TP53 polymorphism, HPV infection, and risk of cervical cancer.

The role of a polymorphism at position 72 of the tumor suppressor gene TP53 in the development of cervical cancer is not well established. The arginine variant of the p53 protein could be more susceptible to degradation by human papillomavirus (HPV) E6 protein than the protein containing proline. Recent studies show controversial results. We investigated a possible association between TP53 polymorphism and cervical cancer in a Peruvian population with high prevalence of HPV infection. HPV status and TP53 polymorphism were determined for 119 cases of invasive cervical cancer and 127 control women from Peru. HPV infection was detected by PCR of cervical cells or tumor biopsies. For determination of TP53 polymorphism, exon 4 of the TP53 gene was amplified by PCR, and DNA was subsequently subjected to restriction enzyme digest. Associations between TP53 polymorphism, HPV infection, and cervical cancer were assessed using logistic regression. Women homozygotes for arginine had a 2.2-fold increased risk (95% confidence interval: 0.6-7.6) for cervical cancer. The odds ratio for women heterozygotes for Arg/Pro was 3.5 (95% confidence interval: 0.9-14). Similarly increased risks were found when restricting analysis to HPV-positive women only. The distribution of TP53 genotypes in this Peruvian population was comparable with that found in Caucasians. Our results cannot rule out an association between the TP53 polymorphism at codon 72, HPV infection, and the etiology of cervical cancer.

Absence of association between HPV DNA, TP53 codon 72 polymorphism, and risk of oesophageal cancer in a high-risk area of China.

We analyzed TP53 codon 72 polymorphism, HPV DNA in 32 subjects with oesophageal cancer and 57 healthy subjects with normal oesophageal cytology from an area of China with a high prevalence for this cancer (Linxian County, Henan Province). The frequency of the proline allele (0.63) was significantly higher in the Chinese population than in most European or American populations. No significant association was found between TP53 codon 72 genotype and cancer. We also found a low prevalence of HPV DNA (6.7%) in both cancer cases and healthy subjects. Our findings do not support the association between risk of oesophageal cancer, human papilloma virus infection, and TP53 codon 72 polymorphism in a Chinese population at high risk of oesophageal cancer.

Mutation at position -12 of intron 45 (c-->t) plays a prevalent role in the partial skipping of exon 46 from the transcript of allele alphaLELY in erythroid cells.

Allele alphaLELY is a common low-expression allele of the erythroid spectrin SPTA1 gene. It results in the aggravated expression of hereditary elliptocytosis due to SPTA1 gene mutations occurring in trans. Allele alphaLELY contains, in particular, mutations in introns 45 and 46, both in polypyrimidine tracts, and causes the partial skipping of exon 46. The corresponding six amino acids belong to the nucleation site where the dimerization process of spectrin begins. In this work we investigated the cause of exon 46 partial skipping. We made four types of constructs with or without the intron 45 mutation, and with or without intron 46 mutation. Intron 45 mutation by itself dramatically triggered partial skipping of exon 46. Intron 46 mutation had no effect by itself. It was not possible to assess whether it modulated, even to a very small extent, the activity of intron 45 mutation. Taken together, intron 45 mutation is the prevalent, if not the exclusive, determinant of the partial skipping of exon 46 in the transcript of allele alphaLELY.

Two distinct truncated variants of ankyrin associated with hereditary spherocytosis.

We present two distinct truncated variants of ankyrin associated with mild to moderate hereditary spherocytosis. Ankyrin Saint-Etienne 1 was manifested by an additional band located between bands 2.1 and 2.2. It was associated with a nonsense mutation in exon 39: TGG-->TGA; W1721X. Ankyrin Saint-Etienne 2 appeared as two faint bands underlining bands 2.1 and 2.2. It was associated with a nonsense mutation in exon 41: CGA-->TGA; R1833X. Overall ankyrin was diminished in splenectomized patients. Messenger RNAs Saint-Etienne 1 and 2 amounted to 20 and 37% of the total ankyrin mRNA, respectively. Ankyrin molecules truncated in their C-terminal region retain some ability to bind to the membrane whereas the bulk of nonsense mutations, located in more upstream regions, result in the mere disappearance of one haploid set of ankyrin. In the present cases, it was not possible to apportion the roles of ankyrin reduction and truncation in the pathogenesis of hereditary spherocytosis.

