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Two variants of smoldering multiple myeloma (SMM) have been recognised: (i) an evolving type, characterised by a progressive increase in the M-protein size and short time to progression to overt multiple myeloma (MM) and (ii) a non-evolving type, with a long-lasting, stable M-protein and longer time to progression. Comparative genomic hybridisation (CGH) analyses in both subtypes of SMM (seven evolving and eight non-evolving SMM) were performed. Evolving SMM showed cytogenetic changes consistent with those found in de novo symptomatic MM (1q gains, chromosome 13 deletions) while the non-evolving variant showed no 1q gains and deletions were uncommon.

Purpose: To allow the longitudinal investigation of molecular events associated with the progression of human hepatocellular carcinoma (HCC), we sought to develop a murine model by orthotopic implantation of tumor fragments obtained from patients diagnosed at early stage. Experimental Design: Tumor pieces (2 × 2 mm) were implanted on the liver surface of nu/nu mice. After xenograft growing, subsequent passages were performed to achieve long-term implant viability. Isolation of tumoral hepatocytes was done to establish new cell lines. HCC characteristics, proliferation rate, apoptotic index (terminal deoxynucleotidyl transferase-mediated nick end labeling), and expression of cell-cycle regulators (cyclins E and A, p21Cip1, p27Kip1, p16INK4a, pRb, and p53) were assessed by Western Blot and immunohistochemistry, to correlate them with tumor progression. Results: Five (50%) of the 10 primary HCCs resulted in small slow-growing liver implants. Three of them are viable after 48 months, whereas the remaining two survived for 15 and 13 months. Xenografts throughout passages exhibited a more aggressive phenotype with a poorer degree of differentiation, intense proliferation, moderate apoptosis, cell-cycle deregulation, p53 alterations, microvascular invasion, and dissemination. In one single passage, we observed critical growth delay, which was associated with significant p27kip1 overexpression. We established the anchor-free growing BCLC-9 cell line from one xenograft. This has gains of chromosomes 7, 5p, 6q, and 9q, is hepatitis B virus-DNA positive, does not secrete α-fetoprotein, and has TP53 missense mutations in codons 192 and 242. Conclusions: The orthotopic implantation of early HCC fragments in nude mice provides a useful model to investigate the mechanisms of human HCC evolution and to establish new cell lines.

T-cell prolymphocytic leukemia (T-PLL) is a rare postthymic T-cell disorder that may show different morphologic variants and a very aggressive clinical behavior. On occasion, patients may present with an indolent clinical course and long survival. The disease is genetically characterized by the presence of complex karyotypes with recurrent alterations involving chromosomes 8, 14, and 11. The possible relationship between genetic alterations and morphologic variants and the clinical course of the disease, however, is not well known. Comparative genomic hybridization (CGH) was used to detect chromosomal imbalances in eight patients diagnosed with T-PLL, including three cases of small-cell variant with an indolent clinical evolution. Abnormal profiles were detected in all cases. The chromosomal regions most often over-represented were 8q (75%), 5p (62%), and 14q (37%), as well as 6p and 21 (both 25%). The chromosomal regions most often underrepresented were 8p and 11q (75%), 13q (37%), and 6q, 7q, 16q, 17p, and 17q (25%). The number of chromosomal imbalances in the three small-cell variants was relatively similar to that of cases with typical morphology. Only gains of 6p and losses of 7q present in three and two cases, respectively, with typical morphology were not detected in any small-cell variant. CGH analysis revealed alterations in 15 chromosomal regions not detected by conventional cytogenetics. These results indicate that T-PLL carry a high number of chromosomal alterations that are not related to the morphologic variants or the clinical behavior of the disease.

