Spectrum of factor VIII mutations in Arab patients with severe haemophilia A.

Haemophilia A is an X-linked recessive bleeding disorder caused by mutations in the factor VIII (FVIII) gene. The mutation spectrum is known in various populations, but not in Arabs. We selected 20 unrelated Arab patients with severe haemophilia A. Those patients underwent detailed clinical examination and their plasma FVIII:C activity was also measured. We extracted DNA from their blood samples and we looked for intron 22 inversion, deletions, insertions and base substitutions in the FVIII gene. Intron 22 inversion was common (detected in 11 patients, 55%), eight base substitutions (six of which are novel) were detected in nine patients (45%) and none had an insertion or deletion. Of eight base substitutions detected, six were potentially pathologic and this was correlated well with the severe clinical phenotype observed. Larger studies with more Arab patients from various Arab countries are needed in order to establish a solid conclusion about the prevalence of various mutations in this unique ethnic group. For the families included in this study, the results obtained can be helpful for carrier testing, prenatal diagnosis or pre-implantation techniques for detection of unaffected embryos.

Association of mitochondrial DNA transversion mutations with familial medullary thyroid carcinoma/multiple endocrine neoplasia type 2 syndrome.

Medullary thyroid carcinoma (MTC) is a malignant tumour of the calcitonin-secreting parafollicular C cells of the thyroid, and occurs sporadically or as a component of the multiple endocrine neoplasia (MEN) type 2/familial medullary thyroid carcinoma (FMTC) syndromes. In the present study, we investigated the frequency of mtDNA mutations in 26 MTC tumour specimens (13 sporadic and 13 familial MTC) and their matched normal tissues by sequencing the entire coding regions of mitochondrial genome. Nonsynonymous mutations were detected in 20 MTC samples (76.9%): nine out of 13 sporadic MTC (69.2%) and 11 out of 13 (84.6%) familial MTC/MEN2. Both transition and transversion types of mutations were found in the samples. Interestingly, 76.2% (16/21) of transversion mutations were found in FMTC/MEN2 patients, whereas 66.7% (12/18) of transition mutations were in sporadic MTC. Synonymous mutations were found in 12 MTC samples. In total, we identified 27 transversion mutations (21 nonsynonymous and six synonymous) in MTC. Of them, 22 (81.5%) were from FMTC/MEN2, and five (18.5%) were from sporadic MTC. The association of transversion mutation with familial MTC/MEN2 was statistically significant (P = 0.0015, binomial test). Majority of the mutations were involved in the genes located in the complex I of the mitochondrial genome, and were often resulting in a change of a moderately or highly conserved amino acid of their corresponding protein. Mitochondrial respiratory function was also compromised in a TT cell line, which carries mtDNA mutation at nt 4917 and 11,720, and in peripheral lymphocytes of MTC patients with mtDNA mutations. These data suggest that mtDNA mutation may be involved in MTC tumourigenesis and progression. Given that mtDNA mutation spectra are different between sporadic and familial MTC, different mechanisms of oxidative DNA damage may occur in the disease process.

LC-MS/MS determination of dibasic amino acids for the diagnosis of cystinuria. Application in a family affected by a novel splice-acceptor site mutation in the SLC7A9 gene.

Cystinuria is an autosomal recessive disorder caused by defective transport of cystine and the dibasic amino acids ornithine, lysine and arginine across cell membranes. Poor solubility of cystine in urine leads to kidney stones and associated symptoms and complications. Mutations of genes SLC3A1 and SLC7A9 encoding for amino acid transport systems are responsible for different types of cystinuria. In this study we describe a new LC-MS/MS assay for these amino acids in urine. Moreover, we report a novel splice-acceptor site mutation in the SLC7A9 gene that we believe is the cause of the phenotype observed in four siblings from a first-cousin marriage. Into the wells of a 96-well microtitre plate, 10 microl of urine was mixed with 90 microl of a solution containing [(2)H4]cystine, [(2)H2]ornithine, [(13)C,(2)H4]arginine and [(2)H5]glutamine that was used as an internal standard for lysine. Chromatographic separation was achieved isocratically and detection was in the selected-reaction monitoring mode. The injection-to-injection time was 8 min. Calibration curves were linear up to 1000 micromol/L. Intra-day (n = 10) and inter-day (n = 6) variations (750 and 10 micromol/L) were less than 11.4%. Urine samples from healthy individuals (n = 135) were analysed and age-matched reference ranges were generated. The method was applied retrospectively and prospectively to analyse samples (n = 13) from nine cystinuria patients. The mutation reported here was not found in 100 controls with similar ethnicity to the studied family and is believed to have consequences for the transcribed mature RNA and protein structure and function.

Increased relative mitochondrial DNA content in leucocytes of patients with NAION.

