Linkage of recessive familial amyotrophic lateral sclerosis to chromosome 2q33-q35.

Amyotrophic lateral sclerosis (ALS) usually presents as a sporadic disorder of motor neurons. However, familial forms of ALS have been described--autosomal dominant forms (ALS1, ALS3), clinically indistinguishable from the sporadic form, and autosomal recessive forms with early onset and slower progression of symptoms (ALS2). To localize the gene for one of the autosomal recessive forms of ALS, we applied linkage analysis to a large inbred family from Tunisia. A lod score maximum of Zmax = 8.2 at theta = 0.00 was obtained with marker D2S72 located on chromosome 2q33-q35. The fine mapping of this region suggested that the ALS2 locus lies in the 8 cM segment flanked by D2S155 and D2S115.

The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS) are neurodegenerative conditions that affect large motor neurons of the central nervous system. We have identified a familial juvenile PLS (JPLS) locus overlapping the previously identified ALS2 locus on chromosome 2q33. We report two deletion mutations in a new gene that are found both in individuals with ALS2 and those with JPLS, indicating that these conditions have a common genetic origin. The predicted sequence of the protein (alsin) may indicate a mechanism for motor-neuron degeneration, as it may include several cell-signaling motifs with known functions, including three associated with guanine-nucleotide exchange factors for GTPases (GEFs).

A questionnaire survey on patients' willingness to pay with reference to the waiting time of public in-vitro fertilization treatment in Hong Kong.

OBJECTIVE: To evaluate patients' willingness to pay (WTP) with reference to the waiting time of public in-vitro fertilisation (IVF) treatment in order to improve the public IVF service in Hong Kong. STUDY DESIGN: A prospective multi-centred questionnaire survey. Infertile women attending infertility clinics of nine public hospitals in Hong Kong between October 2017 and August 2018 were asked to complete a questionnaire in their first clinic visit. RESULTS: Out of 1092 respondents, 10.4 % had private IVF cycles prior to their first visit at public hospitals. In general, patients were willing to pay more for a shorter waiting time for public IVF service. The proportion of respondents who were willing to pay more than HK$10,000 (US$1282) for one IVF cycle increased from 54.6% to 80.7% if the waiting time for public IVF service were hypothetically shortened from four years to one year. Likewise, 22.5 % versus 45.5 % were willing to pay more than HK$ 25,000 (US$3205) with a waiting time of four versus one year respectively. Assuming the cost per IVF cycle was HK$ 25,000 (US$3205), 23.4 % of respondents could afford one IVF cycle, 40.0 % of them could afford two IVF cycles and 31.5 % could afford three IVF cycles. A multivariate regression model demonstrated that only family income and presence of existing child(ren) were significant independent determinants of the maximum amount that an individual was willing to pay for IVF (p < 0.05). Those with family monthly income below HK$100,000 ($12,820) were less than half as likely, and those without existing child(ren) were more than double as likely, to be willing to pay higher for IVF. CONCLUSION: Patients were willing to pay more for a shorter waiting time for public IVF service. Those with family income below HK$100,000 (US$ 12,820) were less than half as likely, and those without existing children were more than double as likely, to be willing to pay higher for IVF.

Mitochondrial dysfunction-induced amphiregulin upregulation mediates chemo-resistance and cell migration in HepG2 cells.

The aim of this study was to investigate the contribution of mitochondrial dysfunction to chemoresistance and migration of hepatoma cells. We found that inhibition of mitochondrial respiration and mitochondrial DNA (mtDNA) depletion resulted in induction of amphiregulin (AR) expression in HepG2 cells. Upon oligomycin treatment of HepG2 cells, the cytosolic Ca(2+) was significantly raised after 30 min, and the intracellular level of reactive oxygen species (ROS) was elevated 2.2-fold after 4 h. Moreover, the condition medium of oligomycin-treated HepG2 cells was found to stimulate the migration of SK-Hep-1 cells. On the other hand, oligomycin-induced cisplatin-resistance and cell migration of HepG2 cells were attenuated by AR-specific RNA interference (#L-017435, Dharmacon) and a neutralizing antibody (MAB262, R&D Systems), respectively. Together, these findings suggest that mitochondrial dysfunction induced Ca(2+) mobilization, and ROS overproduction, which modulated the chemo-resistance and migration of hepatoma cells through the induction and activation of AR.

Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase.