Patients with smoldering multiple myeloma (SMM) meet the diagnostic criteria of multiple myeloma (MM) but are asymptomatic. Between January 1978 and July 2001, 53 patients (median age 63 years) were diagnosed with SMM. The median serum M-protein and proportion of bone marrow plasma cells were 36 g/l and 27% respectively. Two subsets of SMM were identified: (i) evolving SMM (n = 22), characterized by a progressive increase in serum M-protein, a previously recognized monoclonal gammopathy of undetermined significance (MGUS) and a significant higher proportion of IgA type and (ii) non-evolving SMM (n = 26) with stable M-protein that abruptly increases when symptomatic MM develops. Thirty-four patients developed symptomatic MM. The median time to progression in the overall series was 3.2 years and the only feature associated with a shorter time to progression was the evolving versus non-evolving type (1.3 vs. 3.9 years respectively, P = 0.007). The pattern of progression consisted of anaemia, lytic bone lesions or both, without renal failure, hypercalcaemia or extramedullary plasmacytomas. Fifty-seven per cent of patients that required chemotherapy showed no or minimal response. The median survival from diagnosis and from progression was 8.2 and 3.5 years respectively.

Three new cases are reported of cytogenetically Philadelphia-negative (Ph-) chronic myelocytic leukemia (CML), with positive BCR/ABL gene rearrangement according to a reverse transcriptase polymerase chain reaction technique. Fluorescence in situ hybridization (FISH) studies using different probes showed three different situations involving chromosomes 9 and 22 for the masked BCR/ABL fusion gene. With the use of BCR/ABL-extra signal and CEP 9 probes (Vysis, Downers Grove, IL, USA), FISH studies detected the BCR/ABL fusion gene at the end of chromosome 9 in patient 1, a BCR/ABL fusion gene on both chromosomes 22 in patient 2 (who was in an accelerated phase of CML), and a BCR/ABL fusion signal on chromosome 22 in patient 3. Interestingly, FISH interphase signals showed the same pattern in patients 1 and 3, but the BCR/ABL fusion gene was located on different chromosomes. Careful interpretation of the results and a simultaneous study of nuclei and metaphases are therefore recommended in each case. In conclusion, in cases of Ph- CML, FISH studies are of paramount importance since they can detect chromosomal reorganization and its location, and can also provide quantitative follow-up of these patients.

The presence of a polymorphic (GT)(n) repeat, a microsatellite repeat, at the human dopamine beta-hydroxylase (DBH) gene had been previously investigated in healthy people and in schizophrenic patients. The different DBH genotypes had been found to be associated to different DBH biochemical function, but no differences were found in the allelic and genotype frequencies between schizophrenic and control groups. To further clarify the potential involvement of the variation at the DBH gene in schizophrenia we have studied the DBH (GT)(n) repeat in a sample of 47 Spanish schizophrenic patients, in their healthy relatives (n = 72), and in a control population (n = 74). We have been able to identify five different variants of the DBH gene (A1, A2, A3, A4, A5) in the different groups. Subsequent statistical analysis revealed that the genotypes as well as the allele frequencies did not differ significantly among schizophrenic patients and the control population. Interestingly, the allelic variant A2 and the genotype A4/A2 were significantly more frequent in schizophrenic patients as compared with their healthy relatives. However, the association of the A2 allele with schizophrenia was not supported by the haplotype relative risk analysis of transmitted versus nontransmitted alleles. Therefore, although it will be important to extend the present analysis in a larger sample of schizophrenic patients and controls, our results suggest that the (GT)(n) does not seem to play a major role in the genetics of schizophrenia at least in this group of Spanish schizophrenic patients. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:88-92, 2000.

