uncommon in SMA, some of which are due to intragenic SMN2 variants that may influence the amount of complete SMN transcripts and, therefore, of full-length SMN protein. Detection of these variants is crucial to predict SMA phenotypes in the present scenario of therapeutic advances and with the perspective of SMA neonatal screening and early diagnosis to start treatments. Here, we present a novel, affordable, and versatile method for complete sequencing of the SMN2 gene based on long-range polymerase chain reaction and next-generation sequencing. The method was validated by analyzing samples from 53 SMA patients who lack SMN1, allowing to characterize paralogous, rare variants, and single-nucleotide polymorphisms of SMN2 as well as SMN2-SMN1 hybrid genes. The method identifies partial deletions and can be adapted to determine rare pathogenic variants in patients with at least one SMN1 copy.

Practical guidelines to manage discordant situations of SMN2 copy number in patients with spinal muscular atrophy.

OBJECTIVE: Assessment of SMN2 copy number in patients with spinal muscular atrophy (SMA) is essential to establish careful genotype-phenotype correlations and predict disease evolution. This issue is becoming crucial in the present scenario of therapeutic advances with the perspective of SMA neonatal screening and early diagnosis to initiate treatment, as this value is critical to stratify patients for clinical trials and to define those eligible to receive medication. Several technical pitfalls and interindividual variations may account for reported discrepancies in the estimation of SMN2 copy number and establishment of phenotype-genotype correlations. METHODS: We propose a management guide based on a sequence of specified actions once SMN2 copy number is determined for a given patient. Regardless of the method used to estimate the number of SMN2 copies, our approach focuses on the manifestations of the patient to recommend how to proceed in each case. RESULTS: We defined situations according to SMN2 copy number in a presymptomatic scenario of screening, in which we predict the possible evolution, and when a symptomatic patient is genetically confirmed. Unexpected discordant cases include patients having a single SMN2 copy but noncongenital disease forms, 2 SMN2 copies compatible with type II or III SMA, and 3 or 4 copies of the gene showing more severe disease than expected. CONCLUSIONS: Our proposed guideline would help to systematically identify discordant SMA cases that warrant further genetic investigation. The SMN2 gene, as the main modifier of SMA phenotype, deserves a more in-depth study to provide more accurate genotype-phenotype correlations.

Sarcomeric disorganization and nemaline bodies in muscle biopsies of patients with EXOSC3-related type 1 pontocerebellar hypoplasia.

INTRODUCTION: Mutations in the EXOSC3 gene are responsible for type 1 pontocerebellar hypoplasia, an autosomal recessive congenital disorder characterized by cerebellar atrophy, developmental delay, and anterior horn motor neuron degeneration. Muscle biopsies of these patients often show characteristics resembling classic spinal muscle atrophy, but to date, no distinct features have been identified. METHODS: Clinical data and muscle biopsy findings of 3 unrelated patients with EXOSC3 mutations are described. RESULTS: All patients presented as a severe congenital cognitive and neuromuscular phenotype with short survival, harboring the same point mutation (c.92G>C; p.Gly31Ala). Muscle biopsies consistently showed variable degrees of sarcomeric disorganization with myofibrillar remnants, Z-line thickening, and small nemaline bodies. CONCLUSIONS: In this uniform genetic cohort of patients with EXOSC3 mutations, sarcomeric disruption and rod structures were prominent features of muscle biopsies. In the context of neonatal hypotonia, ultrastructural studies might provide early clues for the diagnosis of EXOSC3-related pontocerebellar hypoplasia. Muscle Nerve 59:137-141, 2019.

Utility of two SMN1 variants to improve spinal muscular atrophy carrier diagnosis and genetic counselling.

