Duplex PCR for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

The main difficulty in developing a molecular diagnosis of spinal muscular atrophy (SMA) resides in the specific genomic structure of the locus. Indeed, two highly homologous survival motor neurone genes, SMN1 and SMN2, are present at the locus. The detection of the homozygous deletion of exons 7 and 8 of the SMN1 gene, which is present in 90 to 98% of the patients, is based on methods highlighting 1 of the 8 nucleotidic mismatches existing between these 2 genes. In order to offer preimplantation genetic diagnosis (PGD) for SMA, we developed a new allele-specific amplification method. The main disadvantage of our previously described strategy resided in the possibility of diagnosing, in case of amplification failure, an unaffected embryo as affected. We present here a new PGD-SMA method. We established the conditions for three different duplex PCRs, allowing the specific detection of the SMN1 gene and one polymorphic marker, either D5S629, D5S1977, or D5S641. Of the 60 to 90 single cells tested, the PCR efficiency varied from 98 to 100% with a complete genotype obtained in a range between 81 and 87% with a global allele drop-out rate of 9%. Such a test was used to perform 1 PGD cycle for which 7 embryos could be analysed. All the embryos were fully diagnosed, six as unaffected and one as affected. Four embryos were transferred, but no pregnancy ensued. Copyright © 2003 John Wiley & Sons, Ltd.     

老年患者罗哌卡因与布比卡因蛛网膜下腔阻滞效果的比较

比较老年患者下肢手术罗哌卡因与布比卡因蛛网膜下腔阻滞效果．方法：择期骨科下肢手术的高龄病人80例,随机分为两组,R组（n=40）蛛网膜下腔注入5mg／ml罗哌卡因3ml,B组（n=40）蛛网膜下腔注入5mg／ml布比卡因3m1．采用针刺法评估感觉阻滞效果,记录感觉阻滞起效时间、运动阻滞起效时间、最高感觉阻滞平面、麻醉维持时间、术中麻醉质量与肌松效果及用药后的不良反应．结果：与B组比较,R组运动阻滞起效时间较长,运动阻滞和感觉阻滞维持时间较短（P〈0．05）;感觉阻滞起效时间差异无统计学意义（P〉O．05）;两组麻醉质量和肌松效果均较好,麻醉质量及肌松效果无统计学差异（P〉0．05）;两组不良反应发生率均较低,且差异无统计学意义（P〉0．05）．结论：老年病人下肢手术患者采用罗哌卡因蛛网膜下腔阻滞术中麻醉质量及肌松效果良好,且其运动阻滞维持时间较布比卡因短,有利于术后的恢复和功能锻炼．表4,参7．     

Prenatal prediction in families with autosomal recessive proximal spinal muscular atrophy (5q11.2–q13.3): Molecular genetics and clinical experience in 109 cases

Prenatal prediction in families at risk for autosomal recessive proximal spinal muscular atrophy (SMA) mainly of type I is often requested due to the high incidence and the fatal outcome of the disease. So far, only indirect genotype analysis can be performed in SMA families, since the gene has not yet been identified. We present our experience of 109 prenatal diagnoses obtained in 91 families by use of single- and multi-locus polymorphic microsatellites of the region 5q11.2–q13.3. The marker combinations and specific features of the closest microsatellites are described in detail. From 137 requests for prenatal prediction of SMA between October 1991 and August 1994, 28 families were excluded, mostly because the clinical diagnosis was uncertain or doubtful. Others had to be classified as ‘SMA-variants’ or showed autosomal dominant transmission of SMA. Of the 109 prenatal diagnoses performed, 29 fetuses were diagnosed to be at high risk (>99 per cent) of developing the disease, while in seven additional pregnancies no exact prediction could be made due to a recombination event in one parental haplotype. Altogether, recombinations between closely flanking markers were observed in 14 cases. In 35 cases, the parents decided to terminate the pregnancy. Of the remaining pregnancies, 32 could be followed beyond term. All infants were reported to develop normally without signs of SMA. Two children were born with transverse reduction defects of one hand, which was most likely related to early chorionic villus sampling at 9 and 10 weeks' gestation. No further abnormalities could be detected. The limits of indirect genotype analysis and the problems of diagnostic accuracy and heterogeneity of proximal SMA are discussed.     

Effects of whole spine alignment patterns on neck responses in rear end impact

Objective: The aim of this study was to investigate the whole spine alignment in automotive seated postures for both genders and the effects of the spinal alignment patterns on cervical vertebral motion in rear impact using a human finite element (FE) model.

Methods: Image data for 8 female and 7 male subjects in a seated posture acquired by an upright open magnetic resonance imaging (MRI) system were utilized. Spinal alignment was determined from the centers of the vertebrae and average spinal alignment patterns for both genders were estimated by multidimensional scaling (MDS). An occupant FE model of female average size (162 cm, 62 kg; the AF 50 size model) was developed by scaling THUMS AF 05. The average spinal alignment pattern for females was implemented in the model, and model validation was made with respect to female volunteer sled test data from rear end impacts. Thereafter, the average spinal alignment pattern for males and representative spinal alignments for all subjects were implemented in the validated female model, and additional FE simulations of the sled test were conducted to investigate effects of spinal alignment patterns on cervical vertebral motion.

Results: The estimated average spinal alignment pattern was slight kyphotic, or almost straight cervical and less-kyphotic thoracic spine for the females and lordotic cervical and more pronounced kyphotic thoracic spine for the males. The AF 50 size model with the female average spinal alignment exhibited spine straightening from upper thoracic vertebra level and showed larger intervertebral angular displacements in the cervical spine than the one with the male average spinal alignment.