An increased risk of Alzheimer disease (AD) has been reported in young mothers of Down syndrome (DS) probands. Subsequently, an increased frequency of the apolipoprotein E (apoE) allele epsilon 4 has been found in mothers (or = 32 years) of DS children due to meiosis II (MII) errors providing a potential explanation for the increased risk of AD in DS mothers. In the present study we genotyped apoE and determined the origin of non-disjunction of 132 mothers and the corresponding fathers and DS children from Spain. Unexpectedly no epsilon 4 alleles have been detected in MII mothers ofor = 32 years of age (P = 0.02). Thus our study not only fails to find the effect previously reported, but it detects an opposite correlation. An increase in the epsilon 4 frequency (0.227) is detected in MI mothers28 as compared to the epsilon 4 frequency present in MI mothers28 years of age (0.089), although the differences are not significant if correction for multiple comparisons is applied. The simplest overall interpretation of the previously reported and present findings is that the detected associations are due to random statistical variation rather than to some real effect of the epsilon 4 allele. However the important potential implications of alternative explanations imply that this issue deserves further clarification in independent studies in other populations.

Potential regulatory DNA elements within the rat protamine P1 promoter region have been identified using gel retardation assays and DNase I footprinting analysis with rat nuclear extracts obtained from different tissues. Distinctive gel shift bands are generated by rat nuclear extracts from mature testis, kidney, brain, spleen and liver, which bind to the cis-acting SRE (Serum response element) and Prot1C (Protamine 1 Consensus) oligonucleotide sequences [Queralt R. and Oliva R. Gene. 133 (1993) 197-204]. In vitro DNase I footprinting analysis demonstrates protection of the SRE region at positions -124 to -115. In addition, we have detected protection in two new regions adjacent to the SRE that we called SAP (SRE Adjoining Protection; nt -153 to -141) and SEP (SRE Extended Protection; nt-114 to -100), respectively. These sequences, SAP and SEP, show no apparent consensus homology with cis-acting elements of other known transcription factors. Two additional DNase I protected regions have also been found at positions +27 to +46 and +61 to +88, the first of which contains the sequence +42 TCGNNNNNGCCAA +30 recognized by nuclear factor I (NFI).

In order to detect regulatory conserved DNA elements within the protamine 1-encoding gene ( P 1) promoter, we have sequenced this region from the rat, guinea pig, gorilla, orangutan, anubis baboon and red monkey P 1 genes and compared it to the homologous human, bull, boar and mouse nucleotide (nt) sequences. We demonstrate the presence of a consensus sequence, HSMCYTCAYAAT ( Prot1C : protamine 1 consensus), from nt position — 64 to position — 53 in all P 1 genes whose promoter sequences are now known. We also show that sequences similar to Prot1C are found in the promoter region of other testis-specific genes, such as the transition protein 1-encoding gene promoter which is thought to have derived from the P 1 genes. The relevance of this conserved element in the expression of P 1 genes is strongly supported by the recent demonstration of a mouse testis trans -acting factor [Tet-1; Tamura et al., J. Biol. Chem. 267 (1992) 4327–4332] which binds and matches in the mouse the first 11 bp of the corresponding consensus Prot1C sequence reported here. Another highly conserved element (TGTGAGG) has been identified 20 ± 3 nt upstream from ProtlC . This sequence forms a perfect palindrome with the central 7 nt of Prot1C and is absent in the homologous region of other genes. Further upstream, at positions −113 to −132, a third highly conserved region is present (MATGCCCATATWTGGRCAYG) which is similar to the c- fos SRE (serum-response element) and contains the central core common to all SRE s. This element has not been found in the homologous region of other sperm-specific genes. (H, A or C or T; M, A or C; R, A or G; S, C or G; W, A or T; Y, C or T.)

