Biomedical subjects

S R Lin

Publications and source records attributed to S R Lin.

At least 55 records · Page 3Linked to original sources

PubMed1998

Cytokines in murine lyme carditis: Th1 cytokine expression follows expression of proinflammatory cytokines in a susceptible mouse strain.

The cardiac infiltrate seen in murine Lyme carditis is composed predominantly of macrophages, but small numbers of T cells are also present. To identify the cytokines present in cardiac lesions from susceptible mice, semiquantitative polymerase chain reaction was done on cardiac tissue from mice infected with Borrelia burgdorferi. The temporal expression of proinflammatory and T cell-derived cytokines was characterized in cardiac tissue at days 0, 3, 7, 14, 21, and 42 after infection with B. burgdorferi. Early in the course of infection, up-regulation of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha was detected. The Th1 cytokine interferon-gamma appeared after the expression of the proinflammatory cytokines and remained elevated throughout the study. Interleukin-4 was not detectable at any time in cardiac lesions. These data are the first to identify cytokines expressed at the lesional level in murine Lyme carditis and to demonstrate a Th1 pattern of cytokine expression in this lesion.

Animals↗

PubMed1998

Identification of arg-30 as the essential residue for the enzymatic activity of Taiwan cobra phospholipase A2.

Taiwan cobra (Naja naja atra) phospholipase A2 (PLA2) was inactivated by arginine-specific reagents, phenylglyoxal and 1, 2-cyclohexanedione. Kinetic analyses of the modification reaction revealed that the inactivation of PLA2 followed pseudo-first-order kinetics and the loss of activity was correlated with the incorporation of one molecule of modification reagent per PLA2 molecule. This was confirmed by the results of amino acid composition determination, that showed that a marked decrease in enzymatic activity was associated with the modification of one arginine residue. Tryptic cleavage of the modified protein and microsequencing revealed that Arg-30 was the functionally essential residue. The incorporation of a modifier into the PLA2 did not significantly affect the secondary structure of the enzyme, as revealed by the CD spectrum, and Ca2+-binding of the modified PLA2 was unaffected. Nevertheless, the nonpolarity of the active site of PLA2 markedly decreased with the arginine modification, as evidenced by the decreases in the enhancement of Trp and 8-anilinonaphthalene sulfonate fluorescence. These results, together with those of X-ray crystallographic analysis of N. naja atra PLA2 [Scott et al. (1990) Science 250, 1541-1546], demonstrate that Arg-30 is one of the residues involved in the interfacial binding of a PLA2 molecule with its substrate.

Animals↗

PubMed1998

Alterations of RET oncogene in human adrenal tumors.

Previous studies have revealed specific activations of the RET oncogene in multiple endocrine neoplasia type 2 (MEN 2) and thyroid tumors. To understand the role of the RET proto-oncogene activation in sporadic adrenal tumors, we analyzed the alterations of the RET proto-oncogene in the cysteine-rich extracellular domain (exons 6 and 10), the terminal region of the extracellular domain and transmembrane domain (exon 11) and the tyrosine kinase domain (exons 12-17) in 35 cases of adrenal tumors (including 18 Conn's syndrome, 3 Cushing's syndrome, 2 non-functional adrenocortical tumor and 12 pheochromocytomas by polymerase chain reaction-single strand conformational polymorphism and sequencing methods. One case with pheochromocytoma and one with Conn's syndrome had point mutation. We also detected the rearrangement of the RET gene by reverse transcription-polymerase chain reaction and Southern hybridization. One case with Conn's syndrome and one with Cushing's syndrome were found to harbor RET/PTC1 (RET tyrosine kinase domain rearranged with H4 gene). The above results indicate that RET proto-oncogene mutations and RET/PTC1 are involved in the pathogenesis of sporadic adrenal tumors. Mutations at codon 634 of the RET gene were also found in adrenal tumors. This suggests that the RET oncogene may also play a role in the tumorigenesis of adrenal tumors, and this possibility requires further investigation.

Adrenal Gland Neoplasms↗

PubMed1998

Molecular characterization of Chinese G6PD deficiency by using polymerase chain reaction/single strand conformation polymorphism.

