A male child with the rumpshaker mutation, X-linked spastic paraplegia/Pelizaeus-Merzbacher disease and lysinuria

Of 52 antibiotic-resistant Bordetella bronchiseptica isolates from cats, ten carried plasmids. Only two of these plasmids, pLV1400 and pLV1401, were self-transmissible to Escherichia coli K12; both plasmids encoded resistance to ampicillin, tetracycline, sulphonamides, streptomycin and mercuric chloride, and were of incompatibility group P (IncP). Transferable tetracycline resistance has not been reported in B. bronchiseptica previously. The plasmids were identical in size (c.51 kb), restriction endonuclease digestion pattern and gene sequences (trfA and korA) within the IncP replicon. The trfA and korA sequences differed from those of the archetypal IncP plasmids RP4 and R751. Although the two B. bronchiseptica isolates were from epidemiologically and geographically separated cats, pulsed-field gel electrophoresis of their XbaI- or DraI-digested chromosomal DNA indicated that they were genotypically identical. The plasmid-encoded ampicillin resistance was mediated by a penicillinase of molecular weight 49,000, and pI 8.45 which was inhibited by clavulanate (IC50 = 0.1 mg/L) and tazobactam (IC50 = 0.42 mg/L) but not by parachloromercuribenzoate or EDTA. The high-level tetracycline resistance was mediated by a class C efflux mechanism that has not been described previously in this genus. The presence of transferable multi-drug resistance on a promiscuous plasmid may limit options for therapy of respiratory tract infection in companion and farm animals.     

Clinical and molecular genetic analysis of 4 Swiss families with the pure form of hereditary spastic spinal paralysis

Hereditary spastic paraplegia (HSP) is a rare neurodegenerative disease of the spinal cord with a progressive gait disorder, associated with other neurological abnormalities in the complicated form. A cluster of families with this disorder in the central part of the country has long been known to Swiss neurologists. In the present report, we describe our clinical and molecular findings in four large families originating from this region and suffering from a pure HSP form. Clinical presentation was similar in the four families. The age of onset varied widely from 2 to 70 years with the appearance of a gait disorder, which slowly progressed to wheelchair confinement after 30-70 years. No other neurological abnormality was found except for impairment of the vibration sense and sphincter abnormalities. In three families an association with markers of the SPG4 locus on chromosome 2 was found. In the fourth, the largest one, no linkage could be found with either SPG4, or with the other two known loci, SPG3 on chromosome 14 and SPG6 on chromosome 15. These data demonstrate the genetic heterogeneity in HSP, even in families from the same region. They also suggest the presence of at least one additional locus for the pure form.     

Quality assessment of whole genome mapping data in the refined familial spastic paraplegia interval on chromosome 14q

Autosomal dominant familial spastic paraplegia (AD-FSP) is a genetically heterogeneous neurodegenerative disorder characterized by progressive spasticity of the lower limbs. Three loci on chromosome 14q (SPG3), 2p (SPG4), and 15q (SPG6) were shown to be responsible for AD-FSP. Analysis of recombination events in three SPG3-linked families allowed us to narrow the critical interval from 9 to 5 cM. An approximately 5-Mb YAC contig comprising 32 clones and 90 STSs was built from D14S301 to D14S991, encompassing this region of 14q21. Fifty-six ESTs assigned previously to this region with radiation hybrid (RH) panels Genebridge 4 and G3 were precisely localized on the YAC contig. The 90 STSs positioned on the contig were tested on the TNG RH panel to compare our YAC-based map with an RH map at a high level of resolution. Comparison between our map and the whole genome mapping data on this interval of chromosome 14q is discussed.     