AIM: To investigate possible changes in relative mitochondrial DNA (mtDNA) content in patients with non-arteritic anterior ischaemic optic neuropathy (NAION). METHODS: 19 patients with NAION were compared to 32 controls matched for age, sex distribution, and ethnicity. DNA was extracted from leucocytes and competitive multiplex polymerase chain reaction was carried out with two primer pairs (one pair for mtDNA ND1 gene and the other pair for beta actin nuclear gene) in the presence of a fluorescent dye. RESULTS: The mean relative mtDNA content in controls (0.93 (SD 0.11); 95% CI 0.89 to 0.97) was significantly less than in NAION patients (2.40 (1.05); 95% CI 1.90 to 2.91; p < 0.001). Relative mtDNA content was negatively correlated with Snellen visual acuity (Spearman's rho; r = -0.37; p = 0.022). CONCLUSION: Increased relative mtDNA content in NAION patients may imply a response to oxidative stress, possibly in part because of mitochondrial respiratory chain defects. Significantly more non-synonymous mtDNA nucleotide changes, significantly increased relative mtDNA content, and a significant association between relative mtDNA content and visual acuity all imply that mitochondrial abnormalities may be a risk factor for NAION.

Kinetics and distribution of alcohol oxidising activity in Acholeplasma and Mycoplasma species.

Alcohol metabolism by Acholeplasma and Mycoplasma cell suspensions was determined using changes in dissolved oxygen tension to monitor oxygen uptake. All seven Acholeplasma test species oxidised ethanol and (where tested) propanol, butanol and pentanol. The rate of oxidation, at any particular substrate concentration, decreased with increasing alcohol molecular mass. Amongst 20 Mycoplasma species tested, M. agalactiae, M. bovis, M. dispar, M. gallisepticum, M. pneumoniae and M. ovipneumoniae oxidised ethanol. Propanol was also oxidised by M. dispar and isopropanol by M. agalactiae, M. bovis and M. ovipneumoniae. Isopropanol was oxidised at particularly high rates (V(max)100 nmol O(2) taken up min(-1) mg cell protein(-1)) and with a relatively high affinity (K(m) value<2 mM); oxygen uptake was consistent with oxidation to acetone. The significance of alcohol oxidation is unclear, as it would not be predicted to lead to ATP synthesis.

Status of antituberculosis drug resistance in Saudi Arabia 1979-98.

All published material on the prevalence of drug-resistant tuberculosis within Saudi Arabia over the period 1979-98 was reviewed. The prevalence of single-drug-resistant tuberculosis ranged from 3.4% to 41% for isoniazid, 0% to 23.4% for rifampicin, 0.7% to 22.7% for streptomycin and 0% to 6.9% for ethambutol. The prevalence of multidrug-resistant tuberculosis (defined by WHO as resistance to two or more first-line antituberculosis drugs) ranged from 1.5% to 44% in different regions. No strong conclusions could be drawn owing to variations in the populations studied, geographical origins, site of Mycobacterium tuberculosis isolation (pulmonary or extrapulmonary) and drug sensitivity testing. However, the need to develop a standardized national policy for surveillance of drug-resistant tuberculosis in Saudi Arabia is clear.

Overview of the laboratory accreditation programme of the College of American Pathologists.

Although Saudi medical laboratories have developed enormously over the past 25 years, the absence of a national body for medical laboratory accreditation has meant the number of accredited laboratories (seven) remains low. Of these, five are accredited by the College of American Pathologists' Laboratory Accreditation Program (LAP)--the 'gold standard' of laboratory accreditation. It requires successful performance in the College of American Pathologists' proficiency testing programme as well as passing on-site inspections carried out by practising laboratory technicians, after which the laboratory is accredited for a 2-year period. This article gives an insight into the current situation of laboratory accreditation in Saudi Arabia and an updated overview of the process involved in obtaining laboratory accreditation from the College of American Pathologists.

Evaluation of the COBAS AMPLICOR MTB test for the detection of Mycobacterium tuberculosis complex.

We evaluated the COBAS AMPLICOR polymerase chain reaction (PCR) based test for the detection of Mycobacterium tuberculosis complex in 866 respiratory and non-respiratory samples. Acid-fast staining and culture on Lowenstein-Jensen medium were also performed on all samples. Of the 866 samples tested, 87 (10.0%) were PCR-positive compared to 94 (10.9%) culture positive. There were no false positive results but 7 PCR-negative, culture-positive samples were, considered false negatives after reviewing medical records of patients. A PCR inhibitory rate of 2.0% (17/866) was observed in respiratory samples only. Sensitivity, specificity, and positive and negative predictive values for this test were 92.5%, 100%, 100% and 99.1% respectively. This test is a valuable diagnostic tool for today's mycobacteriology laboratory.

GSTM1 and GSTT1 deletion genotypes in various spontaneous optic neuropathies in Arabs.