Using the polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP), we studied the molecular characterization of 97 (86 male and 11 female) glucose-6-phosphate dehydrogenase (G6PD) deficient Chinese newborn babies and infants in southern Taiwan. Movement shifts were clearly seen in exon 2, 5, 6 and 12, respectively. No movement shift was seen in exon 9. Mutation confirmations were followed by direct sequencing. Using this approach we identified the molecular defect in 90 of the 97 above samples. Our results show a total of seven variants, the most common being at nucleotide (nt) 1376 mutation G-->T 42.3% (41/97) and at nt 1388 mutation G-->A 34.0% (33/97). The other mutation sites were at nucleotide 95 A--G (5.2%), nucleotide 392 G-->T (4.1%), nucleotide 493 A-->G (3.1%) and two new variants with mutation at nucleotide 371 A-->G (2.1%) and nucleotide 519 C-->G (2.1%). Still 7.2% (7/97) remained unidentified. There was no significant difference in G6PD activity among the different mutations. Simple and fast, PCR/SSCP may be suitable for molecular screening for G6PD deficiency in Chinese and other people.

Female↗

PubMed1997

Probing calcium ion-induced conformational changes of Taiwan cobra phospholipase A2 by trinitrophenylation of lysine residues.

Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulfonate (TNBS). Three major derivatives, TNP-1, TNP-2, and TNP-3, were separated by high-performance liquid chromatography (HPLC) from the reaction mixtures in the absence of Ca2+. However, only TNP-2 and TNP-3 were isolated when trinitrophenylated reaction was carried out in the presence of Ca2+. TNP-1 and TNP-2 contained only one TNP group, on Lys-65 and Lys-6, respectively; and both Lys-6 and Lys-65 were modified in TNP-3. The extent of modification on Lys-6 and Lys-65 was calculated from the peak areas of TNP proteins in the HPLC profile. It was found that the susceptibility of Lys-6 toward TNBS markedly increased by the addition of Ca2+ when Ca2+ concentration was higher than 5 mM. With regard to the involvement of Lys-6 in the binding of substrate, the increase in the reactivity of Lys-6 may arise from a conformational change around Lys-6 for binding with substrate in the presence of Ca2+. Alternatively, the nonessentiality of Lys-65 for PLA2 activity was revealed by the finding that TNP-1 still retained 95% activity of native enzyme. Moreover, the reactivity of Lys-65 toward TNBS did not greatly change in either the absence or presence of Ca2+, suggesting that Ca2+ binding did not cause an appreciable change in the microenvironment around Lys-65. These results indicate that the differential reactivities of Lys-6 and Lys-65 toward TNBS as affected by the binding of Ca2+ are well consistent with their functional roles in the catalytic mechanism of PLA2, and suggest that the occurrence of conformational changes with PLA2 could be explored by chemical modification studies.

Amino Acid Sequence↗

PubMed1997

The structural variations of epsilon-amino groups in phospholipase A2 enzymes from Naja naja atra and Bungarus multicinctus venoms.

Comparative studies on Naja naja atra phospholipase A2 (NNA-PLA2), Bungarus multicinctus phospholipase A2 (BM-PLA2), and their Lys-modified derivatives were made to assess the differences in the fine structures around the conserved Lys residues of PLA2 enzymes. It has found that the accessibility of Lys residues of PLA2 enzymes toward modified reagent, trinitrobenzene sulfonate, were not the same. Moreover, the extent of decrease in pI values of PLA2 enzymes that resulted from trinitrophenylation of lysine residues was different between NNA-PLA2 and BM-PLA2. The Lys-6 of BM-PLA2 mostly contributed to the positively charged character of the enzyme molecule, whereas the contribution of Lys-6 of NNA-PLA2 to its molecular charge was not notably different from other Lys residues. A linear relationship was observed by plotting the mobilities of PLA2 enzymes and their TNP derivatives against their pI values. However, native and Lys-modified NNA-PLA2 were not aligned with those of BM-PLA2 in the same line. Apparently the gross conformation of PLA2 enzymes was not notably perturbed by the modification of Lys residues, but the fine structure of NNA-PLA2 was not the same as that of BM-PLA2. These results indicate that the positioning of side chains of the conserved Lys residues in the two PLA2 enzymes is essentially different, and suggest that the variations in the fine structures of homologous proteins could be effectively explored by chemical modification studies and electrophoretic analysis.