Conclusions: The cervical spine alignment is continuous with the thoracic spine, and a trend of the relationship between cervical spine and thoracic spinal alignment was shown in this study. Simulation results suggested that variations in thoracic spinal alignment had a potential impact on cervical spine motion as well as cervical spinal alignment in rear end impact condition.     

Case report: birth after preimplantation genetic diagnosis of a subtle mutation in SMN1 gene

Spinal muscular atrophy (SMA) preimplantation genetic diagnosis (PGD) has been available since 1998. Protocols are based on the detection of the homozygous deletion of exon 7, which are present in 90–98% of SMA patients. A couple where the woman was a heterozygous carrier of the usual SMN1 Del7 mutation and the man was a heterozygous carrier of pMet263Arg substitution in exon 6 of SMN1 gene was referred for PGD. The usual PGD test being unsuitable for this couple, we developed a novel duplex polymerase chain reaction (PCR)-based PGD test for the detection of the mutation pMet263Arg by allele specific amplification, combined with the amplification of D5S641 extragenic polymorphic marker. PCR conditions were established using single control lymphoblasts and lymphocytes from the pMet263Arg substitution carrier. Amplification was obtained in 100% of the 86 single cells tested, amplification refractory mutation system (ARMS) PCR was specific in 100% of single cells tested and a complete genotype (mutation plus D5S641) was achieved in 88% of them. A PGD cycle was performed successfully and a pregnancy was obtained. An unaffected girl was born and postnatal diagnosis confirmed PGD results. This is the first PGD described for SMA because of another mutation than the major homozygous exon 7 deletion of SMN1. In the future, a similar strategy could be adopted for other subtle mutations of this gene. Copyright © 2006 John Wiley & Sons, Ltd.     

Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

We have developed a new allele-specific amplification method for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA; Werdnig-Hoffmann disease) from a single cell. This method is based on the detection of the deletion of exon 7 of the telomeric copy of the survival motor neurone (SMN^t) gene. An oligonucleotide was designed to be specific to the SMN^t nucleotidic sequence with exonic mismatch G (for SMN^t)→A (for SMN^c) at its 3′ end. This test produces reliable PCR products in 95% of single lymphoblasts (85/88) tested as well as in 16/16 blastomeres from normal controls. Specificity analysis showed that we were able to detect homozygous deletion of the SMN^t gene in 99% of single lymphoblasts (103/104) from a SMA patient. No contamination was detected in 68 blanks tested. Multiple cell and DNA dilution analysis revealed that the test is accurate and specific up to 100 pg DNA and should thus also be suitable for PGD at the blastocyst stage. This rapid procedure requires a single round of fluorescent PCR and no restriction digestion, while previously described single cell methods include nested PCR followed by restriction enzyme digestion. Two PGD cycles for SMA using this procedure were performed in our centre. Copyright © 2001 John Wiley & Sons, Ltd.     

Work conditions in agriculture as risk factors of spinal pain in postmenopausal women

The aim of the study was to evaluate the risk factors for spinal pain in Polish postmenopausal women working in agriculture. The study included 1751 randomly selected postmenopausal women aged 45–65?years. The women were exposed to hazardous work conditions, in particular the use of force and heavy lifting, often more than 10?kg and with frequency of at least once a day. Two-thirds of them suffered from spinal pain, mostly in the lumbar spine and less frequently in the cervical and thoracic spine. The prevalence of spinal pain depended on exposure to health risks associated with overwork, early onset of menopause and increasing age. The prevalence of pain in the lumbar spine correlated positively with higher frequency of heavy lifting and using excessive force during agricultural work. The severity of spinal pain correlates positively with the age of pain onset and negatively with level of education.     

The OEIS complex: two case reports that illustrate the spectrum of abnormalities and a review of the literature

We present two cases of OEIS (omphalocele, exstrophy, imperforate anus, spinal defects) complex -MIM 258040 and a review of the literature. Case 1 was a 14-year-old girl who presented at 30 weeks' gestation. An ultrasound examination showed an omphalocele and spina bifida; the bladder was not visualised. She went into spontaneous labour two weeks later and the baby died shortly after birth. A full post-mortem examination was refused, but the mother did agree to an external examination, skin biopsy for fibroblast culture, X rays and MR imaging. The MR imaging showed a pelvic kidney, a large omphalocele containing the other kidney, liver, bowel and a fluid filled structure thought to represent an exstrophy of the bladder (EB). Case 2 was a 30-year-old woman who had an ultrasound examination at 20 weeks' gestation; this showed an omphalocele, but the bladder was not visualised. The pregnancy was subsequently terminated and a post-mortem examination showed a low set umbilical cord associated with a small omphalocele; there was an imperforate anus; a blind ending rectum terminated in the omphalocele. We conclude that these two cases illustrate the variability of the OEIS complex. Copyright © 2006 John Wiley & Sons, Ltd.     

A Whole Body Postural Loading Simulation and Assessment Model for Workplace Analysis and Design

Abstract

This study reports on the development and validation of a new computer model for simulating human postures at work, and assessing the reaction forces and bending moments in 43 human articulation joints. The proposed model estimates the intradiscal pressure in the vertebral column in response to external loading forces encountered during human interactions with work objects or processes. The model was implemented in a self-contained interactive software package. The simulation results compare favorably with the reported experimental data, and provide reasonable confidence in the quality of the model. Its characteristics and its applications in evaluating physical task performance are also discussed