AIM: To investigate whether the prevalence GSTT1 and GSTM1 deletion genotypes (T0M1, T1M0 and T0M0) are increased in certain spontaneous optic neuropathies. METHODS: We compared the prevalence of GSTT1 and GSTM1 deletion genotypes in 108 Arab patients with optic neuritis (ON, 26 patients), LHON-like optic neuropathy (LLON, 35 patients), sporadic bilateral optic neuropathy in children (SBON, 21 patients) and non-arteritic ischaemic optic neuropathy (NAION, 26 patients) to 120 ethnicity-matched controls. Genotypes were determined by multiplex polymerase chain reaction. RESULTS: All three GST deletion genotypes were significantly more prevalent in the entire optic neuropathy group than in controls. When patients were stratified by optic neuropathy type, the prevalence of at least one deletion genotype was significantly increased in each type of optic neuropathy. CONCLUSIONS: These results imply that GST malfunction in the setting of GST deletion genotypes may interfere with metabolism of oxidative intermediates and may exacerbate direct or indirect pathological effects of oxidative stress on the optic nerve in the setting of these spontaneous optic neuropathies. It is possible that these GST polymorphisms are risk factors for the types of optic neuropathies investigated here.

Clinical characterization of the HOXA1 syndrome BSAS variant.

BACKGROUND: The Bosley-Salih-Alorainy syndrome (BSAS) variant of the congenital human HOXA1 syndrome results from autosomal recessive truncating HOXA1 mutations. We describe the currently recognized spectrum of ocular motility, inner ear malformations, cerebrovascular anomalies, and cognitive function. METHODS: We examined nine affected individuals from five consanguineous Saudi Arabian families, all of whom harbored the same I75-I76insG homozygous mutation in the HOXA1 gene. Patients underwent complete neurologic, neuro-ophthalmologic, orthoptic, and neuropsychological examinations. Six individuals had CT, and six had MRI of the head. RESULTS: All nine individuals had bilateral Duane retraction syndrome (DRS) type 3, but extent of abduction and adduction varied between eyes and individuals. Eight patients were deaf with the common cavity deformity of the inner ear, while one patient had normal hearing and skull base development. Six had delayed motor milestones, and two had cognitive and behavioral abnormalities meeting Diagnostic and Statistical Manual of Mental Disorders-IV criteria for autism spectrum disorder. MRI of the orbits, extraocular muscles, brainstem, and supratentorial brain appeared normal. All six appropriately studied patients had cerebrovascular malformations ranging from unilateral internal carotid artery hypoplasia to bilateral agenesis. CONCLUSIONS: This report extends the Bosley-Salih-Alorainy syndrome phenotype and documents the clinical variability resulting from identical HOXA1 mutations within an isolated ethnic population. Similarities between this syndrome and thalidomide embryopathy suggest that the teratogenic effects of early thalidomide exposure in humans may be due to interaction with the HOX cascade.

Cholesterol protects Acholeplasma laidlawii against oxidative damage caused by hydrogen peroxide.

The aim of this study was to determine whether cholesterol, added to the cell growth medium or to cell suspension buffer, could protect Acholeplasma laidlawii cells against the toxic effects of hydrogen peroxide (H(2)O(2)). Variable concentrations of cholesterol (0.05-1.0 mg/ml) were added to the A. laidlawii suspension buffer and to the growth medium. Cells were then washed carefully and incubated with 0.001% (v/v) H(2)O(2) at 37 degrees C for 30 min and the viability was determined. The results indicated that cells were more viable in the presence of cholesterol than were cells grown in the absence of cholesterol. In addition, the oxygen uptake rate resulting from the oxidation of 5.5 mmol/L glucose was 2-fold and 4-fold higher for cells grown in medium supplemented with 0.05 and 0.50 mg/ml cholesterol, respectively, compared to cells grown in a medium with no added cholesterol. These findings indicate that cholesterol might play a role in protecting Mollicutes against the oxidative damage caused by H(2)O(2).

Neurologic features of horizontal gaze palsy and progressive scoliosis with mutations in ROBO3.

OBJECTIVE: To review the neurologic, neuroradiologic, and electrophysiologic features of autosomal recessive horizontal gaze palsy and progressive scoliosis (HGPPS), a syndrome caused by mutation of the ROBO3 gene on chromosome 11 and associated with defective decussation of certain brainstem neuronal systems. METHODS: The authors examined 11 individuals with HGPPS from five genotyped families with HGPPS. Eight individuals had brain MRI, and six had electrophysiologic studies. RESULTS: Horizontal gaze palsy was fully penetrant, present at birth, and total or almost total in all affected individuals. Convergence, ocular alignment, congenital nystagmus, and vertical smooth pursuit defects were variable between individuals. All patients developed progressive scoliosis during early childhood. All appropriately studied patients had hypoplasia of the pons and cerebellar peduncles with both anterior and posterior midline clefts of the pons and medulla and electrophysiologic evidence of ipsilateral corticospinal and dorsal column-medial lemniscus tract innervation. Heterozygotes were unaffected. CONCLUSIONS: The major clinical characteristics of horizontal gaze palsy and progressive scoliosis were congenital horizontal gaze palsy and progressive scoliosis with some variability in both ocular motility and degree of scoliosis. The syndrome also includes a distinctive brainstem malformation and defective crossing of some brainstem neuronal pathways.