Amino Acid Sequence↗

PubMed1997

Amino acid sequence and chemical modification of a novel alpha-neurotoxin (Oh-5) from king cobra (Ophiophagus hannah) venom.

A novel alpha-neurotoxin, Oh-5, was isolated from king cobra (Ophiophagus hannah) venom and purified by successive SP-Sephadex C-25 column chromatography and reversed-phase HPLC. The complete sequence of Oh-5 was determined by Edman degradation of peptide fragments generated by endopeptidases, i.e., trypsin, Saccharomyces aureus V8 protease and lysyl endopeptidase. This novel toxin comprises 72 amino acid residues with 10 cysteines. The sequence shows 89% sequence homology with Oh-4, and 60% with Toxins a and b from the same venom. The tyrosine, tryptophan, lysine and arginine residues in Oh-5 were modified with tetranitromethane (TNM), 2-nitrophenylsulfenyl (NPS) chloride, trinitrobenzene sulfonate (TNBS), and p-hydroxyphenylglyoxal (HPG), respectively. Modification of Tyr-4 or Trp-27 did not affect the lethal toxicity at all, while the Tyr-4 and 23 nitrated derivative retained about 50% of the lethality of native toxin. Selective trinitrophenylation of Lys-51 or 69 resulted in a decrease in lethality by 29%, and 50% lethality was retained after modification of Lys-2, 51, and 69. A drastic decrease in lethality to 26% was observed when both Arg-35 and 37 were modified. The neurotoxicity was further decreased when Arg-9 was additionally modified. These results suggest that the aromatic residues, Tyr-4 and Trp-27, are not crucial for the neurotoxicity, whereas the cationic residues are involved in multipoint contact between the toxin molecule and the nicotinic acetylcholine receptor (nAChR). The residues Tyr-23 and Arg-35 and 37 in the central loop of Oh-5 seem to contribute greatly to the neurotoxicity.

Amino Acid Sequence↗

PubMed1997

A novel neurotoxin, cobrotoxin b, from Naja naja atra (Taiwan cobra) venom: purification, characterization, and gene organization.

A novel neurotoxin, cobrotoxin b, was isolated from Naja naja atra (Taiwan cobra) venom by successive chromatographies on gel filtration and SP-Sephadex C-25 columns. The yield of this novel toxin was 5% of that of cobrotoxin from the same venom. Its neurotoxicity determined as the inhibition of acetylcholine-induced muscle contractions was approximately 50% of that of cobrotoxin. Cobrotoxin b consists of 61 amino acid residues including 8 cysteine residues. Moreover, there are 12 amino acid substitutions between cobrotoxin b and cobrotoxin. The genomic DNA, with a size of 2,386bp, encoding the precursor of cobrotoxin b was isolated from the liver of N. naja atra. The gene consists of three exons separated by two introns. This exon/intron structure is essentially the same as that reported for the cobrotoxin gene. Moreover, the nucleotide sequences of the two neurotoxin genes exhibit 92% identity. These results highly suggest that the cobrotoxin b and cobrotoxin genes are derived from a common ancestor. Comparative analyses of cobrotoxin b and cobrotoxin precursors showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. This indicates that the protein-coding regions may have arised via accelerated evolution. BLAST searches for sequence similarity in the GeneBank databases showed that intron 1 of the cobrotoxin b and cobrotoxin genes encodes a small nucleolar RNA (snoRNA). However, the snoRNA gene is absent from the gene encoding the Laticauda semifasciata erabutoxin c precursor (L. semifasciata and N. naja atra are sea and land snakes, respectively). Since previous studies suggested the potential mobility of snoRNA genes during evolution, we propose that intron insertions or deletions of snoRNA genes occurred with the evolutionary divergence between the sea snake and land snake neurotoxins.