Screening methods in genetic diagnosis of hereditary protein C deficiency

Genomic analysis and detailed blood coagulation examinations of 22 family members of 18 families with repeatedly low protein C activity have been performed. Blood coagulation examinations: INR, fibrinogen, plasminogen, alpha-2-antiplasmin, lupus anticoagulant, APC resistance test, protein C activity and antigen, protein S activity and antithrombin activity. Genetic examinations: the presence of FII G20210A alle and FV:Q506 Leiden mutation were examined and for the mutation screening in the protein C gene combination of polymerase chain reaction (PCR) with denaturing gradient gelelectrophoresis (DGGE) or with single-strand conformation polymorphism (SSCP) analysis has been performed. The amplified DNA fragments with aberrant migration during DGGE and SSCP analysis were sequenced. Nine family members of seven families were identified carrying mutations in the protein C gene: one nonsense mutation in exon VII (Arg 157-Stop), two types of missense mutations in four patients in exon IXA (230 Arg-Lys, 254 Thr-Ile, the latter is a new mutation, Protein C Pécs), one missense mutation in two patients in exon IXB (325 Val-Ala), one missense mutation in exon IXC (359 Asp-Asn) and a rare frameshift deletion in exon IXC (364 Met-Trp, 378 Stop). Nine families were evaluated carrying no mutation in their protein C gene, but other genetic or blood coagulation disturbances have been identified, eight of them had borderline decrease in their protein C activity (60-70%). The presence of FV:Q506 mutation could be diagnosed in eight families (in 3 cases homozygous, in 5 cases heterozygous form), among them combination of the defects could be proved in three of the eight families: FV:Q506 Leiden mutation with antiphospholipoid antibodies in 2 families and the presence of Leiden mutation with prothrombin gene mutation in 1 family. Protein S deficiency in combination with prothrombin gene mutation has been identified in 1 family. There were 2 families where no genetic or blood coagulation alterations could be detected in the background of the repeatedly low protein C activity. Large deletions or insertions which are not detectable by our screening methods could not be excluded in these families and therefore sequencing of the total protein C gene had been performed with negative results. According to the literature and our experience the screening methods that were administered in this study are suitable for the detection of mutations in the protein C gene.     

Methods of analysis and resources available for genetic trait mapping

Stones can be spilled from the gallbladder during laparoscopic cholecystectomy. These stones can be left in the peritoneal cavity or trapped at the trocar site. The potential late sequel and associated morbidity are not well documented. We reviewed the records of four patients who underwent laparoscopic cholecystectomy at Mount Sinai Medical Center in New York City who suffered from late complications attributed to gallstones left in the peritoneal cavity or abdominal wall. Four patients presented 1-14 months after laparoscopic cholecystectomy with intraabdominal and abdominal wall abscesses. The spillage of gallstones was noticed during the initial operation only in one of the patients. Three patients required laparotomy and open drainage of intraabdominal abscesses with drainage of pus and gallstones after failed attempts at percutaneous drainage. Two patients underwent local exploration of an abdominal wall abscess containing stones. Stones left in the abdominal cavity or trapped in trocar sites after laparoscopic cholecystectomy can cause serious late complications requiring repeated surgical interventions. Every effort should be made in order to avoid spillage of stones during dissection of the gallbladder and cystic duct and during retrieval of the gallbladder through the abdominal wall.     

Fine genetic and comparative mapping of the deafness mutation Ames waltzer on mouse chromosome 10

The fundamental properties of a flow cytometer that govern its capacity to detect and resolve dim fluorescent particles are 1) the efficiency with which it can convert a photon emitted by a fluorochrome into a photoelectron at the photocathode of the photomultiplier tube and 2) the amount of optical background noise that is collected along with the fluorescence emission from the particle. Either of these properties alone is insufficient to predict an instrument's performance. Thus, to characterize a flow cytometer, both of these properties need to be determined. To determine these properties, one must not only consider the optical characteristics of the instruments but also understand the impact of the signal processing on the data collected. After the collection efficiency and the optical background noise level are determined, it is possible to predict the instrument's performance. A specific flow cytometer performance level is not unique to only one pair of these properties. To achieve a specific level of instrument performance, whole families of pairs of these properties are possible.     