Single-site mutants in the Cu,Zn superoxide dismutase (SOD) gene (SOD1) occur in patients with the fatal neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). Complete screening of the SOD1 coding region revealed that the mutation Ala4 to Val in exon 1 was the most frequent one; mutations were identified in exons 2, 4, and 5 but not in the active site region formed by exon 3. The 2.4 A crystal structure of human SOD, along with two other SOD structures, established that all 12 observed FALS mutant sites alter conserved interactions critical to the beta-barrel fold and dimer contact, rather than catalysis. Red cells from heterozygotes had less than 50 percent normal SOD activity, consistent with a structurally defective SOD dimer. Thus, defective SOD is linked to motor neuron death and carries implications for understanding and possible treatment of FALS.

A new probe for the diagnosis of myotonic muscular dystrophy.

Myotonic muscular dystrophy (DM) is the most common muscular dystrophy, affecting adults as well as children. It is inherited as an autosomal dominant trait and is characterized by variable expressivity and late age-of-onset. Linkage studies have established the locus on chromosome 19. In order to identify tightly linked probes for diagnosis as well as to define in detail the DM gene region, chromosome 19 libraries were constructed and screened for restriction fragment length polymorphisms tightly linked to DM. A genomic clone, LDR152 (D19S19), was isolated that is tightly linked to DM; recombination fraction = 0.0 (95% confidence limits 0.0-0.03); lod score, 15.4.

Changes of microstructure and mineralized tissue in the middle and late phase of osteoporotic fracture healing in rats.

BACKGROUND: With osteoporosis emerged as one of the most important health issues, more and more investigations are focusing on osteoporotic fracture healing. However, there are few studies on the changes of microstructure and mineralized tissue of newly formed callus. OBJECTIVE: We established an osteoporotic fracture rat model to evaluate the changes of microstructure and mineralized tissue during osteoporotic fracture healing. MATERIALS AND METHODS: A mid-shaft femur fracture model was established 12 weeks after ovariectomy as an osteoporotic fracture group (OPF group). Femurs were then harvested at 4 weeks, 8 weeks and 12 weeks after fracture for peripheral quantitative computed tomography (pQCT), micro-computed tomography (MicroCT), histology and biomechanical test. A sham-operated group was used for comparison, i.e. the normal fracture group (NF group). RESULTS: The pQCT-derived total external callus area in the OPF group was smaller than that in the NF group at 4 weeks after fracture (P<0.05), whereas it was 21% larger in the OPF group than that in the NF group at 12 weeks after fracture (P<0.01). The pQCT-derived bone mineral density in the OPF group was significantly inferior to the NF group at all the time points (P<0.05 for all the time points, respectively). MicroCT data, at 12 weeks after fracture, showed the total callus, bony callus, and newly formed bone was approximately 20% lower in the OPF group than that in the NP group, and the total connectivity was 56% lower in the OPF group as compared to the NF group. Biomechanical test data, at 12 weeks after fracture, showed that the failure load of the left femur of OPF group was 17% less compared to that of the NF group (P<0.01), and 15% lower bending stiffness (P<0.05), 20% lower bending stress (P<0.01), and 28% lower energy at failure (P<0.01) were observed in the OPF group as compared to the NF group. CONCLUSION: The decrease in mineralized tissue and the not well connected microstructure in newly formed callus may explain the decline of mechanical impairment of fracture healing in the ovariectomized rats.

Flavonoids derived from herbal Epimedium Brevicornum Maxim prevent OVX-induced osteoporosis in rats independent of its enhancement in intestinal calcium absorption.

