Combinatorial ASO-mediated therapy with low dose SMN and the protective modifier Chp1 is not sufficient to ameliorate SMA pathology hallmarks.

Spinal muscular atrophy (SMA) is a devastating genetically inherited neuromuscular disorder characterized by the progressive loss of motor neurons in the spinal cord, leading to muscle atrophy and weakness. Although SMA is caused by homozygous mutations in SMN1, the disease severity is mainly determined by the copy number of SMN2, an almost identical gene that produces ~10% correctly spliced SMN transcripts. Recently, three FDA- and EMA-approved therapies that either increase correctly spliced SMN2 transcripts (nusinersen and risdiplam) or replace SMN1 (onasemnogen abeparvovec-xioi) have revolutionized the clinical outcome in SMA patients. However, for severely affected SMA individuals carrying only two SMN2 copies even a presymptomatic therapy might be insufficient to fully counteract disease development. Therefore, SMN-independent compounds supporting SMN-dependent therapies represent a promising therapeutic approach. Recently, we have shown a significant amelioration of SMA disease hallmarks in a severely affected SMA mouse carrying a mutant Chp1 allele when combined with low-dose of SMN antisense oligonucleotide (ASO) treatment. CHP1 is a direct interacting partner of PLS3, a strong protective modifier of SMA. Both proteins ameliorate impaired endocytosis in SMA and significantly restore pathological hallmarks in mice. Here, we aimed to pharmacologically reduce CHP1 levels in an ASO-based combinatorial therapy targeting SMN and Chp1. Chp1 modulation is a major challenge since its genetic reduction to ~50% has shown to ameliorate SMA pathology, while the downregulation below that level causes cerebellar ataxia. Efficacy and tolerability studies determined that a single injection of 30 µg Chp1-ASO4 in the CNS is a safe dosage that significantly reduced CHP1 levels to ~50% at postnatal day (PND)14. Unfortunately, neither electrophysiological predictors such as compound muscle action potential (CMAP) or motor unit number estimation (MUNE) nor histological hallmarks of SMA in neuromuscular junction (NMJ), spinal cord or muscle were ameliorated in SMA mice treated with Chp1-ASO4 compared to CTRL-ASO at PND21. Surprisingly, CHP1 levels were almost at control level 4-weeks post injection, indicating a rather short-term effect of the ASO. Therefore, we re-administrated Chp1-ASO4 by i.c.v. bolus injection at PND28. However, no significant improvement of SMA hallmarks were seen at 2 month-of-age either. In conclusion, in contrast to the protective effect of genetically-induced Chp1 reduction on SMA, combinatorial therapy with Chp1- and SMN-ASOs failed to significantly ameliorate the SMA pathology. Chp1-ASOs compared to SMN-ASO proved to have rather short-term effect and even reinjection had no significant impact on SMA progression, suggesting that further optimization of the ASO may be required to fully explore the combination.

Clinical, electrophysiological and genetic characteristics of childhood hereditary polyneuropathies.

BACKGROUND: Hereditary polyneuropathies are heterogeneous group of diseases of the peripheral nervous system. In this study, we investigated the demographic, clinical, electrophysiological, and genetic characteristics of hereditary polyneuropathy patients diagnosed and followed up in our tertiary center clinic in Izmir, Turkey. METHODS: Patients who were diagnosed with hereditary polyneuropathies during nerve conduction studies in our center were evaluated retrospectively. RESULTS: In a total of 1484 nerve conduction studies, 207 patients were diagnosed with polyneuropathy. Ninety-nine of those patients were determined to have hereditary polyneuropathy, 52 of which were male and 47 were female. Sixty-nine patients with hereditary polyneuropathy were compatible with axonal and 30 were compatible with demyelinating polyneuropathy. Genetic analysis was performed in 69 patients, and 49 of those patients were genetically diagnosed, leading to a diagnosis rate of 71%. CONCLUSIONS: Advances in genetics have led to an increase in the heterogeneity of hereditary polyneuropathies, causing difficulties in the use of existing classifications. Although typical mutations expected in childhood-onset polyneuropathies are seen less frequently, polyneuropathies are frequently encountered as findings of complex, multisystemic diseases.