Spinal muscular atrophy (SMA) is caused by deletions/mutations in SMN1. Most heterozygous SMA carriers have only one SMN1 copy in one of the alleles (1/0 carriers). However, a few carriers lack SMN1 in one of their chromosomes, but present two gene copies in the other. These "2/0 carriers" are undistinguishable from non-carrier individuals (1/1) with currently available methods. Previous association of SMN1 variants c.*3 + 80 T > G and c.*211_*212del with two SMN1 copies in cis in Ashkenazi population prompted us to analyze them in 270 Spanish individuals (SMA carriers, patients and general population). Both variants were much more frequently detected in chromosomes with 2 SMN1 copies in cis in comparison with chromosomes carrying one copy (17.9 vs. 0.7%; p < 0.001). In particular, one-fifth of 2/0 SMA carriers harboured one or both variants compared to none of 99 non-carriers with two SMN1 copies (p < 0.001). The c.*211_*212del variant was also much more frequent in exon 8 of SMN2-SMN1 hybrids than in that of intact SMN1 genes (20 vs. 0.83%, p < 0.001), suggesting its association with chromosomal rearrangements. Although absence of these variants does not exclude that a particular individual is a 2/0 SMA carrier, their presence is valuable to substantially increase residual risk in putative carriers, thus improving genetic counselling.

Next-generation sequencing reveals a new mutation in the LTBP2 gene associated with microspherophakia in a Spanish family.

BACKGROUND: Microspherophakia is a rare autosomal recessive eye disorder characterized by small spherical lens. It may present as an isolated finding or in association with other ocular and/or systemic disorders. This clinical and genetic heterogeneity requires the study of large genes (ADAMTSL4, FBN1, LTBP2, ADAMTSL-10 and ADAMTSL17). The purpose of the present study is to identify the genetic cause of this pathology in a consanguineous Spanish family. METHODS: A clinical exome sequencing experiment was executed by the TruSight One® Sequencing Panel (TSO) from Illumina©. Sanger sequencing was used to validate the NGS results. RESULTS: Only the insertion of an adenine in exon 36 of the LTBP2 gene (c.5439_5440insA) was associated with pathogenicity. This new mutation was validated by Sanger sequencing and segregation analysis was also performed. Haplotype analyses using the polymorphic markers D14S1025, D14S43 and D14S999 close to the LTBP2 gene indicated identity by descent in this family. CONCLUSION: We describe the first case of a microspherophakia phenotype associated with a novel homozygous mutation in the LTBP2 gene in a consanguineous Caucasian family by means of NGS technology.

Correlation between SMA type and SMN2 copy number revisited: An analysis of 625 unrelated Spanish patients and a compilation of 2834 reported cases.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss or mutations in SMN1. According to age of onset, achieved motor abilities, and life span, SMA patients are classified into type I (never sit), II (never walk unaided) or III (achieve independent walking abilities). SMN2, the highly homologous copy of SMN1, is considered the most important phenotypic modifier of the disease. Determination of SMN2 copy number is essential to establish careful genotype-phenotype correlations, predict disease evolution, and to stratify patients for clinical trials. We have determined SMN2 copy numbers in 625 unrelated Spanish SMA patients with loss or mutation of both copies of SMN1 and a clear assignation of the SMA type by clinical criteria. Furthermore, we compiled data from relevant worldwide reports that link SMN2 copy number with SMA severity published from 1999 to date (2834 patients with different ethnic and geographic backgrounds). Altogether, we have assembled a database with a total of 3459 patients to delineate more universal prognostic rules regarding the influence of SMN2 copy number on SMA phenotype. This issue is crucial in the present scenario of therapeutic advances with the perspective of SMA neonatal screening and early diagnosis to initiate treatments.

Genotype-phenotype correlation of SMN locus genes in spinal muscular atrophy children from Argentina.