AIM: Factorial design was used to test our hypothesis whether a group of flavonoids (FE) derived from herbal Epimedium Brevicornum Maxim exerted its preventive effects on estrogen-deficiency-induced osteoporosis mainly through an enhancement in intestinal calcium absorption. MATERIALS AND METHODS: Forty-five 12-month-old female Wistar rats were used and randomly assigned into sham-operated group and four ovariectomy (OVX) subgroups, i.e. OVX with vehicle (OVX group), OVX with FE (FE group), OVX with calcium supplement (CS group), and OVX with FE and CS (FE + CS group). Daily oral administration of FE (10 mg/kg/day) and/or CS (56 mg/kg/day) started on day 4 after OVX for 12 weeks. Before sacrificing the animals, urine and serum samples were collected for assaying indicators related to intestinal calcium absorption, regulator for calcium homeostasis, and markers of bone turnover. The left proximal femur was dissected for evaluation of the primary end-point (failure force), the second end-points (pQCT-quantified densitometry, geometry, and micro-CT-quantified 3-D trabecula micro-architecture), and pQCT-defined cross-sectional envelope. RESULTS: FE was found to be able to prevent OVX-induced reduction in failure force as well as the above second end-points, without resulting in an increased uterus weight. CS had no preventive effect on OVX-induced reduction in failure force. Two-way factorial interaction analysis between FE and CS showed that the un-enhanced suppression of parathyroid hormone for calcium homeostasis did not provide link between the enhanced intestinal calcium absorption and the enhanced inhibition of bone resorption in the present study. Furthermore, the discrepancies between the enhanced intestinal calcium absorption and the un-enhanced end-point measures as well as anabolic effect were also revealed by the interaction analysis. CONCLUSION: The present study suggested that FE inhibited bone resorption, stimulated bone formation, and accordingly prevented osteoporosis without hyperplastic effect on uterus in the OVX rat model, which was however independent of an enhancement in intestinal calcium absorption.

Intense superoxide dismutase-1 immunoreactivity in intracytoplasmic hyaline inclusions of familial amyotrophic lateral sclerosis with posterior column involvement.

This report concerns retrospective immunohistochemical and immunoelectron microscopic studies on superoxide dismutase-1 (SOD1) in intracytoplasmic hyaline inclusions (IHIs) of the anterior horn cells of three patients with familial amyotrophic lateral sclerosis (ALS) with posterior column involvement. All of the patients were members of the American "C" family. Almost all of the IHIs, present in the soma and cordlike swollen neurites of some affected neurons of the three patients, were intensely stained by an antibody to human SOD1. By contrast, the cytoplasm of anterior horn cells of the ALS patients and of ten control individuals reacted only weakly with the antibody or not at all. Immunoelectron microscopy revealed that the granule-associated thick linear structures that composed the IHIs were intensely labeled by the antibody to SOD1. The IHIs were also positively stained by antibodies to ubiquitin and phosphorylated neurofilament protein, with the distribution of immunoreactivity resembling that seen with the anti-SOD1 antibody. The DNA analysis disclosed a single-site GCC to GTC substitution at codon 4 (Ala4 --> Val) in the SOD1 gene from the brain samples of the patients and from the peripheral blood of their family members. Our results suggest that SOD1 is a component of IHIs and may interact with Ubiquitin and neurofilament protein, and point to the possibility that the presence of intense SOD1 immunoreactivity in the IHIs may be of relevance in processes involving structurally altered SOD1 molecules encoded by the mutated gene.

A novel mutation in the sterol 27-hydroxylase gene of a Pakistani family with autosomal recessive cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder of lipid storage with prominent neurologic features. The disease is associated with mutations in CYP27, which encodes mitochondrial sterol 27-hydroxylase, an enzyme that catalyzes the oxidation of sterol intermediates during bile acid synthesis. The loss of this enzyme results in accumulation of cholestanol in the nervous system and other tissues. Six different mutations have been previously described in CTX. We analyzed a Pakistani family, which included four affected individuals with clinical characteristics of CTX, for mutations in CYP27. The exons of CYP27 in the family DNA were amplified by polymerase chain reaction (PCR) and analyzed for mutations by band shifts (single stranded conformational polymorphism [SSCP]) and DNA sequencing. The PCR product for exon 4 showed an SSCP change in this family. The DNA of affected individuals showed an abnormal mobility pattern interpreted as homozygous for the mutation. One non-affected sibling was homozygous for the normal migrating pattern, whereas the parents and another non-affected sibling were heterozygous. The sequence of exon 4 of affected individuals showed a substitution of C to T in codon 237, thus substituting arginine to a stop codon. This mutation would terminate the translation, which may result in a protein half the size of the wild type rendering it practically inactive.

Presymptomatic and prenatal diagnosis in myotonic dystrophy by genetic linkage studies.

Myotonic dystrophy (DM) is an autosomal dominant disorder with age-dependent penetrance and extremely variable expressivity. With the genetic markers CKMM and ApoC2, both of which are tightly linked and centromeric to DM, presymptomatic and prenatal diagnosis for myotonic dystrophy is available. We present the results of 4 families tested for carrier status of myotonic dystrophy by genetic linkage studies and define potential limitations of these studies. A protocol for genetic linkage studies in DM is outlined.