The exon 46-encoded sequence is essential for stability of human erythroid alpha-spectrin and heterodimer formation.

Human erythroid alpha-spectrin alleles responsible for hereditary elliptocytosis (alphaHE alleles) undergo increased incorporation into red blood cell membranes when the polymorphism alphaLELY (LELY: Low Expression LYon) occurs in trans. The alphaLELY polymorphism is characterized by a mutation in exon 40 at codon 1857 (CTA --> GTA, Leu --> Val) and the partial (50%) skipping of exon 46, which encodes residues 2177-2182 (Wilmotte et al, J Clin Invest 91:2091, 1993). Both of these peptide sequence alterations are located within the region of the alpha-chain involved in initiating heterodimer assembly, and either or both mutations could potentially contribute to decreased incorporation of alpha-chains from the alphaLELY allele in heterozygotes into red blood cell membranes. These possibilities were evaluated by testing the protease resistance and in vitro binding properties of normal and mutant recombinant 4-motif alpha subunit peptides containing the dimer initiation region. The two forms of alpha spectrin produced by alternative mRNA splicing of the alphaLELY allele were represented by alpha18-21(1857), a peptide with the codon 1857 mutation and retaining the exon 46 encoded sequence, and alpha18-21(1857-Delta46), a peptide carrying both the 1857 codon mutation and the exon 46 deletion. The properties of these two recombinant peptides were compared with alpha18-21, a peptide with the normal sequence at codon 1857 and retaining the exon 46 encoded sequence. The codon 1857 mutation does not adversely affect dimer formation, but it is responsible for the increased trypsin cleavage between the alphaIV and alphaV domains that was the characteristic feature initially used to identify the alphaLELY (SpalphaV/41) polymorphism (Alloisio et al, J Clin Invest 87:2169, 1991). Deletion of the six amino acids encoded by exon 46 perturbs folding of the alpha21 motif, because this region of the alpha18-21(1857-Delta46) peptide is rapidly degraded and this recombinant peptide is unusually prone to self-aggregation. Exon 46 deletion reduces, but does not eliminate, dimerization. Comparison of mild trypsin proteolytic products from an alphaLELY homozygote and the two alphaLELY recombinant peptides strongly suggests that little, if any, of the 50% of the alpha chains from the alphaLELY allele that contain the exon 46 deletion are incorporated into the mature erythroid membrane. Based on the in vitro analysis of recombinant alphaLELY peptides, the inability of detectable amounts of exon 46(-) alpha chains to assemble into the mature membrane skeleton in vivo is probably due to a combination of decreased dimer binding affinity and increased proteolytic degradation of these mutant chains.

Detection of Pneumocystis carinii DNA by PCR amplification in various rat organs in experimental pneumocystosis.

Pneumocystosis is usually a disease of the lungs, but the number of cases of extrapulmonary pneumocystosis has greatly increased during the AIDS epidemic. Much remains unknown about the frequency and mechanisms of dissemination. In the present study, a systematic search for Pneumocystis carinii by PCR with primers specific for mitochondrial rRNA was performed in the lung, liver, spleen and kidney of 12 immunosuppressed rats and two immunocompetent rats. The amplified products were analysed by Southern hybridisation with a digoxigenin-11-dUTP labeled probe. P. carinii DNA was found in lungs in all 14 rats and in at least one organ other than lung in 11 immunosuppressed rats and the two control rats. We suggest that extrapulmonary dissemination may not be an exceptional phenomenon in the course of pneumocystosis, but rather part of the natural evolution of the disease.

Mycobacterium avium complex strains from AIDS patients belong to a homogeneous group ascribed by rRNA typing methods.

16S rRNA RFLP analysis of Mycobacterium avium complex (MAC) strains isolated from 25 AIDS patients led to identification of seven ribotypes. The same ribotype was determined for strains from 19 patients with and without disseminated disease. When isolates representing the seven ribotypes were examined for their internal transcribed spacer (ITS) between the 16S and 23S rRNA gene nucleotide sequence, four different sequences, including a new ITS type, were recovered. All isolates with the most common ribotype belonged to the sequevar Mav-B. When MAC strains from AIDS patients were compared by ITS sequencing and ribotyping, a significant degree of homogeneity was observed. The discriminatory level reached with ribotyping might be useful for grouping isolates from different clinical sources.