Nanoscope based on nanowaveguides.

The far field spatial resolution of conventional optical lenses is of the order of the wavelength of light, due to loss in the far field of evanescent, near electromagnetic field components. We show that subwavelength details can be restored in the far field with an array of divergent nanowaveguides, which map the discretized, subwavelength image of an object into a magnified image observable with a conventional optical microscope. We demonstrate in simulations that metallic nanowires, nanocoaxes, and nanogrooves can be used as such nanowaveguides. Thus, an optical microscope capable of subwavelength resolution - a nanoscope - can be produced, with possible applications in a variety of fields where nanoscale optical imaging is of value.

Identification of a candidate modifying gene for spinal muscular atrophy by comparative genomics.

Spinal muscular atrophy (SMA) is a common recessive disorder characterized by the loss of lower motor neurons in the spinal cord. The disease has been classified into three types based on age of onset and severity. SMA I-III all map to chromosome 5q13 (refs 2,3), and nearly all patients display deletions or gene conversions of the survival motor neuron (SMN1) gene. Some correlation has been established between SMN protein levels and disease course; nevertheless, the genetic basis for SMA phenotypic variability remains unclear, and it has been postulated that the loss of an additional modifying factor contributes to the severity of type I SMA. Using comparative genomics to screen for such a factor among evolutionarily conserved sequences between mouse and human, we have identified a novel transcript, H4F5, which lies closer to SMN1 than any previously identified gene in the region. A multi-copy microsatellite marker that is deleted in more than 90% of type I SMA chromosomes is embedded in an intron of this gene, indicating that H4F5 is also highly deleted in type I SMA chromosomes, and thus is a candidate phenotypic modifier for SMA.

SMN oligomerization defect correlates with spinal muscular atrophy severity.

Spinal muscular atrophy (SMA) is a motor-neuron disorder resulting from anterior-horn-cell death. The autosomal recessive form has a carrier frequency of 1 in 50 and is the most common genetic cause of infant death. SMA is categorized as types I-III, ranging from severe to mild, based upon age of onset and clinical course. Two closely flanking copies of the survival motor neuron (SMN) gene are on chromosome 5q13 (ref. 1). The telomeric SMN (SMN1) copy is homozygously deleted or converted in >95% of SMA patients, while a small number of SMA disease alleles contain missense mutations within the carboxy terminus. We have identified a modular oligomerization domain within exon 6 of SMN1. All previously identified missense mutations map within or immediately adjacent to this domain. Comparison of wild-type to mutant SMN proteins of type I, II and III SMA patients showed a direct correlation between oligomerization and clinical type. Moreover, the most abundant centromeric SMN product, which encodes exons 1-6 but not 7, demonstrated reduced self-association. These findings identify decreased SMN self-association as a biochemical defect in SMA, and imply that disease severity is proportional to the intracellular concentration of oligomerization-competent SMN proteins.

Objective measurement of oral function in adults with spinal muscular atrophy.

BACKGROUND: Impairment of bulbar function in adult individuals with spinal muscular atrophy (SMA) usually is not assessed by established motor scores. Measurements of oral function including quantitative muscle and endurance tests are able to detect subtle changes. The aim of this study was to systematically evaluate the measurement of maximum bite force and endurance, maximum tongue pressure and endurance, as well as maximum mouth opening in adult individuals with SMA types 2 and 3. METHODS: Data from oral function tests in 43 individuals were analyzed. Differences in oral function between individuals with different SMA types and numbers of SMN2 copies were tested. Spearman´s rho correlations among oral function measures themselves as well as with established clinical outcome scales were analyzed. RESULTS: The absolute maximum measures of oral function (maximum bite force, maximum tongue pressure, maximum mouth opening) were able to discriminate between individuals with different SMA types, individuals with a different number of SMN2 copies and with different walking abilities. The pairwise correlations of the absolute maximum measures of oral function were fair to moderate in size; the same was true for their correlations with the established motor scores. All correlations assessing endurance measures of oral function were weaker and statistically insignificant. CONCLUSIONS: Among the oral function tests maximum tongue pressure and maximum mouth opening are particulary promising as clinical and sensitive outcome measures for clinical trials. Oral function tests may supplement existing motor scores, in particular concerning specific questions about bulbar function or in severely affected non-ambulatory individuals where mild (treatment-related) changes would otherwise remain undetected. Trial registration DRKS, DRKS00015842. Registered 30 July 2019, https://drks.de/search/de/trial/DRKS00015842.