Tight linkage of apolipoprotein C2 to myotonic dystrophy on chromosome 19.

The cDNA and genomic probes for apolipoprotein C2 detect two restriction fragment length polymorphisms on chromosome 19. The combined estimated percentage of heterozygosity, assuming equilibrium, is approximately 75%, ie, apolipoprotein C2 is informative in 75% of matings. We have analyzed over 350 individuals in large multigenerational families for linkage of apolipoprotein C2 to myotonic muscular dystrophy. The maximum lod score was 16.29 with the maximum recombination fraction (theta) of 0.02, with 95% confidence limits for theta of 0.001 to 0.065. Thus, apolipoprotein C2 is useful in carrier detection and prenatal diagnosis with an accuracy of about 98%.

Tight linkage of creatine kinase (CKMM) to myotonic dystrophy on chromosome 19.

The myotonic dystrophy (DM) gene is localized to the proximal long arm of chromosome 19. There have been reports of tight linkage to a number of chromosome 19 markers, including APOC2 and creatine kinase muscle type (CKMM), but they did not establish orientation of the 2 markers to DM. We screened several large multi-generational DM families for linkage to a series of chromosome 19 markers including CKMM. CKMM is tightly linked to DM in these data with z(theta) = 28.41; theta = 0.01. Analysis of cross-over data indicates CKMM is on the same side and closer to DM than APOC2. Thus, CKMM is a useful probe for carrier detection studies in presymptomatic individuals as well as for prenatal diagnosis.

Duchenne muscular dystrophy: high frequency of deletions.

DNA probes are available for Duchenne muscular dystrophy (DMD) carrier detection and prenatal diagnosis. With probes for about 25% of the proximal portion of the gene, we found the proximal probes detected deletions in 23% of nonselected DMD boys, while a single distal probe detected 17% more as deletions. The combined percentage was 39% for all probes tested. Prenatal diagnosis and carrier detection are more accurate if deletions are mapped rather than by use of restriction fragment length polymorphism analysis. The effort involved in screening all affected boys for deletions is considerably less, and provides an accurate genetic marker for subsequent prenatal diagnosis in the family and prospective counseling for female relatives. It seems likely that, once the entire gene (cDNA) is available for screening, most DMD boys will show deletions.

Linkage analysis in familial amyotrophic lateral sclerosis.

Familial amyotrophic lateral sclerosis (FALS) constitutes 5 to 10% of cases of ALS and, in most families, its inheritance is consistent with an autosomal dominant trait with age-dependent penetrance. The biochemical abnormality underlying the disorder is unknown. We analyzed DNA from 131 members of 6 multigenerational ALS families, which included 13 affected members, for genetic linkage to 39 expressed and DNA markers, using the techniques of 2-point linkage analysis, multilocus linkage analysis, and exclusion mapping. We identified FALS families with structures suitable for linkage, by computer simulation techniques. A DNA bank established to provide optimum use of available FALS families provided DNA from immortalized lymphoblast cell lines and frozen postmortem tissue. We could not link FALS to any of the markers studied, but excluded chromosome regions unlikely to be a locus of the FALS gene. With the help of this exclusion data, we will concentrate on regions of the human genome that remain unexcluded.

Novel mutations in spastin gene and absence of correlation with age at onset of symptoms.

Autosomal dominant hereditary spastic paraplegia is genetically heterogeneous, with at least five loci identified by linkage analysis. Recently, mutations in spastin were identified in SPG4, the most common locus for dominant hereditary spastic paraplegia that was previously mapped to chromosome 2p22. We identified five novel mutations in the spastin gene in five families with SPG4 mutations from North America and Tunisia and showed the absence of correlation between the predicted mutant spastin protein and age at onset of symptoms.

Linkage studies in tuberous sclerosis. Chromosome 9?, 11?, or maybe 14!