BACKGROUND/PURPOSE: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder, considered one of the leading causes of infant mortality. It is caused by mutations in the SMN1 gene. A highly homologous copy of this gene named SMN2 and other neighbouring genes, SERF1A and NAIP, are considered phenotypic modifiers of the disease. In recent years, notable advances have been made in SMA research regarding evaluation, prognosis, and therapeutic options. Thus, genotype-phenotype studies in SMA are important to stratify patients for motor function tests and for envisaged clinical trials. The aim of this study was to provide clinical and molecular data of a series of Argentinean children with SMA to establish a comprehensive genotype-phenotype correlation. METHODS: 144 Argentinean children with SMA (56 children with type I, 58 with type II, and 30 with type III) were evaluated. The copy number of SMN2, SERF1A, and NAIP genes was established using MLPA (Multiplex Ligation-dependent Probe Amplification) and then correlated with the patients clinical subtypes. To improve clinical characterization we considered the initial symptoms that prompted the consultation, age of acquisition of motor abilities to independent walking and age at loss of gait. We also evaluated clinical and molecular features of sibling pairs in seven families. RESULTS: A strong correlation was observed between the SMN2 copy number and SMA phenotype while SERF1A and NAIP copy number showed a moderate correlation. We observed intra- and inter-family differences among the SMA types. CONCLUSION: This first genotype-phenotype correlation study in Argentinean SMA children provides data to improve patient stratification and define more adequate follow-up parameters.

Decay in survival motor neuron and plastin 3 levels during differentiation of iPSC-derived human motor neurons.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in Survival Motor Neuron 1 (SMN1), leading to degeneration of alpha motor neurons (MNs) but also affecting other cell types. Induced pluripotent stem cell (iPSC)-derived human MN models from severe SMA patients have shown relevant phenotypes. We have produced and fully characterized iPSCs from members of a discordant consanguineous family with chronic SMA. We differentiated the iPSC clones into ISL-1+/ChAT+ MNs and performed a comparative study during the differentiation process, observing significant differences in neurite length and number between family members. Analyses of samples from wild-type, severe SMA type I and the type IIIa/IV family showed a progressive decay in SMN protein levels during iPSC-MN differentiation, recapitulating previous observations in developmental studies. PLS3 underwent parallel reductions at both the transcriptional and translational levels. The underlying, progressive developmental decay in SMN and PLS3 levels may lead to the increased vulnerability of MNs in SMA disease. Measurements of SMN and PLS3 transcript and protein levels in iPSC-derived MNs show limited value as SMA biomarkers.

Analysis of the C9orf72 gene in spinal muscular atrophy patients.

Spinal muscular atrophy and amyotrophic lateral sclerosis are both motor neuron disorders. Several studies have tried to establish a link between the two diseases but the subject is still under debate. In amyotrophic lateral sclerosis, large expansions of the hexanucleotide GGGGCC in intron 1 of the C9orf72 gene are responsible for a variable percentage of familial and sporadic cases. We investigated whether the number of the hexanucleotide repeat in C9orf72 was associated with the phenotype and the number of SMN2 copies in a group of 162 SMA patients. Conventional PCR, repeat primed-PCR and Southern blot were used to determine repeat number and characterize large expansions. Results showed that no pathological (> 30 repeats) or premutated alleles (20-30 repeats) were found. The allelic distribution of the C9orf72 gene in spinal muscular atrophy patients overlapped with the data obtained in our control population, discarding putative repeats that may be associated with the disease. No association was observed with either the SMA phenotype or the number of SMN2 copies. In conclusion, the involvement of C9orf72 as a genetic modifier in spinal muscular atrophy is unlikely. Current investigation of modifier genes in SMA and of the link between ALS and SMA should consider other possible candidates.

Abnormalities in early markers of muscle involvement support a delay in myogenesis in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is characterized by loss of motor neurons in the spinal cord that results in muscle denervation and profound weakness in affected patients. We sought evidence for primary muscle involvement in the disease during human development by analyzing the expression of several muscle cytoskeletal components (i.e. slow, fast, and developmental myosin, desmin, and vimentin) in fetal or postnatal skeletal muscle samples from 5 SMA cases and 6 controls. At 14 weeks' gestation, SMA samples had higher percentages of myotubes expressing fast myosin and lower percentages of myotubes expressing slow myosin versus control samples. Desmin and vimentin were highly expressed at prenatal stages without notable differences between control and SMA samples, although both proteins showed persistent immunostaining in atrophic fibers in postnatal SMA samples. We also studied the expression of Pax7-positive nuclei as a marker of satellite cells and found no differences between control and SMA prenatal samples. There was, however, a significant increase in satellite cells in postnatal atrophic SMA fibers, suggesting an abnormal myogenic process. Together, these results support the hypothesis of a delay in muscle maturation as one of the primary pathologic components of SMA. Furthermore, myosins and Pax7 may be useful research markers of muscle involvement in this disease.