Is a positive clinical outcome after exercise therapy for chronic non-specific low back pain contingent upon a corresponding improvement in the targeted aspect(s) of performance? A systematic review.

INTRODUCTION: The effect size for exercise therapy in the treatment of chronic non-specific low back pain (cLBP) is only modest. This review aims to analyse the specificity of the effect by examining the relationship between the changes in clinical outcome (pain, disability) and the changes in the targeted aspects of physical function (muscle strength, mobility, muscular endurance) after exercise therapy. METHODS: We searched for exercise therapy trials for cLBP published up to 15 April 2010 in Medline, Embase, Cochrane Library, Cinahl, and PEDro. Two independent reviewers selected studies according to the inclusion criteria. DATA EXTRACTION: one author extracted the data of the articles. DATA SYNTHESIS: 16 studies with a total of 1,476 participants met the inclusion criteria. There was little evidence supporting a relationship between the changes in pain or physical function and the changes in performance for the following measures: mobility (no correlation in 9 studies, weak correlation in 1 study), trunk extension strength (7 and 2, respectively), trunk flexion strength (4 and 1, respectively) and back muscle endurance (7 and 0, respectively). Changes in disability showed no correlation with changes in mobility in three studies and a weak correlation in two; for strength, the numbers were four (no correlation) and two (weak correlation), respectively. CONCLUSIONS: The findings do not support the notion that the treatment effects of exercise therapy in cLBP are directly attributable to changes in the musculoskeletal system. Future research aimed at increasing the effectiveness of exercise therapy in cLBP should explore the coincidental factors influencing symptom improvement.

Whole body vibration added to endurance training in obese women - a pilot study.

Whole body vibration (WBV) training is an increasingly popular training method that is strongly promoted for weight loss, but scientific data on its effectiveness, particularly in obese subjects, are sparse. 14 obese women (BMI: 37.4 ± 1.3 kg/m2) randomized to 2 different groups (each n=7) participated in a 6-week endurance training program that was either combined or not combined with additional WBV training. Anthropometric measures, phase angle and body composition (assessed by bioelectrical impedance analysis; BIA), and resting energy expenditure (REE) were obtained before and after the training program. Body weight did not change during the training period (P=0.87), but waist circumference decreased in both groups (P=0.007; WBV: -3.4 ± 1.4 cm; no-WBV: -1.7 ± 0.7 cm) independent of WBV training (P=0.29 for group×time interaction). BIA revealed an enhancing effect of WBV training in comparison to no-WBV training on the phase angle (+0.20 ± 0.12° vs. -0.19 ± 0.12°; P=0.04) and calculated body cell mass (+0.8 ± 0.2 vs. -0.3 ± 0.4 kg; P=0.02), while calculated percentage fat mass decreased in both conditions (P=0.05) to similar extent (P=0.59; WBV: -0.8 ± 0.2%; no-WBV: -0.4 ± 0.5%). REE increased across the training (P=0.01; WBV: +77 ± 33 kcal/24 h; no-WBV: +68 ± 34 kcal/24 h), with this increase again not depending on WBV condition (P=0.85). Results of our pilot study in obese women provide preliminary evidence for a beneficial effect of WBV, when added to endurance training, on the bioelectrical phase angle, an increasingly recognized marker of individual's health status.

Wakefulness impairs selective consolidation of relevant trauma-associated memories resulting in more frequent intrusions.