Refined genetic localization of the Best disease gene in 11q13 and physical mapping of linked markers on radiation hybrids

In the framework of the system of monitoring the environmental impact on population health, the concentration of lead, cadmium and selenium in blood and cadmium in urine was measured in adults (n = 670), children (n = 599) and umbilical blood (n = 549) using atomic absorption spectrophotometry. Furthermore, cytogenetic analysis of peripheral lymphocytes in all population groups under study was investigated. The median blood Pb level for the overall group of adults (47.8 micrograms/l, i.e. 0.23 mumol/1) was significantly higher in men (51.5 micrograms/l, i.e. 0.25 mumol/l). Smoking significantly influenced the blood Pb level in women. The 90th percentile in no group exceeded the value of 100 micrograms/l (0.48 mumol/l). The median blood Cd level in adults (0.9 microgram/l, i.e. 0.008 mumol/l) depends on smoking habit (1.25 micrograms/l, i.e. 0.01 mumol/l). The median urine Cd level was 0.585 microgram/g creatinine (0.59 mumol/mole creatinine) in adults and 0.37 microgram/g creatinine (0.37 mumol/mole creatinine) in children. The median blood Se level (53.5 micrograms/l, i.e. 0.68 mumol/l) was found to be higher in the group of non-smokers (57.5 micrograms/l, i.e 0.73 mumol/l). Lead and selenium level were significantly lower in the umbilical blood. Cytogenetic analysis results showed age-dependent average percentages of aberrant cells: 1.1% in umbilical blood, 1.27% in children and 1.71 in adults in line with the reference values for the Czech population.     

Refined mapping and YAC contig construction of the X-linked cleft palate and ankyloglossia locus (CPX) including the proximal X-Y homology breakpoint within Xq21.3

STUDY OBJECTIVES: To investigate topographical changes in electroencephalographic (EEG) frequencies and spectral power density in relation to different surgical procedures (abdominal hysterectomy versus mastectomy) during steady-state isoflurane-nitrous oxide (N2O) anesthesia. DESIGN: Prospective, nonrandomized, open study. SETTING: University hospital. PATIENTS: 34 ASA status I and II patients scheduled for elective abdominal hysterectomy or mastectomy. INTERVENTIONS: 12 patients were studied without surgery (Group I, control). 22 patients were studied for the first 14 minutes following skin incision during hysterectomy (Group 2, n = 11) or mastectomy (Group 3, n = 11). MEASUREMENTS AND MAIN RESULTS: Anesthesia was maintained with 0.6% isoflurane in 66% N2O in oxygen (O2). EEG was recorded via 17 channels followed by calculation of spectral power densities in selected frequency bands for each recording site. In addition, heart rate, mean arterial pressure (MAP), end-tidal carbon dioxide tensions, and isoflurane concentration were recorded. Total observation time was 20 minutes in all groups. At baseline, EEG variables were comparable in all groups. The EEG demonstrated slow wave activity superimposed with alpha waves. Start of surgery resulted in increases of slower waves and decreases in alpha activity. In both surgical groups, these EEG changes were most pronounced at frontal recording sites (p < 0.05) with differences in the frequency content. In Group 2 (hysterectomy), delta-activity became dominant, whereas in Group 3 (mastectomy), a shift to theta waves was observed. During surgery MAP was increased by 40% (Group 2; p < 0.05) and 21% (Group 3; p < 0.05), respectively. CONCLUSIONS: These results show that specific surgical procedures may induce EEG slow wave activity to a different degree. The EEG response varied in relation to the surgical procedure and/or the intensity of noxious stimulation. Mastectomy resulted in the appearance of theta activity whereas, during laparotomy, the EEG frequency content was shifted to delta waves. The topographical analysis indicates spatial inhomogeneities in the EEG responses with a dominance at frontal areas. From this findings, it may be concluded that the electrode montage used for intraoperative EEG recordings has to be carefully selected.     

