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Despite significant advances in basic research, the treatment of degenerative diseases of the nervous system remains one of the greatest challenges for translational medicine. The childhood onset motor neuron disorder spinal muscular atrophy (SMA) has been viewed as one of the more tractable targets for molecular therapy due to a detailed understanding of the molecular genetic basis of the disease. In SMA, inactivating mutations in the SMN1 gene can be partially compensated for by limited expression of SMN protein from a variable number of copies of the SMN2 gene, which provides both a molecular explanation for phenotypic severity and a target for therapy. The advent of the first tailored molecular therapy for SMA, based on modulating the splicing behaviour of the SMN2 gene provides, for the first time, a treatment which alters the natural history of motor neuron degeneration. Here we consider how this will change the landscape for diagnosis, clinical management and future therapeutic trials in SMA, as well as the implications for the molecular therapy of other neurological diseases.

Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by mutations in the survival motor neuron1 gene (SMN1). Global carrier frequency is around 1 in 50 and carrier detection is crucial to define couples at risk to have SMA offspring. Most SMA carriers have one SMN1 copy and are currently detected using quantitative methods. A few, however, have two SMN1 genes in cis (2/0 carriers), complicating carrier diagnosis in SMA. We analyzed our experience in detecting 2/0 carriers from a cohort of 1562 individuals, including SMA parents, SMA relatives, and unrelated individuals of the general population. Interestingly, in three couples who had an SMA child, both the parents had two SMN1 copies. Families of this type have not been previously reported. Our results emphasize the importance of performing a detailed carrier study in SMA parents with two SMN1 copies. Expanding the analysis to other key family members might confirm potential 2/0 carriers. Finally, when a partner of a known carrier presents two SMN1 copies, the study of both parents will provide a more accurate diagnosis, thus optimizing genetic counseling.

The authors present a complete SMN gene analysis in four type I unrelated spinal muscular atrophy patients who retained one copy of the SMN1 gene. Two intragenic point mutations were identified in exon 3 (I116F, Q136E), affecting a very conserved region with the Tudor domain of SMN1. The remaining two patients showed no alterations in the SMN1 coding sequences although a transcription defect was detected in one of them, corroborating the existence of non-functional SMN1 genes.

We performed molecular analysis of the factor 8 gene (F8) in 272 unrelated Spanish patients with haemophilia A (HA) and detected a mutation by routine analysis in 267 of them (98.1%). No mutation was detected in the remaining five patients despite clinical and laboratory confirmation of HA. The aim is to describe the molecular alterations in F8 discovered by gene dosage methodologies in three of these patients. For methodology, F8 sequencing, intragenic marker analysis, multiplex ligation-dependent probe amplification and quantitative real time-PCR were followed. One patient had Klinefelter syndrome (47,XXY) and a large deletion spanning exons 1-12 masked by the other F8 allele; the second patient showed a large duplication spanning exons 2-10 and the third patient revealed a non-contiguous double duplication of exons 14 and 23-25. The remaining two patients had mild HA and dosage results were normal. The application of gene dosage methods is useful to define haemophilic patients in whom mutations are not detected using other routine methods. Nevertheless, in a small percentage of patients (1%), no molecular pathology can be identified after testing several genetic methodologies.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder. Carrier frequency studies of SMA have been reported for various populations. Although no large-scale population-based studies of SMA have been performed in Iran, previous estimates have indicated that the incidence of autosomal recessive disorder partly because of the high prevalence of consanguineous marriage is much higher in the Iranian population than in other populations.In this study, we used a reliable and highly sensitive quantitative real-time PCR assay with SYBR green I dye to detect the copy number of the SMN1 gene to determine the carrier frequency of SMA in 200 healthy unrelated, non-consanguineous couples from different part of Iran.To validate the method in our samples, we determined the relative quantification (RQ) of patients with homozygous deletion (0.00) and hemyzygous carriers (0.29-0.55). The RQ in 10 of 200 normal individuals were within the carrier range of 0.31-0.57, estimating a carrier frequency of 5% in the Iranian population.Our data show that the SMA carrier frequency in Iran is higher than in the European population and that further programs of population carrier detection and prenatal testing should be implemented.

We present a 16 years old female with a chromosomal mixoploidy and multiple phenotypic anomalies. Peripheral blood G-band karyotype was 47,XXX and her skin fibroblast karyotype revealed a mosaic with a 47,XXX cell line in 88% of metaphases and a 94,XXXXXX cell line in 12% of metaphases, consistent with a hypertetraploidy. The most prominent clinical signs were: short stature, left upper limb asymmetry, senile-like appearance, generalized hypertrichosis, and small hands and feet. Radiological examination showed bone dysplasia. The result of molecular studies demonstrated that the patient inherited the two X chromosomes from the mother and one from the father, indicating that her 47,XXX trisomy resulted from an oogenesis error in the first meiotic division. The 94,XXXXXX cell line was likely the result of a cytokinesis error. To our knowledge, this is the first documented patient with a trisomy and a hypertetraploidy.

Inversion of intron 22, the most frequent mutation event in haemophilia A (HA), was tested in our HA families to diagnose the females at risk of being carriers, to trace the origin of the mutation and to investigate the presence of germinal or somatic mosaicism. A total of 166 females belonging to 54 families with inversion, were analysed. All but one of the mothers tested were carriers and the inversion originated almost exclusively in male germ cells. Somatic or germline mosaicisms were excluded in 53 of these women and in 20 grandfathers, suggesting that such mosaicisms may be a rare event in families with inversion of intron 22.

We report here a mentally retarded 32-month-old boy whose initial diagnosis was Angelman syndrome based on his clinical features. Cytogenetic studies showed a normal karyotype. Due to an elevated level of serum creatine kinase activity, we performed analyses to rule out a myopathic process. Although the electromyogram was normal, a few scattered necrotic fibres were seen in the muscle biopsy. DNA and dystrophin studies revealed an in-frame deletion in the 5' region of the dystrophin gene and an abnormal form of the protein product, consistent with a diagnosis of dystrophinopathy. We cannot totally rule out the possibility that this boy has the two separate conditions.

Spinal muscular atrophy is an autosomal recessive disorder characterized by the progressive loss or degeneration of the motor neurons. To investigate the expression of survival motor neuron (SMN), the spinal muscular atrophy-determining gene, and its relationship with the pathogenesis of the disease, we analyzed by means of in situ hybridization the location of SMN mRNA in fetal, newborn, infant, and adult human central nervous system tissues. The large motor neurons of the spinal cord are the main cells that express SMN together with the neurons of the medulla oblongata, the pyramidal cells of the cortex, and the Purkinje cells of the cerebellum. Some sensory neurons from the posterior horn and dorsal root ganglia express SMN to a lesser degree. Furthermore, strong SMN expression is detected in the ependymal cells of the central canal. The expression is present in the spinal cord at 8 weeks of fetal life throughout postnatal and adult life. The sharp expression of SMN in the motor neurons of the human spinal cord, the target cells in spinal muscular atrophy, suggests that this gene is implicated in neuronal development and in the pathogenesis of the disease. The location of the SMN gene expression in other neuronal structures not clearly or directly associated with clinical manifestations or pathological findings of spinal muscular atrophy may indicate a varying sensitivity to the absence or dysfunction of the SMN gene in motor neurons.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).