Recent studies show that sleep reduces intrusive memories after analog trauma. This effect is assumed to be caused by sleep's impact on memory consolidation. However, the underlying processes of this phenomenon have not been uncovered. Thus, the current study investigates the hypothesis that sleep reduces intrusive memories by supporting the selective consolidation of relevant memories. Seventy-five participants were exposed to traumatic picture stories before nocturnal sleep or wakefulness during daytime. Memory for relevant and irrelevant trauma-associated stimuli was assessed prior to and after the retention period. Consistent with the hypothesis, results demonstrate reduced memory loss for relevant as opposed to irrelevant trauma-associated stimuli after sleep but not after wakefulness. Moreover, an incremental retention benefit for relevant trauma-associated stimuli was negatively correlated with the number of intrusive trauma memories after wakefulness. These results suggest that lack of sleep impairs selective gating of relevant trauma-associated memories, thereby enhancing intrusion development after trauma.

Genotype-phenotype studies in infantile spinal muscular atrophy (SMA) type I in Germany: implications for clinical trials and genetic counselling.

We reviewed the natural history and assessed the SMN2 copy number of 66 patients with infantile spinal muscular atrophy (SMA) type I born between 2000 and 2005 in Germany whose diagnosis was confirmed by a homozygous SMN1 deletion in the first 6 months of life. After excluding patients who had received valproic acid, the median/mean age at disease endpoint was 6.1/7.3 months (range 0.0-34.0). Four (6.1%) patients with one SMN2 copy had severe SMA type '0' with joint contractures and respiratory distress from birth. Median/mean age at onset (months) in 57 (86.3%) patients with two SMN2 copies was 1.2/1.3, and 3.5/3.4 in 5 (7.6%) patients with three SMN2 copies. Median/mean age at disease endpoint was 6.5/7.8 months (range 0.5-30) in patients with two SMN2 copies. All patients with three SMN2 copies were still alive at 10-55 months, two of them under permanent ventilation. Our data are relevant for prognostication and genetic counselling. The observed clinical variability, especially in the group with two SMN2 copies, might be important for clinical trials in SMA I where a possible control group could be defined as follows: age at onset within 4-5 months, age at genetic diagnosis <6 months, two SMN2 copies present, head control in less than 10%, no respiratory distress from birth, disease endpoint either age at death or age at permanent ventilation.

Congenital heart disease is a feature of severe infantile spinal muscular atrophy.

OBJECTIVE: Homozygous deletions/mutations of the SMN1 gene cause infantile spinal muscular atrophy (SMA). The presence of at least one SMN2 gene copy is required for normal embryogenesis. Lack of SMN protein results in degeneration of motor neurons, while extraneuronal manifestations have been regarded as a chance association with SMA. We report on heart defects in the subgroup of congenital SMA type I patients. METHODS: Data were recruited from 65 unselected SMA I patients whose diagnosis had been confirmed genetically within the first 6 months of age. SMN2 copy numbers were analysed retrospectively and correlated with clinical findings including heart malformations. RESULTS: Four (6%) patients had one copy of SMN2, 56 (86%) had two and five (8%) had three SMN2 copies. Three out of four (75%) patients with a single SMN2 copy had congenital SMA with haemodynamically relevant atrial or ventricular septal defects. CONCLUSIONS: Previous case reports of SMA I patients with congenital heart defects did not clarify whether the cardiac malformations were coincidental. Given the respective incidences of congenitally lethal SMA with a single SMN2 copy and of cardiac septal defects in humans, a chance association of both conditions would occur in less than one out of 50 million individuals. Our findings suggest that the SMN protein is relevant for normal cardiogenesis.

Hydrogen interactions with low-index surface orientations of tungsten.