Applications of capillary electrophoresis in DNA mutation analysis of genetic disorders

AIM: To facilitate DNA mutation analysis by use of capillary electrophoresis. METHODS: The usefulness and applications of capillary electrophoresis in DNA fragment sizing and sequencing were evaluated. RESULTS: DNA mutation testing in disorders such as cystic fibrosis, Huntington disease, alpha thalassaemia, and hereditary fructose intolerance were undertaken effectively. However, sizing the (CAG)n repeat in the case of Huntington disease was a potential problem when using capillary electrophoresis. Separation polymers used in capillary electrophoresis are still in the developmental phase, with improved ones being released regularly. CONCLUSIONS: In the DNA diagnostic setting, capillary electrophoresis is a valuable development because it expands the scope for automation and has useful analytical properties. The potential to perform complex multiplexing within one electrophoresis run facilitates DNA diagnosis. The different mobility of DNA fragments in capillary electrophoresis compared with conventional gel electrophoresis will require, in some circumstances, additional care when results are being interpreted or reported. Capillary electrophoresis is a cheap alternative for combined automated sequencing and fragment analysis that utilises multicolour fluorescence capability. However, in its present form, it is not useful for large scale sequencing.     

Role of myelin basic protein and proteolipid protein genes in multiple sclerosis: single strand conformation polymorphism analysis of the human sequences

Susceptibility to multiple sclerosis (MS) is widely held to have a strong genetic component. While the identities of genes conferring susceptibility are currently unknown, possible candidates include those genes coding for proteins which function in central nervous system (CNS) myelin. Two such genes are the human myelin basic protein (MBP) and proteolipid protein (PLP) genes, whose products make up approximately 80% of the total protein in CNS myelin. The association of a variable number tandem repeat (VNTR) 5' to the human MBP gene with MS has been the subject of conflicting reports. Here we test the hypothesis that mutations in the human MBP and PLP genes might be associated with MS by examining the entire expressed sequence of both genes by single strand conformation polymorphism (SSCP) analysis, using a panel of 71 MS patients and 71 controls. We have also re-examined the VNTR region in patients and controls. Three base changes were found in the human PLP gene and nine base changes in the human MBP gene; these were essentially equally distributed between patients and controls. No preferential distribution of various alleles of the VNTR between patients and controls was found. Although intronic and regulatory regions have not been examined, it would appear unlikely that mutations in these genes coding for the two major CNS myelin proteins contribute significantly to genetic susceptibility to MS.     

Expression of the genes encoding myelin basic protein and proteolipid protein in human malignant gliomas

Pathological differentiation of oligodendroglioma and mixed oligoastrocytoma from astrocytoma is difficult, relying on morphological characteristics due to the lack of reliable immunohistochemical stains. Oligodendrocytes, the presumed cell of origin of oligodendrogliomas, highly express the genes encoding myelin basic protein (MBP) and proteolipid protein (PLP). We analyzed the expression of these genes to determine whether they might be useful molecular markers of oligodendrocytic tumors. MBP and PLP were highly expressed in all oligodendrogliomas and minimally expressed in glioblastomas multiforme. MBP was highly expressed in mixed oligoastrocytomas, whereas PLP expression was minimal. The association between tumor classification and expression of the MBP and PLP genes was statistically significant. Expression of these genes may serve as a useful molecular marker for some subtypes of human gliomas.     

Leber hereditary optic neuropathy. Electron microscopy and molecular genetic analysis of a case