Male individuals with a 46,XX karyotype have been designated as XX males. In 80% of the cases, the presence of Yp sequences, including the male sex-determining gene, SRY, has been demonstrated by molecular and/or fluorescence in situ hybridization (FISH) analyses. In most cases, Yp sequences are located on the short arm of the X chromosome, resulting from unequal recombination between Yp and Xp during paternal meiosis. Much less frequent in XX males is the localization of the SRY gene to an autosome. Here we report on the genetic investigation of an atypical XX male in which the SRY gene was located at the end of the long arm of chromosome 1. The patient, with a normal male phenotype, was referred for azoospermia. Conventional cytogenetic analysis showed a 46,XX karyotype. Molecular-cytogenetics (FISH) and molecular (PCR and MLPA) studies identified not only Yp-specific sequences located on the distal long arm of chromosome 1 but also the deletion of the subtelomeric 1qter region. A specific phenotype has been reported for a deletion of the 1qter region associated with mental retardation. The molecular investigation of the 1qter region showed that in our patient the microdeletion is more telomeric than in patients reported with mental retardation. To our knowledge, this is the first report of a XX male with the Yp region transferred to the terminal long arm of chromosome 1. This is also the first microdeletion of the subtelomeric 1qter region not associated with mental retardation.

To allow the longitudinal investigation of molecular events associated with the progression of human hepatocellular carcinoma (HCC), we sought to develop a murine model by orthotopic implantation of tumor fragments obtained from patients diagnosed at early stage.Tumor pieces (2 x 2 mm) were implanted on the liver surface of nu/nu mice. After xenograft growing, subsequent passages were performed to achieve long-term implant viability. Isolation of tumoral hepatocytes was done to establish new cell lines. HCC characteristics, proliferation rate, apoptotic index (terminal deoxynucleotidyl transferase-mediated nick end labeling), and expression of cell-cycle regulators (cyclins E and A, p21(Cip1), p27(Kip1), p16(INK4a), pRb, and p53) were assessed by Western Blot and immunohistochemistry, to correlate them with tumor progression.Five (50%) of the 10 primary HCCs resulted in small slow-growing liver implants. Three of them are viable after 48 months, whereas the remaining two survived for 15 and 13 months. Xenografts throughout passages exhibited a more aggressive phenotype with a poorer degree of differentiation, intense proliferation, moderate apoptosis, cell-cycle deregulation, p53 alterations, microvascular invasion, and dissemination. In one single passage, we observed critical growth delay, which was associated with significant p27(kip1) overexpression. We established the anchor-free growing BCLC-9 cell line from one xenograft. This has gains of chromosomes 7, 5p, 6q, and 9q, is hepatitis B virus-DNA positive, does not secrete alpha-fetoprotein, and has TP53 missense mutations in codons 192 and 242.The orthotopic implantation of early HCC fragments in nude mice provides a useful model to investigate the mechanisms of human HCC evolution and to establish new cell lines.

Background Autosomal dominant early-onset Alzheimer disease is a heterogeneous condition that has been associated with mutations in 3 different genes: the amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) genes. Most cases are due to mutations in thePSEN1gene, whereas mutations in theAPPandPSEN2genes are rare. Objective To describe a novel mutation in thePSEN2gene associated with early-onset autosomal dominant Alzheimer disease. Patients and Methods The proband was a 49-year-old individual who displayed progressive dementia beginning at age 45 years. One of the parents and one of the grandparents had developed dementia at ages 64 years and 60 years, respectively, and 1 sibling had mild cognitive impairment. Some family members also had Tourette syndrome. Mutation analysis of theAPP,PSEN1,PSEN2,and tau (TAU) genes was performed. Apolipoprotein E (APOE) was also genotyped. Results We found a missense mutation at codon 430 of thePSEN2gene that predicts a threonine-to-methionine substitution. This mutation was detected in the affected individuals and in 1 cognitively healthy sibling. The mutation was absent in 260 control chromosomes. The normal amino acid was conserved in the human and mousePSEN1and mousePSEN2homologues. No influence of theAPOEgenotype was observed. Conclusions We have found a novel mutation in thePSEN2gene in a family with early-onset Alzheimer disease. The variation in the age at onset confirms thatPSEN2mutations are associated with variable clinical expression.

A novel mutation (V89L) in the presenilin 1 (PSEN1) gene is described in a family with pathologically confirmed Alzheimer's disease. The mutation was identified in two affected members with early onset Alzheimer's disease characterised by early and marked behavioural disturbances. The mutation is located on the same side of the helix as other described mutations in the first transmembrane domain and its relation to other mutations in this helix suggests that they share a common pathogenic mechanism.