Published reports show linkage of tuberous sclerosis (TSC) to either chromosome 9 in some families or chromosome 11 in other families. We studied 243 individuals (82 with TSC) from 16 multigenerational TSC families. The diagnosis of TSC conformed to the criteria of Gomez. Penetrance was estimated at 0.90. DNA markers were analyzed using Southern blotting, probe hybridization, autoradiography, and genetic linkage analysis. Two-point lod scores for TSC were calculated for 43 genetic markers distributed over 11 chromosomes. Tests for homogeneity rejected the null hypothesis of homogeneity. Linkage to TSC was excluded (z less than or equal to -2, theta greater than or equal to 0.05) for 23 of these markers including 9q34 and 11q markers. One family gave z(theta max) = 1.8, theta max = 0.001 with ABO (on 9q34), and two other families attained lod scores greater than 1 for 9q34-region markers. The lod score for TSC versus chromosome 14 marker pAW101 (D14S1) was z(theta max) = 1.98, theta max = 0.15. A single large family has overall negative lod scores for markers localized to both chromosome 9 and chromosome 11. These data confirm genetic heterogeneity in TSC and suggest linkage of some families to 9q34. Furthermore, the data suggest that 14q may be an interesting area.

Notexin-induced muscle injury in the dog.

Notexin, a myotoxic phospholipase, was used to induce focal necrosis in the sartorius muscles of normal mixed-breed adult dogs and in 12-week-old beagles. Notexin injury caused pathologic changes similar to those of Duchenne muscular dystrophy (DMD) and its canine homologue, golden retriever muscular dystrophy (GRMD). All three conditions are characterized by increased serum creatine kinase (CK) levels, sarcolemmal defects, delta lesions, hyaline degeneration of myofibers, calcium-positive myofibers, and minimal effects on neurovascular structures. Four and 24 h after exposure to notexin, serum CK levels were elevated, and many myofibers were necrotic. In addition, by 24 h the necrotic areas were heavily invaded by mononuclear cells, and calcium-positive myofibers were prominent. Capillaries appeared intact even in areas of marked myonecrosis. Massive cellular infiltrate and myotube formation was evident at 3 days post injury. By 7 days, most affected fascicles were occupied by small immature myofibers. Regeneration was largely complete at 21 days. Our results suggest that notexin-induced muscle injury in dogs will be useful in the evaluation of potential therapies for DMD such as myoblast transplantation.

Increased reactive oxygen species in familial amyotrophic lateral sclerosis with mutations in SOD1.

Amyotrophic lateral sclerosis (ALS) is a paralytic disorder characterized by degeneration of large motor neurons of the brain and spinal cord. A subset of ALS is inherited (familial ALS, FALS) and is associated with more than 70 different mutations in the SOD1 gene. Here we report that lymphoblast cell lines derived from FALS patients with 16 different mutations in SOD1 gene exhibit significant increase of intracellular reactive oxygen species (ROS) compared with sporadic ALS (SALS) and normal controls (spouses of ALS patients). The ROS generation did not correlate with SOD1 activity. Further, cells incubated with vitamin C, catalase or the flavinoid quercetin significantly reduced ROS in all groups. The catalase inhibitor 3-amino-1,2,4-triazole resulted in a ten-fold increase of ROS in all groups. Neither L-nitroarginine, a nitric oxide synthase inhibitor or vitamin E altered the ROS levels. Thus, these studies suggest that hydrogen peroxide (H(2)O(2)) is a major ROS elevated in FALS lymphoblasts and it may contribute to the degeneration of susceptible cells. Further, we postulate a mechanism by which increased H(2)O(2) could be generated by mutant SOD1.

The tumor suppressor, TAX1BP2, is a novel substrate of ATM kinase.

DNA damage repair response is a crucial process for cancer prevention. One of the key regulators of this process is ataxia telangiectasia mutated (ATM) kinase, which modulates the p53 level by direct and indirect phosphorylation. Recent data showed that ATM also localizes at the centrosome, but the function remains elusive. TAX1BP2 was initially identified as a novel centrosomal protein that interacts directly with the human T-cell leukemia virus type 1 (HTLV-1)-encoded oncoprotein, Tax, and inhibits centrosome overduplication. Subsequently, TAX1BP2 was found to be a tumor suppressor in hepatocellular carcinoma, and accumulation of TAX1BP2 was observed upon chemotherapeutic drug treatment. Here, we provide evidence that TAX1BP2 is a direct phosphorylation substrate of ATM. The protein level of TAX1BP2 is significantly upregulated in response to DNA damaging agents. Serine-922 of TAX1BP2 is the phosphorylation site of ATM, and such phosphorylation modulates the protein stability, ubiquitination and tumor suppressor activity of TAX1BP2. Taken together, we demonstrate for the first time that TAX1BP2 is a novel effector of ATM in DNA damage response and delineated a new mechanism by which ATM stabilizes the tumor suppressor TAX1BP2.