Molecular genetics of hereditary elliptocytosis and hereditary spherocytosis.

Red cells ow their mechanical properties, that is, their resistance and their elastic deformability, to a protein network that laminates the lipid bilayer and to proteins spanning the latter. All proteins are interconnected. Their structure, as well as the structure of the corresponding genes, will be outlined. Numerous mutations have allowed to reclassify hereditary elliptocytosis (HE) and poikilocytosis (HP), and, more recently, hereditary spherocytosis (HS) into well defined subsets of hereditary hemolytic anemias. HE stems from changes in the SPTA1, SPTB, EL1 and (exceptionally) GPYC genes that encode spectrin alpha- and beta- chains, protein 4.1 and glycophorin C/D, respectively. HS derives from altercations in the ANK1, EPB3 and ELB42 genes, encoding ankyrin, band 3 and protein 4.2, respectively, and also in the SPTA1 and SPTB genes. We will present a repertory of the known mutations. Innumerable polymorphisms will not be considered here, except for a few remarkable ones. Some general points must be stressed on. (a) Clinically conspicuous disorders are often the result of two alleles interacting in trans to one another. Whereas one allele causes moderate symptoms by itself, the other one is usually silent in the simple heterozygous (and exceptionally in the homozygous) state. As a result, the number of potentially pathogenic alleles is much more important than had been initially suspected. (b) The reduction or the loss of a protein within multiprotein assemblies are frequently encountered in red cell membrane genetic diseases; it leads to the disruption of the complexes with the possible disappearance of the other proteins than the mutated protein. (c) The above genes being also expressed in nonerythroid tissues, one starts finding multisyndromic conditions adding non-hematological manifestations to hemolysis. It is puzzling, though, that such situations are not more frequent. (d) In practice, the molecular diagnosis of HE and HS has reached a semi-routine stage that helps very much the paediatricians and haematologists.

Ethnic distribution of allele alpha LELY, a low-expression allele of red-cell spectrin alpha-gene.

Allele alpha LELY is a low-expression allele of erythroid spectrin alpha-chain. It carries mutations both in exon 40 and intron 45 and is associated with partial skipping of exon 46. Allele alpha LELY remains asymptomatic by itself. In contrast, it enhances the expression level of deleterious alpha-alleles occurring in trans, and as such has clinical importance. The aim of this study was to evaluate the incidence of allele alpha LELY in various ethnic groups, i.e. Caucasians, African Blacks, Japanese and Chinese. Allele alpha LELY occurred in all groups investigated with a fairly uniform frequency: 31%, 21%, 20% and 22%, respectively. Mutations in exon 40 and intron 45 appeared linked to one another without exception. Partial skipping of exon 46 or the low-expression feature, whenever they could be assessed, were invariably observed. Allele alpha LELY appears to be an ancient and stable allele.

Band 4.2 Komatsu: 523 GAT-->TAT (175 Asp-->Tyr) in exon 4 of the band 4.2 gene associated with total deficiency of band 4.2, hemolytic anemia with ovalostomatocytosis and marked disruption of the cytoskeletal network.

A novel mutation of 523 GAT-->TAT (175 Asp-->Tyr) in exon 4 of the band 4.2 gene was detected in a 37-year-old Japanese patient with total lack of band 4.2 protein, designated as allele 4.2 Komatsu. In this patient, moderate uncompensated hemolytic anemia (red cell count 3.38 x 10(6)/microliters, hemoglobin 10.8 g/dl, hematocrit 30.9%, reticulocytes 12.4%, indirect bilirubin 1.84 mg/dl) with ovalostomatocytosis and increased osmotic fragility had been noted since birth. Family studies revealed no overt hemolytic anemia in other family members, essentially normal red cell morphology, and a normal profile of red cell membrane proteins including band 4.2. Genetic studies proved that the proband was homozygous and all the family members studied were heterozygous with respect to the mutation of 523 GAT-->TAT of the band 4.2 gene. Although band 4.2 was completely absent in the proband, trace amounts of 72 kDa and 74 kDa peptides were detected in the red cells of all the family members, in which the mutation of 424 GCT-->ACT at exon 3 of the band 4.2 gene (Nippon type) was not present. Electron microscopic studies with the surface replica method and the quick-freeze deep-etching method showed the most marked disorganization of the cytoskeletal network in the patient's red cells in situ among the cases of band 4.2 deficiencies we have studied. This suggests that the amino acid of the band 4.2 protein, which was affected by the present mutation in exon 4, is much more crucial for the functioning of band 4.2 protein than that at codon 142 in exon 3. The cytoplasmic domain of band 3 in the proband's red cells was essentially normal in protein chemistry and in gene analysis with single-stranded conformation polymorphism (SSCP).