We report on density functional theory calculations that have been performed to systematically investigate the hydrogen-surface interaction as a function of surface orientation. The interactions that were analyzed include stable atomic adsorption sites, molecular hydrogen dissociation and absorption energies, migration pathways and barriers on tungsten surfaces, and the saturation coverage limits on the (1 1 1) surface. Stable hydrogen adsorption sites were found for all surfaces. For the reconstructed W(1 0 0), there are two primary adsorption sites: namely, the long-bridge and short-bridge sites. The threefold hollow site (3F) was found to be the most stable for W(1 1 0), while the bond-centered site between the first and second layer was found to be most stable for the W(1 1 1) surface. No bound adsorption sites for H2 molecules were found for the W surfaces. Hydrogen (H) migration on both the (1 0 0) and (1 1 0) surfaces is found to have preferred pathways for 1D motion, whereas the smallest migration barrier for net migration of H on the W(1 1 1) surface leads to 2D migration. Although weaker H interactions are predicted for the W(1 1 1) surface compared to the (1 0 0) or (1 1 0) surfaces, we observe higher H surface concentrations of Θ = 4.0 at zero K, possibly due to the corrugated surface structure. These results provide insight into H adsorption, surface saturation coverage and migration mechanisms necessary to describe the evolution from the dilute limit to concentrated coverages of H.

Differential evolution of the Saccharomyces cerevisiae DUP240 paralogs and implication of recombination in phylogeny.

Multigene families are observed in all genomes sequenced so far and are the reflection of key evolutionary mechanisms. The DUP240 family, identified in Saccharomyces cerevisiae strain S288C, is composed of 10 paralogs: seven are organized as two tandem repeats and three are solo ORFs. To investigate the evolution of the three solo paralogs, YAR023c, YCR007c and YHL044w, we performed a comparative analysis between 15 S.cerevisiae strains. These three ORFs are present in all strains and the conservation of synteny indicates that they are not frequently involved in chromosomal reshaping, in contrast to the DUP240 ORFs organized in tandem repeats. Our analysis of nucleotide and amino acid variations indicates that YAR023c and YHL044w fix mutations more easily than YCR007c, although they all belong to the same multigene family. This comparative analysis was also conducted with five arbitrarily chosen Ascomycetes-specific genes and five arbitrarily chosen common genes (genes that have a homolog in at least one non-Ascomycetes organism). Ascomycetes-specific genes appear to be diverging faster than common genes in the S.cerevisiae species, a situation that was previously described between different yeast species. Our results point to the strong contribution, during DNA sequence evolution, of allelic recombination besides nucleotide substitution.

Respiratory muscle endurance training reduces chronic neck pain: A pilot study.

BACKGROUND: Patients with chronic neck pain show also respiratory dysfunctions. OBJECTIVE: To investigate the effects of respiratory muscle endurance training (RMET) on chronic neck pain. METHODS: In this pilot study (single-subject design: 3 baseline measurements, 4 measurements during RMET), 15 neck patients (49.3 ± 13.7 years; 13 females) conducted 20 sessions of home-based RMET using a SpiroTiger® (normocapnic hyperpnoea). Maximal voluntary ventilation (MVV), maximal inspiratory (Pimax) and expiratory (Pemax) pressure were measured before and after RMET. Neck flexor endurance, cervical and thoracic mobility, forward head posture, chest wall expansion and self-assessed neck disability [Neck Disability Index (NDI), Bournemouth questionnaire] were weekly assessed. Repeated measure ANOVA (Bonferroni correction) compared the first and last baseline and the last measurement after RMET. RESULTS: RMET significantly increased MVV (p= 0.025), Pimax (p= 0.001) and Pemax (p< 0.001). During RMET, neck disability significantly decreased (NDI: p= 0.001; Bournemouth questionnaire: p= 0.002), while neck flexor endurance (p< 0.001) and chest wall expansion (p< 0.001) increased. The changes in respiratory and musculoskeletal parameters did not correlate. CONCLUSIONS: RMET emerged from this pilot study as a feasible and effective therapy for reducing disability in patients with chronic neck pain. The underlying mechanisms, including blood gas analyses, need further investigation in a randomized controlled study.

Molecular and functional analysis of intragenic SMN1 mutations in patients with spinal muscular atrophy.