Amino Acid Sequence↗

PubMed1997

[Seroepidemiology of Japanese encephalitis viral infection among 3-6 years old children from mountainous and plains townships located in the northern, central, southern and eastern Taiwan].

In order to evaluate the Japanese encephalitis virus (JEV) vaccination program in rural Taiwan, we conducted a seroepidemiological survey of JEV among rural children 3 to 6 years of age in Taiwan. The children were selected through a systemic sampling following stratification by age of children in 4 selected aboriginal villages and 4 adjacent nonaboriginal villages. The overall vaccine coverage rate for the primary (2 doses) dose was 81.2% (1853/2281) with higher rates (87.7%-87.9%) found among the more recent birth cohort of 3 to 4 years of age. The neutralizing antibody (NT) against JEV was measured with plaque reduction neutralization test (PRNT) using Nakayama strain as the virus. With a positive NT antibody defined as > or = 1:10 dilution of serum yielding more than 50% plaque reduction, the overall JEV NT antibody positive rate among children receiving 3 doses of vaccine was 67%. However, the age-specific positive rates varied significantly with varying ages; the lowest of 47% being among children 4 years of age which was lower than the rates of 68%, 76% and 87% among children of 3, 5 and 6 years of age, respectively. This trend of rising seropositive rates of JEV antibody with increasing age among 4 and 6 years of age was also noted among children who had received no vaccine, suggesting the importance of natural infection among rural Taiwanese children. Despite the high frequency of natural infection, the seropositive rates of JEV antibody still correlated well with the dose of vaccine received, i.e., 67% (1122/1664), 66% (65/97), 33% (4/12) and 40% (19/47) for children receiving 3, 2, 1, and 0 dose of JE vaccines, respectively (P < 0.0001 Chi-square for trend test). When stratified analysis by dose and by type of vaccines was conducted, a significantly higher seropositive rate of JEV NT antibody was noted among children receiving JE vaccine of Beijing type (87%) than children receiving Nakayama type (39%) (p < 0.0001, Chi-square test). Our data indicated that the JEV vaccination, in conjunction with JEV natural infection, has maintained high JEV NT antibody level among rural children of Taiwan.

Age Factors↗

PubMed1997

Heparin-binding epidermal growth factor-like growth factor and transforming growth factor-alpha in human non-small cell lung cancers.

Transforming growth factor-alpha (TGF-alpha), a member of the epidermal growth factor (EGF) family that binds to the EGF receptor (EGFR), is thought to function in an autocrine manner in non-small cell lung cancers (NSCLC). Heparin-binding EGF-like growth factor (HB-EGF), a novel member of the EGF family, also binds to EGFR. To compare the expression of HB-EGF, TGF-alpha and EGFR genes in NSCLC and normal lung tissue, we measured the levels of messenger RNA (mRNA) for these genes in human NSCLC and normal lung tissues by Northern hybridization, reverse transcription-polymerase chain reaction (RT-PCR), and in situ hybridization. A total of eight specimens (paired tumor tissue and normal lung tissue) were harvested from four patients who underwent resection of primary resectable NSCLC. HB-EGF was not expressed in either tumor tissue or normal lung tissue, while EGFR and TGF-alpha were expressed in all samples. TGF-alpha was overexpressed in all tumor tissue samples by several hundred-fold, while the expression of EGFR was not significantly different in tumor tissue and normal lung tissue. There was no correlation between the expression of TGF-alpha and EGFR. In situ hybridization showed that TGF-alpha mRNA was localized mainly in the cancer cells of tumor tissues and in the macrophages of alveoli in normal lung tissue. Our results showed that HB-EGF plays no role in the growth of NSCLC, and that there was no significant overexpression of EGFR in tumor tissue. TGF-alpha may play a major role in the growth of NSCLC. This supports a new direction in rational NSCLC treatment.

Carcinoma, Non-Small-Cell Lung↗

PubMed1997

Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis--a case report of corneal ulcer.