A deletional frameshift mutation in spectrin beta-gene associated with hereditary elliptocytosis in spectrin Napoli.

We studied a clinically manifest, dominantly transmitted elliptocytosis in an Italian family. We found a new spectrin variant, designated spectrin Napoli. Its beta-chain was truncated in its C-terminal region (apparent MW 216 kD). It displayed a low expression level (15%). There was a 8 nt deletion: CTTTTGAGAAGT-->CTGT (nt 6255-6262), starting after codon 2053. This deletion was followed by a 54 nt (18 amino acids) missense sequence and terminated by the TGA triplet which normally overlaps codons 2074 and 2075 (CTTGAG). The overall length of the mutated beta-chain was comparable to that found in spectrin Nice, spectrin Tokyo and spectrin Tandil, which are other variants with truncated beta-chains; however, a distinct nonsense codon was used in spectrin Napoli.

Hereditary elliptocytosis associated with spectrin Le Puy in a Japanese family: ultrastructural aspect of the red cell skeleton.

A dominantly-inherited hereditary elliptocytosis of intermediate severity was recorded in a Japanese family from Yamagata. The condition was associated with a spectrin truncated beta-chain (MW: 214 kD; 31% of total beta-spectrin), and a defect of mutant spectrin as regards tetramerization and phosphorylation. cDNA analysis revealed skipping of exon X, the third-to-last exon of the spectrin beta-gene. At the gene level, a one-base substitution (A-->G) changed position +4 of the 5' donor splice site consensus sequence of intron X. This mutation has been described before in a French kindred, defining spectrin Le Puy. Electron micrographs following quick-freeze deep-etching showed that the skeletal network was disorganized.

Low expression allele alpha LELY of red cell spectrin is associated with mutations in exon 40 (alpha V/41 polymorphism) and intron 45 and with partial skipping of exon 46.

The alpha V/41 polymorphism of erythroid alpha-spectrin has been characterized initially by an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction (Alloisio N., L. Morlé, J. Maréchal, A.-F. Roux, M.-T. Ducluzeau, D. Guetarni, B. Pothier, F. Baklouti, A. Ghanem, R. Kastally, et al. 1991. J. Clin. Invest. 87:2169-2177). Until now, it has been found associated invariably with a low expression level of the corresponding alpha chain. Among 61 chromosomes investigated in French and North African individuals or kindreds, we observed 19 chromosomes with the alpha V/41 polymorphism. With no single exception, the latter displayed a point mutation in exon 40 (Leu-->Val; CTA-->GTA) at position alpha 1857. According to the triple helical model of spectrin structure, this change accounts for the peptide maps' abnormalities. Sequencing the entire alpha V domain cDNA disclosed, in addition, a partial skipping of exon 46. At the gene level, a substitution (C-->T) was evidenced at nucleotide -12 of intron 45. This mutation appeared linked to the exon 40 mutation in 17 chromosomes, again with no single exception, among 53 examined chromosomes. We hypothesized that the lack of exon 46 would hamper the nucleation process and eventually account for the low expression feature. The present doubly mutated allele was renamed allele alpha LELY (low expression, Lyon).

A splice site mutation of alpha-spectrin gene causing skipping of exon 18 in hereditary elliptocytosis.