BACKGROUND: Leber hereditary optic neuropathy (LHON) is a mitochondrial genetic disorder characterized by bilateral central visual loss typically in early adulthood. Few histopathologic studies, including ultrastructural and molecular genetic analysis, have been reported. METHODS: Ocular tissue was obtained postmortem from an 81-year-old woman with LHON from the Queensland 1 pedigree characterized by mutations at nucleotide positions 4160 and 14484. Routine histopathologic studies, electron microscopy, electron-probe analysis, and molecular genetic analysis were performed. RESULTS: Marked atrophy of the nerve fiber and retinal ganglion cell layers and optic nerves was present. Results of electron microscopic examination demonstrated 1.2 microns electron-dense, double-membrane-bound inclusions, consisting of calcium by electron-probe analysis, in retinal ganglion cells. The optic nerve was homoplasmic for mutations 4160 and 14484. CONCLUSION: Optic nerve and inner retinal atrophy in LHON may be a result of metabolic mitochondrial dysfunction leading to intramitochondrial calcification. Homoplasmy for mitochondrial mutations 4160 and 14484 in the leukocyte/platelet fraction of whole blood may correlate with homoplasmy in the optic nerve.     

A novel mutation causing an aberrant splicing in the protein 4.2 gene associated with hereditary spherocytosis (protein 4.2Notame)

We investigated a Japanese patient with protein 4.2 deficiency. SDS-PAGE showed a complete deficiency of protein 4.2, while Western blot analysis revealed a marked decrease in the amount of protein 4.2, and the existence of a doublet of 74 and 72 kDa bands. Direct sequencing and dot-blot hybridization with allele-specific oligonucleotide probes indicated that the proband was compound heterozygous for a missense mutation in codon 142 with Ala-->Thr (GCT-->ACT) and a single nucleotide substitution (G-->A) of the first base of intron 6 (G-->A) of the protein 4.2 gene. The former is the commonest mutation observed in cases of protein 4.2 deficiency, whereas the latter is a novel mutation, located within the consensus sequence of the 5' splicing site (AGGU) (Protein 4.2Notame). RT-PCR analysis using total RNA isolated from reticulocytes of the proband revealed that the intron 6 donor site mutation causes an abnormal splicing; exon 6 is spliced out with intron 6. The abnormal mRNA has a premature termination codon, as the result of a frameshift, and this instability may lead to degradation. Thus, there is a close relation between this mutation and the molecular pathogenesis of protein 4.2 deficiency.     

No mutations found by RET mutation scanning in sporadic and hereditary neuroblastoma

Neuroblastoma occasionally occurs in diseases associated with abnormal neurocrest differentiation, e.g. Hirschsprung disease. Expression studies in developing mice suggest that the proto-oncogene RET plays a role in neurocrest differentiation. In humans expression of RET is limited to certain tumor types, including neuroblastoma, that derive from migrating neural crest cells. Mutations of RET are found associated with Hirschsprung disease. These data prompted us to investigate expression of RET and to search for gene mutations in neuroblastoma. Out of 16 neuroblastoma cell lines analyzed, 9 show clear expression of RET in a Northern blot analysis. In a single strandt conformation polymorphism (SSCP) analysis of all exons, no mutations were detected other than neutral polymorphisms. In a patient with neuroblastoma, from a family in which different neurocrestopathies, including neuroblastoma and Hirschsprung disease, had occurred, we also failed to detect RET mutations. Possibly, expression of RET in neuroblastoma merely reflects the differentiation status of the tumor cells. The absence of mutations suggests that RET does not play a crucial role in the tumorigenesis of neuroblastoma.     

A novel nonsense mutation associated with an exon skipping in a patient with hereditary protein S deficiency type I

The genetic defect in a patient with hereditary type I protein S (PS) deficiency was investigated. All the exons and intron-exon junctions of the patient's PS gene were amplified by PCR and subjected to heteroduplex screening. Only the PCR product of exon 4 revealed heteroduplex bands. A novel nonsense mutation, Ser62 (TCA) to Stop (TGA) was found in exon 4. RT-PCR detected the aberrant mRNA in the patient's platelets, which was markedly reduced in amount and lacked the region of exon 4, suggesting that the nonsense mutation affected the mutated mRNA metabolism and induced exon skipping. The skipping of exon 4 causes an in-frame deletion of 29 amino acids which just construct the thrombin-sensitive region of the PS molecule. The loss of such an important domain as well as the quantitative decrease in the mutated mRNA appear to be responsible for the type I PS deficiency in this patient.