We report a case of corneal ulcer caused by nontuberculous mycobacteria which was confirmed by smear and culture. We attempted a new method for the rapid identification of mycobacteria to the species level on the basis of evaluation by the polymerase chain reaction of the gene encoding. The method is involved with restriction enzyme analysis of PCR product obtained with primers common to all mycobacteria. Using the restriction enzyme Bst EII and Hae III, clinically relevant and other frequent laboratory isolates were differentiated to the species or subspecies level by PCR-restriction enzyme analysis. The main prevalence of pattern analysis is Mycobacterium chelonae subsp. abscessus in this case. The outcome suggests that PCR-restriction enzyme analysis should be a useful method for early diagnosis concerning nontuberculous mycobacterial keratitis.

Corneal Ulcer↗

PubMed1996

Ethnic differences in immune responses to hepatitis B vaccine.

A national vaccination program against hepatitis B virus (HBV) to immunize every newborn was initiated in Taiwan in 1986. A serologic survey of 1,812 fully vaccinated children residing in four aboriginal villages and four adjacent nonaboriginal Han Chinese rural villages was conducted in 1993. Children in three of the four aboriginal villages had significantly lower titers of antibody against hepatitis B surface antigen (anti-HBs) than did children in the nonaboriginal villages. Evaluation of cold chain operation for vaccine storage and transport suggested that cold chain failure was not responsible for the fact that children residing in the more remote aboriginal villages had lower mean titers of anti-HBs. However, children whose parents were both aborigines had lower anti-HBs mean titer than did children whose parents were both ethnic Han Chinese. Children of mixed parental origins had intermediate mean titer of anti-HBs. Serologic responses to Japanese encephalitis virus and diphtheria vaccines did not show such correlation with ethnic groups, indicating that the determinant for HBV hyporesponsiveness among the aboriginal children is distinct from that of other childhood vaccines. It was therefore concluded that host factors pertaining to ethnic origin might be responsible for the hyporesponsiveness to HBV vaccine in the aboriginal populations. This finding, if substantiated with further prospective studies, might provide possible means for more targeted trials to improve vaccine response and to reduce vaccine failure among these well-defined ethnic groups.

Child↗

PubMed1996

Chemical modification of cationic residues in toxin a from king cobra (Ophiophagus hannah) venom.

The cationic groups of arginine and lysine residues in alpha-neurotoxin, Toxin a, isolated from king cobra (Ophiophagus hannah) venom were subjected to modification with trinitrobenzene sulfonate (TNBS) and p-hydroxyphenylglyoxal (HPG), respectively. The trinitrophenylated (TNP) derivatives of Toxin a at Lys-10, 56, or 71 showed approximately 25% residual lethality, and modifications on Lys-10 and 56 or Lys-10 and 50 resulted in a decrease of lethality by 84% and 86%, respectively. Modifications on Arg-34, 37, and 70 and Arg-34, 37, and 72 in Toxin a caused a decrease in lethality by 92% and 93%, respectively, and it almost completely lost its lethality and binding activity to nicotinic acetylcholine receptor (nAChR) when all four arginine residues were modified. These results indicate that in addition to the cationic residues on loop II (Arg-34, 37), loop III (Lys-50, 56), and the C-terminal tail (Arg-70, 72; Lys-71), Lys-10 on loop I is also related to the neurotoxicity of Toxin a.

Amino Acid Sequence↗

PubMed1996

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2.

In order to address the mechanism whereby Ca2+ wad crucial for the manifestation of the enzymatic activity of phospholipase A2 (PLA2), four divalent cations were used to assess their influences on the catalytic activity and the fine structures of Naja naja atra PLA2. It was found that substitution of Mg2+ or Sr2+ for Ca2+ in the substrate solution caused a decrease in the PLA2 activity to 77.5% or 54.5%, respectively, of that in the presence of Ca2+. However, no PLA2 activity was observed with the addition of Ba2+. With the exception of Mg2+, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA2 markedly increased with the binding of cations to PLA2. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate and p-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca2+, Sr2+, and Ba2+, but not by Mg2+. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba2+ > Sr2+ > Ca2+ > Mg2+, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA2 and that Tyr-3, Lys-6, and Tyr-63 of PLA2 are involved in the binding with the substrate, suggest that the binding of Ca2+ to PLA2 induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg2+ or the bigger atomic radii with Sr2+ and Ba2+ might render the conformation improperly rearranged after their binding to PLA2 molecule.