Spectrin Oran (alpha II/21) has been reported previously as a variant of the alpha II domain. Its expression level is low (10% of total spectrin) in heterozygotes denoting a major disadvantage of the mutated alpha-chain dimer or tetramer with respect to their normal counterparts. Spectrin Oran is associated with symptomatic elliptocytosis in the homozygous state. A 1-minute digestion time allowed to perceive a fast trypsin cleavage (not existing normally) after Arg 890 (helix 3 of repeating segment alpha 9). The responsible change was the lack of amino acids 822 to 862 (helix 2 of repeating segment alpha 8). Such a situation fits with the phasing of spectrin according to which mutated helix 2 and distorted helix 3 are adjacent to one another. The internal position of the structural change accounts for the slight self-association defect. The ultimate genetic lesion was a G to A substitution (intronic position-1) in the acceptor splice site of intron 17 resulting in skipping of exon 18. The substitution also created an acceptor splice site 1 base downstream, but the latter was used at a low grade.

An alpha-spectrin mutation responsible for hereditary elliptocytosis associated in cis with the alpha v/41 polymorphism.

The alpha 207 Leu-->Pro mutation in spectrin has recently been identified as a cause of alpha I/50-46a hereditary elliptocytosis (HE) or pyropoikilocytosis among Black people. We have found this mutation in a Moroccan family in both the heterozygous and homozygous states. The mutated alpha-spectrin allele carried, in cis, the alpha V/41 polymorphism, a common polymorphism altering the peptide maps and associated with a low-expression level. This is the first report of the cis combination of an HE mutation and the alpha V/41 polymorphism. Presumably, such a combination accounts for the very low expression of the abnormal allele in the heterozygous state.

Prevalent skipping of an individual exon accounts for shortened protein 4.1 Presles.

An asymptomatic shortened variant of protein 4.1 (-8.5 Kd) was first recognized in the red blood cells and designated protein 4.1 Presles. We show here that the missing segment belongs to the 22/24 Kd domain. Protein 4.1 cDNA from reticulocytes was amplified, mapped, and sequenced. The truncation appeared to result from the prevalent skipping of an individual and alternatively spliced exon, also called motif II, whereas this motif is preferentially retained under normal conditions. The same phenomenon was observed in lympho-blastoid cells. Sequencing over 80 bp of intronic sequences 5' and 3' of motif II failed to reveal any change. A new alternative splice site was incidently found 81 nucleotide downstream of motif II in both normal and truncated 4.1 mRNA.

A deletional frameshift mutation of the beta-spectrin gene associated with elliptocytosis in spectrin Tokyo (beta 220/216).

A novel spectrin variant carrying a truncated beta-chain and designated Spectrin Tokyo (beta 220/216) is presented. It was associated with elliptocytosis and moderate uncompensated hemolysis. The dimer self-association was reduced. An increase of the alpha I 74-Kd fragment was detected upon partial trypsin digestion. Analysis of cDNA and genomic DNA showed a 1-base deletion in codon 2059 (GCC AGC-->GCA GCT; Ala-Ser-->Ala-Ala) that belongs to exon X of spectrin beta-gene. A missense sequence extended down to (new) codon 2075. Serine 2060, a potential phosphorylation site, was replaced by alanine. The shortened beta-chain failed to undergo phosphorylation in vitro. Spectrin Tokyo shared the same stop codon, overlapping normal codons 2076 and 2077 (CTG AAA), as Spectrin Nice (beta 220/216), which is caused by a dinucleotide insertion in codon 2046 and contains 2076 amino acids. However, for some reason, Spectrin Tokyo had a lower incorporation level into the membrane than Spectrin Nice.

Spectrin Jendouba: an alpha II/31 spectrin variant that is associated with elliptocytosis and carries a mutation distant from the dimer self-association site.

Spectrin Jendouba (alpha II/31) was found in a Tunisian family. In the heterozygous state, it is associated with asymptomatic elliptocytosis and a minimal defect in spectrin dimer self-association. On partial digestion of spectrin with trypsin, an abnormal cleavage appeared following Lys 788. Peptide and DNA sequencing indicated that the responsible mutation is alpha 791 Asp----Glu (GAC----GAA). As in most alpha-spectrin variants associated with elliptocytosis, the change alters helix 3 of the proposed triple helical model of spectrin structure. Modified helix 3 in repeat alpha 8 is the most distant from the N-terminus of alpha-spectrin in known variants associated with elliptocytosis.