The autosomal recessive spinal muscular atrophy (SMA), a neuromuscular disease and frequent cause of early death in childhood, is caused in 96% of patients by homozygous absence of the survival motor neuron gene (SMN1). The severity of the disease is mainly determined by the copy number of SMN2, a copy gene which predominantly produces exon 7-skipped transcripts and only low amount of full-length transcripts that encode for a protein identical to SMN1. Only about 4% of SMA patients bear one SMN1 copy with an intragenic mutation. A comprehensive molecular genetic analysis of 34 SMA patients who carry one SMN1 gene is presented, including 18 that were previously published. Haplotype analysis with the microsatellite markers Ag1-CA and C212 in these SMA families turned out to be a reliable accessory method in predicting known SMN1 mutations in SMA patients carrying one SMN1 copy. Five novel missense mutations were identified that are localized in: exon 2a c.88G>A (p.D30N) and c.131A>T (p.D44V); exon 3 c.283G>C (p.G95R) and c.332C>G (p.A111G); and exon 6 c.784A>G (p.S262G), respectively. The survival motor neuron (SMN) protein has been shown to be a component of a large complex (termed the SMN complex) that promotes the formation of spliceosomal U small nuclear ribonucleoproteins (snRNPs). Within this complex, SMN forms oligomers and directly interacts via its N-terminus with SMN-interacting protein 1 (SIP1) and via its central Tudor domain with spliceosomal (Sm) proteins. We performed in vitro interaction studies to test whether SMA-causing missense mutations identified in this study interfere with the reported interactions of SMN. Our results show that mutations p.G95R and p.A111G reduce SMN binding to Sm proteins, further confirming the previous finding that the Tudor domain is the essential binding site of SMN to Sm-proteins. However, all mutations, including those in exon 2a, a region shown to be important for the binding of SMN to SIP1, do not disturb the interaction of SMN to SIP1.

[Prostate biopsy: Procedure in the clinical routine]. / Prostatabiopsie : Durchführung im klinischen Alltag.

Over the last decade there has been a 25% decrease in the mortality rates for prostate cancer. The reasons for this significant decrease are most likely associated with the application of urological screening tests. The main tools for early detection are currently increased public awareness of the disease, prostate-specific antigen (PSA) tests and transrectal ultrasound (TRUS) guided topographically assignable biopsy sampling. Together with the histopathological results these features provide essential information for risk stratification, diagnostics and therapy decisions. The evolution of prostate biopsy techniques as well as the use of PSA testing has led to an increased identification of asymptomatic men, where further clarification is necessary. Significant efforts and increased clinical research focus on determining the appropriate indications for a prostate biopsy and the optimal technique to achieve better detection rates. The most widely used imaging modality for the prostate is TRUS; however, there are no clearly defined standards for the clinical approach for each individual biopsy procedure, dealing with continuous technical optimization and in particular the developments in imaging. In this review the current principles, techniques, new approaches and instrumentation of prostate biopsy imaging control are presented within the framework of the structured educational approach.

Best practice guidelines for molecular analysis in spinal muscular atrophy.

With a prevalence of approximately 1/10 000, and a carrier frequency of 1/40-1/60 the proximal spinal muscular atrophies (SMAs) are among the most frequent autosomal recessive hereditary disorders. Patients can be classified clinically into four groups: acute, intermediate, mild, and adult (SMA types I, II, III, and IV, respectively). The complexity and instability of the genomic region at chromosome 5q13 harbouring the disease-causing survival motor neuron 1 (SMN1) gene hamper molecular diagnosis in SMA. In addition, affected individuals with SMA-like phenotypes not caused by SMN1, and asymptomatic individuals with two mutant alleles exist. The SMN gene is present in at least one telomeric (SMN1) and one centromeric copy (SMN2) per chromosome in normal (non-carrier) individuals, although chromosomes containing more copies of SMN1 and/or SMN2 exist. Moreover, the two SMN genes (SMN1 and SMN2) are highly homologous and contain only five base-pair differences within their 3' ends. Also, a relatively high de novo frequency is present in SMA. Guidelines for molecular analysis in diagnostic applications, carrier detection, and prenatal analysis using direct and indirect approaches are described. Overviews of materials used in the molecular diagnosis as well as Internet resources are included.

