Mj Barcelo

SMN2 copy number predicts acute or chronic spinal muscular atrophy but does not account for intrafamilial variability in siblings

AbstractSpinal muscular atrophy (SMA) is an autosomal recessive disorder that affects motor neurons. It is caused by mutations in the survival motor neuron gene 1 (SMN1). The SMN2 gene, which is the highly homologous SMN1 copy that is present in all the patients, is unable to prevent the disease. An SMN2 dosage method was applied to 45 patients with the three SMA types (I–III) and to four pairs of siblings with chronic SMA (II–III) and different phenotypes. Our results confirm that the SMN2 copy number plays a key role in predicting acute or chronic SMA. However, siblings with different SMA phenotypes show an identical SMN2 copy number and identical markers, indicating that the genetic background around the SMA locus is insufficient to account for the intrafamilial variability. In our results, age of onset appears to be the most important predictor of disease severity in affected members of the same family.Given that SMN2 is regarded as a target for potential pharmacological therapies in SMA, the identification of genetic factors other than the SMN genes is necessary to better understand the pathogenesis of the disease in order to implement additional therapeutic approaches.

Prevalence of hemochromatosis related HFE gene mutations in patients with acute myeloid leukemia

High serum iron saturation and high ferritin levels have been widely observed in patients with acute leukemias either before or after chemotherapy [1–3]. In a recent study we investigated the serum iron indices in 235 inpatients with hematological malignancies (91 with acute leukemias) in a single analysis, at diagnosis or during evolution, at different intervals in relation to transfusions and chemotherapy courses. We found both raised iron saturation and ferritin levels in 77% of the analyses, whereas this pattern was observed only in 10% of control inpatients of similar sex and age [4]. Some patients presented these biochemical changes before chemotherapy, transfusion or bone marrow transplantation and without apparent liver disease. Although these alterations could be associated with non-specific neoplastic manifestations, their etiology is poorly understood. A differential diagnosis of liver disease, tumoral cytolysis and secondary or primary iron overload is therefore advisable. In these patients, iron metabolism dyscontrol, which could be related to remission failure, susceptibility to infection and radical oxygen toxicities [1–3], should not be regarded as a minor disorder with respect to other clinical problems and complications. Hereditary hemochromatosis (HH) has a high prevalence in populations of European origin. Both serum transferrin saturation and ferritin are usually raised in symptomatic patients [5]. It has been well established that liver cancer is more frequent among advanced HH patients, whereas extrahepatic neoplasias are not [6]. However, it has been reported that patients with heterozygous HH run an increased risk for hematological malignancies and for colorectal and gastric cancer [7]. Owing to the recent detection of two HH associated mutations in the HH related HFE gene [8], we tested the possible association between acute myeloid leukemia (AML) and the disease. We screened the HFE genotype in 36 patients who had been diagnosed and classified in accordance with the FAB criteria [9] as follows: M1, two cases,; M2, five cases; M3, two cases; M4, five cases; M5a, three cases; M5b, six cases; RAEB, six cases; RAEB-t, two cases; and relapsed AML, five cases. Hemochromatosis associated C282Y mutation in the HFE gene (resulting in the replacement of cysteine by tyrosine at codon 282) and H63D indirectly associated mutation in the same gene (resulting in the replacement of histidine by aspartic acid at codon 63) were investigated. The study was carried out, making full use of small amounts of DNA samples previously obtained for immunogenetic diagnoses. We used enzymatic digestion of PCR products encompassing the mutation sites [8]. The C282Y mutation creates a new RsaI restriction site. The 390 bp PCR reaction product (forward primer 5%-TGGCAAGGGTAAACAGATCC-3% and reverse primer 5%-CTCAGGCACTCCTCTCAACC-3%) digested with RsaI shows two fragments of 249 and 141 bp in normal DNA, whereas mutant DNA generates two new fragments (112 and 29 bp). The H63D mutation destroys an MboI site in the 294 bp PCR product (forward primer 5%-ACATGGTTAAGGCCTGTTGC-3% and reverse primer 5%-CTTGCTGTGGTTGTGATTTTCC-3%), whereas normal DNA generates three fragments of 138, 99, and 57 bp. The genotype patterns and the allele frequencies found in the 36 patients are shown in Table 1. The * Corresponding author.

Prenatal diagnosis for risk of spinal muscular atrophy

Objectives Prenatal diagnosis of spinal muscular atrophy is usually performed in high risk couples by detection of a homozygous deletion in the survival motor neurone gene (SMN1). However, other relatives at risk of being carriers very often request genetic counselling and the possibility of prenatal diagnosis. The aim of this study was to validate a SMN1 gene quantitative test to help the couples formed by one spinal muscular atrophy carrier and a partner of the general population (1/200 potential risk) to achieve a less ambiguous risk result for the pregnancy.