Synaptic defects in type I spinal muscular atrophy in human development.

Childhood spinal muscular atrophy is an autosomal recessive neuromuscular disorder caused by alterations in the Survival Motor Neuron 1 gene that triggers degeneration of motor neurons within the spinal cord. Spinal muscular atrophy is the second most common severe hereditary disease of infancy and early childhood. In the most severe cases (type I), the disease appears in the first months of life, suggesting defects in fetal development. However, it is not yet known how motor neurons, neuromuscular junctions, and muscle interact in the neuropathology of the disease. We report the structure of presynaptic and postsynaptic apparatus of the neuromuscular junctions in control and spinal muscular atrophy prenatal and postnatal human samples. Qualitative and quantitative data from confocal and electron microscopy studies revealed changes in acetylcholine receptor clustering, abnormal preterminal accumulation of vesicles, and aberrant ultrastructure of nerve terminals in the motor endplates of prenatal type I spinal muscular atrophy samples. Fetuses predicted to develop milder type II disease had a similar appearance to controls. Postnatal muscle of type I spinal muscular atrophy patients showed persistence of the fetal subunit of acetylcholine receptors, suggesting a delay in maturation of neuromuscular junctions. We observed that pathology in the severe form of the disease starts in fetal development and that a defect in maintaining the initial innervation is an early finding of neuromuscular dysfunction. These results will improve our understanding of the spinal muscular atrophy pathogenesis and help to define targets for possible presymptomatic therapy for this disease.

Evaluation of fetal nuchal translucency in 98 pregnancies at risk for severe spinal muscular atrophy: possible relevance of the SMN2 copy number.

OBJECTIVE: To study fetal nuchal translucency (NT) thickness as a possible early marker in fetuses at risk for severe spinal muscular atrophy (SMA). To investigate the significance of the survival motor neuron (SMN) 2 gene copy number in affected fetuses. METHODS: We performed 2D-ultrasound in 98 pregnancies at risk for SMA, all of which underwent prenatal molecular testing of the SMN1 gene. Crown-rump length (CRL) and NT measurements were obtained in all cases before chorionic villus sampling. Fetuses were diagnosed as healthy, carriers or affected according to the SMN1 molecular testing results. SMN2 copies were also tested in all affected fetuses. RESULTS: Nineteen fetuses were predicted to be affected due to the absence of the SMN1 gene, 18 of which had two SMN2 copies. Mean CRL and NT values did not differ between healthy, carrier and affected fetuses. In the remaining affected case who had only one SMN2 copy, the ultrasound examination showed a NT value of 4.98 mm and findings compatible with hypoplastic left heart. CONCLUSIONS: Most affected SMA fetuses have normal NT values. Our findings support the idea that SMN2 copy number in SMA fetuses is relevant for the development of congenital heart defects and increased NT values.

Treatment of spinal muscular atrophy cells with drugs that upregulate SMN expression reveals inter- and intra-patient variability.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by mutations in the SMN1 gene. The homologous copy (SMN2) is always present in SMA patients. SMN1 gene transcripts are usually full-length (FL), but exon 7 is spliced out in a high proportion of SMN2 transcripts (delta7) (Δ7). Advances in drug therapy for SMA have shown that an increase in SMN mRNA and protein levels can be achieved in vitro. We performed a systematic analysis of SMN expression in primary fibroblasts and EBV-transformed lymphoblasts from seven SMA patients with varying clinical severity and different SMN1 genotypes to determine expression differences in two accessible tissues (skin and blood). The basal expression of SMN mRNA FL and Δ7 in fibroblasts and lymphoblasts was analyzed by quantitative real-time PCR. The FL-SMN and FL/Δ7 SMN ratios were higher in control cells than in patients. Furthermore, we investigated the response of these cell lines to hydroxyurea, valproate and phenylbutyrate, drugs previously reported to upregulate SMN2. The response to treatments with these compounds was heterogeneous. We found both intra-patient and inter-patient variability even within haploidentical siblings, suggesting that tissue and individual factors may affect the response to these compounds. To optimize the stratification of patients in clinical trials, in vitro studies should be performed before enrolment so as to define each patient as a responder or non-responder to the compound under investigation.