An essential SMN interacting protein (SIP1) is not involved in the phenotypic variability of spinal muscular atrophy (SMA).

The survival motor neuron (SMN) protein and the SMN interacting protein 1 (SIP1) are part of a 300 kD protein complex with a crucial role in snRNP biogenesis and pre-mRNA splicing. Both proteins are colocalised in nuclear structures called gems and in the cytoplasm. Approximately 96% of patients with autosomal recessive spinal muscular atrophy (SMA) show mutations in the SMN1 gene, while about 4% fail to show any mutation, despite a typical SMA phenotype. Additionally, sibs with identical 5q13 homologs and homozygous absence of SMN1 can show variable phenotypes which suggest that SMA is modified by other, yet unknown factors. Since both genes, SMN1 and SIP1, belong to the same pathway and are part of the same protein complex, it is obvious to ask whether mutations within SIP1 are responsible for both the phenotypic variability and the appearance of non-SMN mutated SMA patients. First, we identified the chromosomal location of SIP1 and assigned it to chromosomal region 14q13-q21 by fluorescence in situ hybridisation. No SMA related disorder has yet been assigned to this chromosomal region. Next, we determined the exon-intron structure of the SIP1 gene which encompasses 10 exons and identified five transcription isoforms. We sequenced either RT-PCR products or genomic DNA covering the complete coding region from 23 typical SMA patients who had failed to show any SMN1 mutation. No mutation and no polymorphism was found within SIP1. Additionally, we sequenced RT-PCR products or genomic fragments of the entire SIP1 coding region from 26 sibs of 11 SMA families with identical genotypes (delta7SMN/delta7SMN or delta7SMN/other mutation) but different phenotypes and again no mutation was found. Finally, we performed quantitative analysis of RT-PCR products from the same 26 sibs. No difference in expression level of the five isoforms among phenotypically variable sibs was observed. Based on these data, we suggest that neither the phenotypic variability nor the 5q-unlinked SMA are caused by mutations within SIP1.

Mapping of the spinal muscular atrophy (SMA) gene to a 750-kb interval flanked by two new microsatellites.

The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has recently been mapped between D5S629 and D5S557. We report here a new single-locus microsatellite A31 (D5S823) and two multicopy microsatellites 97T-CA and 95/23-CA. The marker A31 maps to the region of overlap between YACs y116, y55 and y122, distal to D5S629; 97T-CA originates from a cosmid corresponding to the STS 97T, localized distally to A31, while 95/23-CA derives from a cosmid corresponding to the STS 97U, localized proximally to D5S557. We tested all our key recombinant families with these markers. In one type I/II SMA family, a recombinant was found that placed the SMA locus distal to D5S823. Homozygosity mapping in a consanguineous type I SMA family indicates that the SMA gene lies proximal to 95/23-CA. Thus, the two new markers, A31 and 95/23-CA further refine the SMA gene to an approximately 750-kb interval.

Efficiency of various strategies and materials to generate new markers: saturating the region 5q11.2-q13.3 with 30 new randomly distributed clones.

Several different strategies and materials were used for saturating the region 5q11.2-q13.3 with new, randomly distributed markers: isolation of human clones from three chromosome-5-specific libraries (a BssHII endclone phage library from the somatic cell hybrid H64 and two total genomic phage libraries from radiation hybrids IH12 and IH132), as well as Alu-PCR from chromosome-5-specific radiation hybrids with overlapping fragments in the region around the spinal muscular atrophy locus, followed either by direct isolation of Alu-PCR products or hybridization of Alu-PCR products to chromosome-5-gridded cosmid libraries. 253 human phage and cosmid clones were mapped to various parts of chromosome 5 by deletion mapping to somatic cell hybrid panels. 30 of these clones were mapped into the region 5q11.2-q13.3, 9 of which are flanking rate cutting BssHII-sites, known to be, often, starting points for genes. They represent excellent starting material for the development of new polymorphic markers and sequence-tagged sites, for YAC screening and building of contigs, as well as for direct isolation of genes.