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.

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.

Rapid identification of female haemophilia A carriers with deletions in the factor VIII gene by quantitative Real-Time PCR analysis

Large deletions of the factorVIII gene account for approximately 5% of severe haemophilia A patients. Although deletions are readily detectable in males, the identification of heterozygosity in possible carriers of these families still constitutes a challenge. In order to identify a deleted allele over the background of the normal allele in these carriers, we developed a rapid real-time quantitative PCR approach by means of LightCycler technology and SYBR green I for monitoring product formation. The method was applied to families with independent deletions (one in exon 14 and the other in exons 23-24) of the Factor VIII gene, thereby allowing a reliable determination of carrier or non-carrier status. The method is extremely versatile and can be adapted to other deletions within the factorVIII gene as well as to other diseases whose molecular pathology consists of deletions or duplications.

Should gamete donors be tested for spinal muscular atrophy

OBJECTIVE To report two cases of spinal muscular atrophy (SMA) after artificial insemination and to discuss why genetic screening of the disease may be justified in gamete donors. DESIGN Case report. SETTING Academic departments of genetics and obstetrics. PATIENT(S) A 32-year-old woman with two successive assisted pregnancies. INTERVENTION(S) Molecular studies of the SMN1 (survival motor neuron), the determining gene of the disease. MAIN OUTCOME MEASURE(S); Prenatal testing to detect a homozygous deletion of the SMN1 gene; carrier diagnosis by quantitative analysis to detect a single or double dose of exon 7 in the SMN1 gene. RESULT(S) After a first assisted pregnancy, an SMA child with a homozygous deletion of the SMN1 gene was born. In the second assisted pregnancy, using sperm from a different donor, a fetus with a homozygous deletion of SMN1 was detected. Carrier status in the donor was confirmed by a single dose of SMN1 in the quantitative analysis. CONCLUSION(S) Genetic screening of SMA carrier status by quantitative analysis of the SMN1 gene should be performed in gamete donors when the recipient is a known carrier. Cost-benefit analysis should be made to consider the inclusion of the test in prospective gamete donor programs.

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.

Investigation of the role of SMN1 and SMN2 haploinsufficiency as a risk factor for Hirayama's disease: Clinical, neurophysiological and genetic characteristics in a Spanish series of 13 patients

OBJECTIVE The effect of the number of copies in the SMN1 and SMN2 genes - the most extensively studied susceptibility and modifying genetic factors in adult onset motor neuron diseases - as a genetic risk factor for Hirayamas disease (HirD) has never been studied. The purpose of this study was to investigate the influence of the number of copies of the SMN1/SMN2 genes on the resulting phenotype in 13 HirD Spanish patients. PATIENTS AND METHODS We performed a qualitative and quantitative SMN1/SMN2 gene analysis in 13 unrelated HirD patients. The phenotype-genotype correlation was investigated, paying particular attention to the effect of the SMN1/SMN2 copy number on the diseases phenotype. RESULTS No patient had a homozygous deletion of the SMN1 or SMN2. No differences were found when comparing the SMN1 and SMN2 copy number distributions of the healthy population and HirD patients, and they do not therefore appear to be a susceptibility factor. There was also no correlation found between the number of copies of the SMN1 and SMN2 and the severity of the resulting phenotype. CONCLUSION Our results suggest that SMN1 and SMN2 are not predisposing factors for HirD and therefore support a lack of association between these genes and the resulting phenotype.

Evidence of a segregation ratio distortion of SMN1 alleles in spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterised by degeneration and loss of the motor neurons of the anterior horn of the spinal cord. The absence of SMN1 is determinant to have SMA and parents of SMA patients are regarded as carriers of the disease. We compared the segregation ratio of the mutated allele and the wild-type allele of all the confirmed carrier parents assuming Mendelian proportions. Results of transmissions in 235 prenatal tests and in 128 unaffected siblings showed a statistically significant deviation in favour of the wild-type SMN1 allele. The number of affected foetuses and carriers were lower than that expected. No significant differences in the sex ratio or in the progenitor origin of the transmitted allele to the carriers were found. One hypothesis that has been advanced to account for the distortion observed in affected foetuses is the negative postzygote selection due to early miscarriage. However, given that the number of carriers in our series was lower than expected, prezygote events such as meiotic drive, survival of gametes or preferential fertilisation should also be considered.