Accuracy of marker analysis, quantitative real-time polymerase chain reaction, and multiple ligation-dependent probe amplification to determine SMN2 copy number in patients with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by absence of or mutations in the survival motor neuron1 gene (SMN1). All SMA patients have a highly homologous copy of SMN1, the SMN2 gene. Severe (type I) SMA patients present one or two SMN2 copies, whereas milder chronic forms (type II-III) usually have three or four SMN2 copies. SMN2 dosage is important to stratify patients for motor function tests and clinical trials. Our aim was to compare three methods, marker analysis, real-time quantitative polymerase chain reaction using the LightCycler instrument, and multiple ligation-dependent probe amplification (MLPA), to characterize their accuracy in quantifying SMN2 genes. We studied a group of 62 genetically confirmed SMA patients, 54 with homozygous absence of exons 7 and 8 of SMN1 and 8 with SMN2-SMN1 hybrid genes. A complete correlation using the three methods was observed in 32 patients (51.6%). In the remaining 30 patients, discordances between the three methods were found, including under or overestimation of SMN2 copies by marker analysis with respect to the quantitative methods (LightCycler and MLPA) because of lack of informativeness of markers, 3' deletions of SMN genes, and breakpoints in SMN2-SMN1 hybrid genes. The technical limitations and advantages and disadvantages of these methods are discussed. We conclude that the three methods complement each other in estimating the SMN2 copy number in most cases. However, MLPA offers additional information to characterize SMA cases with particular rearrangements such as partial deletions and hybrid genes.

Plastin 3 expression in discordant spinal muscular atrophy (SMA) siblings.

Spinal muscular atrophy (SMA) is caused by loss or mutations of the survival motor neuron 1 gene (SMN1). Its highly homologous copy, SMN2, is present in all SMA cases and is a phenotypic modifier. There are cases where asymptomatic siblings of typical SMA patients possess a homozygous deletion of SMN1 just like their symptomatic brothers or sisters. Plastin 3 (PLS3) when over expressed in lymphoblasts from females has been suggested to act as a genetic modifier of SMA. We studied PLS3 expression in four Spanish SMA families with discordant siblings haploidentical for the SMA locus. We excluded PLS3 as a possible modifier in two of our families with female discordant siblings. In the remaining two, we observed small differences in PLS3 expression between male and female discordant siblings. Indeed, we found that values of PLS3 expression in lymphoblasts and peripheral blood ranged from 12 to 200-fold less than those in fibroblasts. These findings warrant further investigation in motor neurons derived from induced pluripotential stem cells of these patients.

Ultrasound evaluation of fetal movements in pregnancies at risk for severe spinal muscular atrophy.

We studied spinal muscular atrophy (SMA) during human development to identify possible delays or alterations in fetal movements detectable by ultrasound. We evaluated 29 pregnancies at risk for severe SMA performing 2D-ultrasound around 11-14 weeks, prior to prenatal molecular testing of the SMN1 gene. We charted the occurrence of generalized body movements, isolated movements of arms and legs, head movements, startle and hiccup. Fetuses were diagnosed as healthy (n=12), carriers (n=10) or affected (n=7) according to the SMN1 molecular testing results obtained. SMN2 copies were also tested in the seven affected fetuses, six of whom showed two SMN2 copies and one a unique SMN2 copy. The movements under study were observed in all recordings, regardless of group and the SMN2 copies. At the gestational age examined, we did not observe a qualitative early limitation of movements in fetuses with SMA, even in cases predicted to develop a severe neonatal form.