Anilino Naphthalenesulfonates↗

PubMed1996

Determination of urinary zinc, chromium, and copper in steel production workers.

The aim of our investigation was to determine the concentrations of Cu, Zn, and Cr in urine samples under routine clinical laboratory conditions. To assess the reliability of these methods, critical factors such as detection limit(s), calibration range(s), cost, accuracy, and precision were studied. Our method was employed for the quantitative determination of zinc, chromium, and copper in urine samples from steel production and quality control (QC) workers and healthy unexposed controls. After pretreatment with acids, the samples were digested via a microwave oven. Zinc was determined by flame absorption spectrophotometry (FAAS), whereas chromium and copper were determined by a graphite-furnace atomic absorption spectrophotometry (GFAAS). Our results indicate that urinary zinc, chromium, and copper levels of the exposed workers are significantly higher than those of the controls. The possibility that these metals are involved in the etiology of diseases is discussed and recommendations are made to improve workplace ventilation and industrial hygiene practices.

Calibration↗

PubMed1996

The interaction of 8-anilinonaphthalene-1-sulfonate with His-47 of Taiwan cobra phospholipase A2 perturbing by the binding of calcium ion.

The 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence intensity of ANS-Naja naja atra (Taiwan cobra) phospholipase A2 (PLA2) complex increased with the addition of Ca2+, but the observed fluorescence enhancement markedly decreased after methylation of His-47 in PLA2 molecule. However, the binding affinities of methylated PLA2 for ANS and Ca2+ were similar to or even greater than those observed with native PLA2. These results, together with the finding that ANS electrostatically interacted with His-47 of PLA2, suggest that the increase in the intensity of ANS fluorescence upon the addition of Ca2+, in part, arises from the ionic interaction of His-47 with ANS being perturbed by the binding of Ca2+.

Anilino Naphthalenesulfonates↗

PubMed1996

A significant decrease of the transcriptional activity of p53 mutants deriving from human functional adrenal tumors.

Recently, our laboratory has found a high incidence (77%) of p53 gene mutations in human functional adrenal tumors. Furthermore, the majority of mutant sites were assembled at codons 100, 102, and 249. These mutation sites are not common, and there have been no studies addressing whether or not these mutants points or mutant styles cause the p53 protein to lose function. It has been well known that p53 is a transcription factor. To examine the transcriptional activities of these mutant p53 genes from patients with functional adrenal tumors, we constructed p53 expression plasmids from tumors and paired adjacent normal adrenal gland tissues, using a transient co-transfection assay with a reporter gene in H358 cells. Wild-type p53 from normal adrenal gland tissues specifically trans-activates the expression of a chloramphenicol acetyltransferase (CAT) reporter gene in H358 cells. Three mutant p53 proteins (at codons 100, 102, and 249, respectively) from tumors showed a >90% loss of transcriptional activity. One mutant at codon 68, other than at hot spots, remained at approximately 65% transcriptional activity. An immunoprecipitation assay showed that the mutant proteins of codon 68 and codon 102 could respond to the three monoclonal antibodies (PAbDO-1, PAb1620, and PAb421), indicating that there were no obvious changes in the antigenicity of the proteins. However, the mutant protein of codon 249 could not respond to the carboxy-terminus-specific antibody PAb421 and conformation-specific antibody PAb1620, indicating that there were some obvious changes in the conformation of the mutant proteins. The mutant protein of codon 100 could not be detected by immunoprecipitation assay but could be analyzed by Western blot. In a further study using a DNA-binding assay, it was shown that the loss of transcriptional activity was caused by the loss of DNA-binding ability. These results show that the p53 mutants, derived from functional adrenal tumors, actually lost DNA-binding ability and decreased the transcriptional activity. However, the role of the mutant protein in the tumorigenesis of functional adrenal tumors requires further investigation.

Adrenal Gland Neoplasms↗

PubMed1995

Factor VIII gene inversions in severe hemophilia A: results of an international consortium study.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

Blotting, Southern↗