The authors describe a novel missense mutation in the presenilin 2 (PSEN2) gene at residue 439 that predicts an aspartate-to-alanine substitution (D439A). This mutation was found in a 58-year old patient who displayed a progressive dementia at the age of 52. The mutation was absent in his cognitively normal relatives. Haplotype analysis indicated that his affected mother was the most probable mutation carrier. The D439A mutation is located near the C-terminal end of the PS2 protein, a region critical for endoproteolytic processing.

In a family with early-onset Alzheimer disease (EOAD) from Spain we found a mutation in the presenilin 1 (PS1) gene that predicts a methionine-to-threonine change at the PS1 residue 139 (M139T). This mutation was previously reported in a independent French family. The age of onset of the disease was similar in the affected members from both families, suggesting a specific age of expression (range 47-50 years). The detection of the M139T mutation in an independent EOAD family strongly supports the pathogenicity of this mutation in familial Alzheimer disease (AD).

Maternal and paternal uniparental disomy of chromosome 13 have been associated with normal phenotypes. We report on a new case of paternal isodisomy 13 in a phenotypically normal girl. Prenatal diagnosis had shown a 46,XX,-13,der(13;13) karyotype in chorionic villi and a 45,XX,der(13;13) karyotype in amniocytes and fetal blood. Molecular studies demonstrated that the de novo der(13;13) was an isochromosome 13 of paternal origin. This observation supports the lack of imprinting effects on chromosome 13 and trisomy rescue as a mechanism leading to uniparental disomy in cases involving isochromosomes.

We have cloned and sequenced 1398 bp of the rat HFE gene promoter region. The alignment of the rat promoter HFE sequence with the HFE promoter sequence from human and mouse detected several highly conserved sequences present at orthologous or heterologous positions in the three species. Subsequent analysis of the conserved promoter sequences identified the presence of 10 novel transcription elements present in the promoter regions of the human, mouse and rat HFE genes (GATA, NF-IL6, AP1, AP2, CREB, PEA3, γ -IRE, GFI1, HNF-3 β , HFH2). Different gel retardation analyses performed with rat-liver nuclear extracts have confirmed the presence of factors binding to some of these transcription elements. This represents the first data concerning the identification of potential transcriptional elements of the HFE promoter in these three species. The expression pattern of the transcription factors corresponding to the novel elements identified in the HFE promoter is consistent with the potential role of the HFE promoter in the transcription regulation and function of the HFE gene. Knowledge of the identified conserved elements in the HFE promoter from human, mouse and rat provides the basis for subsequent in-vitro or in-vivo studies leading to identification of the detailed mechanisms involved in the regulation of the iron metabolism and the design of potential future alternative therapies.

Prolamine P1 genes have been sequenced following PCR amplification from 11 mammals representing five major mammalian orders: Rodentia (rat and guinea pig), Carnivora (cat and bear), Proboscidea (elephant), Perissodactyla (horse), and Artiodactyla (camel, deer, elk, moose, and gazelle). The predicted amino acid sequence for these genes together with previously reported sequences results in a data set of 25 different P1 genes and 30 different P1 amino acid sequences. The alignment of all these sequences reveals that prolamines are amongst the most rapidly diverging proteins studied. In spite of the large number of differences there are conserved motifs that are also common to birds such as the N-terminal ARYR followed by the triple alternating SRSRSR phosphorylation site. The central region contains 3 arginine clusters consisting of 5–6 arginines each. The C-terminus appears to be the most variable region of the protamines. Overall the molecular evolution of P1 genes is in agreement with the expected species evolution supporting that these genes have evolved vertically.