Alternative to fluorescence assays to monitor fusion between Acholeplasma laidlawii cells and liposomes.

AIMS: To develop a new technique as an alternative to the fluorescence assays and electron microscopy for the purpose of monitoring the cell-liposome fusion. METHODS AND RESULTS: Acholeplasma laidlawii whole cells did not oxidize Glucose-6-phosphate (G6P) or Fructose-1,6 diphosphate (F1,6DP) as free (unentrapped) substrates, at concentrations 47 and >270 mM, respectively. Lysed A. laidlawii cells oxidized G6P and F1,6DP at lower concentration of 0.8 and 15 mM, respectively. When these substrates were entrapped inside liposomes, at a final concentration of 1.5 mM, and interacted with A. laidlawii whole cells, in an oxygen electrode chamber, an increase in oxygen uptake was evident. This interaction does not have any effect on cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: The experimental system described here is advantageous over classical fluorescence assays in determining the fate of liposome-entrapped material and raises the possibility of studying the kinetics of metabolic substrates, which are normally excluded from the cell by the cell membrane.

Nisin resistance distinguishes Mycoplasma spp. from Acholeplasma spp. and provides a basis for selective growth media.

The sensitivity of 11 Mycoplasma and 5 Acholeplasma species to the bacteriocin nisin was determined. When applied on filter paper discs to lawns of acholeplasma cells, nisin (20 nmol per disc) gave 3.5- to 7.0-mm zones of growth inhibition. The inclusion of 0.2 mM nisin in agar medium reduced the number of Acholeplasma laidlawii colonies by a factor of more than 10(6), and in a salts solution, 75 microM nisin killed more than 99.9% of cells within 1 min. Under similar conditions, nisin had no significant effect upon the growth or survival of Mycoplasma species. At low concentrations (1 to 3 microM), nisin stimulated glucose oxidation by A. laidlawii and Acholeplasma oculi. However, in comparison with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a recognized protonophore and uncoupler of respiration, the maximum extent of stimulation was low, < or = 20%, compared with up to 180% for CCCP. Also, in contrast to results obtained with CCCP, at concentrations only slightly above those causing stimulation of acholeplasma oxygen uptake, nisin strongly inhibited respiration. Inhibition of oxygen uptake was greater for A. laidlawii cells grown in the absence of cholesterol, and on agar medium, growth inhibition by nisin decreased with increasing concentrations of cholesterol. Nisin resistance may be a valuable characteristic in the selection and identification of Mycoplasma spp.

An analysis of common isodisomic regions in five mUPD 16 probands.

Intrauterine growth retardation (IUGR) with or without additional abnormalities is recognised as a common feature of maternal uniparental disomy for chromosome 16 (mUPD 16) and is usually associated with confined placental mosaicism (CPM). Although it is likely that the CPM largely contributes to the IUGR, postnatal growth retardation and other common abnormalities may also be attributed to the mUPD. Five cases with mUPD 16 and CPM were analysed for common regions of isodisomy using polymorphic markers distributed along the length of the chromosome. In each case the aberration was consistent with a maternal meiosis I error. Complete isodisomy was not detected in any of the patients although two patients were found to be mixed with both iso- and heterodisomy. Interestingly, the patient with the greater region of isodisomy was the most severely affected. The fact that there were no common regions of isodisomy in any of the patients supports the hypothesis that imprinted genes, rather than recessive mutations, may play a role in the shared phenotypes.

An analysis of the distribution of hetero- and isodisomic regions of chromosome 7 in five mUPD7 Silver-Russell syndrome probands.

Silver-Russell syndrome (SRS) shares common features of intrauterine growth retardation (IUGR) and a number of dysmorphic features including lateral asymmetry in about 50% of subjects. Its genetic aetiology is complex and most probably heterogeneous. Approximately 7% of patients with SRS have been found to have maternal uniparental disomy of chromosome 7 (mUPD7). Genomic DNA samples from five SRS patients with mUPD7 have been analysed for common regions of isodisomy using 40 polymorphic markers distributed along the length of chromosome 7. No regions of common isodisomy were found among the five patients. It is most likely that imprinted gene(s) rather than recessive mutations cause the common phenotype. Heterodisomy of markers around the centromere indicated that the underlying cause of the mUPD7 is a maternal meiosis I non-disjunction error in these five subjects.