Rosa Queralt

Smoldering multiple myeloma: natural history and recognition of an evolving type

Summary.  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.

Evolution of protamine P1 genes in mammals

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.

Evolution of protamine P1 genes in primates

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.

Comparative genomic hybridisation identifies two variants of smoldering multiple myeloma.

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.

A novel presenilin 2 gene mutation (D439A) in a patient with early-onset Alzheimer’s disease

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.

Cloning, sequencing and characterization of the rat hereditary hemochromatosis promoter: comparison of the human, mouse and rat HFE promoter regions

We have cloned and sequenced 1398bp 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, gamma-IRE, GFI1, HNF-3beta, 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.

Identification of conserved potential regulatory sequences of the protamine-encoding P1 genes from ten different mammals.

In order to detect regulatory conserved DNA elements within the protamine 1-encoding gene (P1) promoter, we have sequenced this region from the rat, guinea pig, gorilla, orangutan, anubis baboon and red monkey P1 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 P1 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 P1 genes. The relevance of this conserved element in the expression of P1 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 Prot1C. 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 SREs. This element has not been found in the homologous region of other sperm-specific genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Paternal isodisomy 13 in a normal newborn infant after trisomy rescue evidenced by prenatal diagnosis

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.

High levels of chromosomal imbalances in typical and small-cell variants of T-cell prolymphocytic leukemia

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.

Orthotopic implantation of human hepatocellular carcinoma in mice: analysis of tumor progression and establishment of the BCLC-9 cell line.

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.