Protamine P1 genes have been sequenced by PCR amplification and direct DNA sequencing from 9 primates representing 5 major families, Cebidae (new world monkeys), Cercopithecidae (old world monkeys), Hylobatidae (gibbons), Pongidae (gorilla, orangutan, and chimpanzee), and Hominidae (human). In this recently diverged group of primates these genes are clearly orthologous but very variable, both at the DNA level and in their expressed amino acid sequences. The rate of variation amongst the protamine Pls indicates that they are amongst the most rapidly diverging polypeptides studied. However, some regions are conserved both in primates and generally in other placental mammals. These are the 13 N-terminal residues (including a region of alternating serine and arginine residues (the motif SRSR, res. 10–13) susceptible to Ser phosphorylation), a tract of six Arg residues (res. 24–29) in the center of the molecule, and a six-residue region (RCCRRR, res. 39–44), consisting of a pair of cysteines flanked by arginines. Detailed consideration of nearest neighbor matrices and trees based on maximum parsimony indicates that PI genes from humans, gorillas, and chimpanzees are very similar. The amino acid and nucleotide differences between humans and gorillas. are fewer than those between humans and chimpanzees. This finding is at variance with data from DNA-DNA hybridization and extensive globin and mitochondrial DNA sequences which place human and chimpanzee as closest relatives in the super family, Hominoidea. This may be related to the fact that protamine Pls are expressed in germ line rather than somatic cells. In contrast to the variability of the exon regions of the protamine P1 genes, the sequence of the single intron is highly conserved.

Background The relative contribution of mutations in the presenilin ( PSEN ) and amyloid precursor protein genes to autosomal dominant and other early-onset Alzheimer disease (AD) cases is not well established. Objectives To clarify the respective contribution of the amyloid precursor protein and PSEN mutations to autosomal dominant AD and to determine its contribution to sporadic and familial nonautosomal dominant early-onset AD and familial late-onset AD in a referral-based Spanish population. Subjects and Methods Ninety-four patients with AD (60 with early-onset AD and 34 with late-onset AD) from 82 independent families were studied. According to the family history, patients were classified into the following groups: autosomal dominant, familial nonautosomal dominant, and sporadic. Mutational analysis of the coding regions of the amyloid precursor protein, presenilin 1, and presenilin 2 was performed in all patients. Apolipoprotein E was also genotyped. Results Of the 60 early-onset cases, 44 from 36 families had a positive family history (11 with autosomal dominant AD and 25 with familial nonautosomal dominant AD) and 16 were sporadic. The frequency of mutations was 54.6% (6/11) among the autosomal dominant group, 6.2% (1/16) among the sporadic group, and 4% (1/25) among the familial nonautosomal dominant AD group. Most PSEN mutations (92%) would have been detected using a cutoff age of 58 years. The apolipoprotein E ϵ4 allele frequency was increased among early-onset AD without PSEN mutations. Conclusions More than half of the families with autosomal dominant early-onset AD can be explained by coding mutations in the PSEN genes. In the familial and sporadic early-onset groups mutations are rare. When family history is unavailable, an age of 58 years may be used as a cutoff point for genetic analysis. The increased apolipoprotein E ϵ4 allele in patients without PSEN mutations confirms that it is an important risk factor in the cause of non-PSEN early-onset AD.

Protamines are among the most variable nuclear proteins known in eukaryotes. In order to learn more about their evolution and function in humans and to explore the possibility of potential applications in forensic medicine we have developed a rapid method to amplify and directly sequence the protamine P1 gene simultaneously in many different samples. The method takes only 3.5 h from genomic DNA to the sequencing reactions. Despite the high variability of these genes only one polymorphic site was detected at the coding region level in different individuals. This polymorphic variation does not create a change in the amino-acid sequence of the protamine. Because all the protamine genes sequenced from different species are markedly different among them as well as to the human sequence, amplification and direct sequencing of this gene can be used to unequivocally identify the human or animal origin of biological specimens. Furthermore, the single polymorphic site detected in the human P1 gene could be useful in conjunction with other markers in identification studies in humans.