Thrombopoietin receptor (Mpl) expression by megakaryocytes in myeloproliferative disorders

The thrombopoietin receptor (Mpl) is involved in the pathogenesis of chronic myeloproliferative disorders (CMPD). In this study, we determined Mpl expression by bone marrow cells and megakaryocytes in CMPD by applying laser microdissection, real-time RT-PCR, and immunohistochemistry. Mpl mRNA expression was significantly increased up to 9-fold in total bone marrow cells (p < 0.001) and up to 4-fold in megakaryocytes in chronic myeloproliferative disorders (n = 73) compared to normal controls (n = 26, p = 0.01). Immunohistochemistry revealed heterogeneous Mpl expression by megakaryocytes in CMPD with a stronger accentuation in idiopathic myelofibrosis (IMF) in comparison to polycythaemia vera (PV) and essential thrombocythemia (ET). In addition to megakaryocytes, the erythropoietic lineage was prominently labelled by Mpl antiserum, with considerably stronger staining in polycythaemia vera. We conclude that, in CMPD, megakaryocytes and erythroid cells exhibit increased Mpl expression levels which may contribute to the sustained proliferation of both cell lineages in CMPD.     

Phylogenomics of the dog and fox family (Canidae,Carnivora) revealed by chromosome painting

Canid species (dogs and foxes) have highly rearranged karyotypes and thus represent a challenge for conventional comparative cytogenetic studies. Among them, the domestic dog is one of the best-mapped species in mammals, constituting an ideal reference genome for comparative genomic study. Here we report the results of genome-wide comparative mapping of dog chromosome-specific probes onto chromosomes of the dhole, fennec fox, and gray fox, as well as the mapping of red fox chromosome-specific probes onto chromosomes of the corsac fox. We also present an integrated comparative chromosome map between the species studied here and all canids studied previously. The integrated map demonstrates an extensive conservation of whole chromosome arms across different canid species. In addition, we have generated a comprehensive genome phylogeny for the Canidae on the basis of the chromosome rearrangements revealed by comparative painting. This genome phylogeny has provided new insights into the karyotypic relationships among the canids. Our results, together with published data, allow the formulation of a likely Canidae ancestral karyotype (CAK, 2n = 82), and reveal that at least 6–24 chromosomal fission/fusion events are needed to convert the CAK karyotype to that of the modern canids. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regional differences in the compaction of chromatin in human G0/G1 interphase nuclei

The large-scale structure of chromatin corresponding to G- and R-bands in human G₀/G₁ interphase nuclei was compared. Fluorescence in situ hybridization (FISH) was used to measure the interphase distance between 42 pairs of probes separated by 0.1–1.5Mbp. The probe pairs were derived from 21q22.2 and Xp21.3, G-band positive regions, and from 4p16.3, 6p21.3, and Xq28, R-band positive regions. Distributions of measured interphase distances in all regions approximated a Rayleigh distribution, suggesting that the chromatin follows a random-walk path over this range. A linear correlation of mean-square interphase distance and genomic separation, also indicative of random-walk folding, was observed in all regions. The slope of the correlation observed using probes from G-band regions was systematically lower than that from R-band regions. The difference in the slope between Xp21.3 and Xq28 was particularly striking and was observed in normal fibroblast cells, fixed alternatively with methanol and acetic acid or paraformaldehyde, and HeLa cells. These results demonstrate regional differences in large-scale chromosome structure during interphase, with the more openly configured chromatin corresponding to R-bands.This revised version was published online in November 2005 with corrections to the Cover Date.     

Combined analysis of T cell receptor gamma and immunoglobulin heavy chain gene rearrangements at the single-cell level in lymphomas with dual genotype

By prospectively studying immunoglobulin heavy chain gene (IgH) and T cell receptor gamma (TCRgamma) gene rearrangements in 398 lymphoma cases, a dual genotype was observed in 13% of B cell and 11% of T cell lymphomas. According to histological subtype, the highest incidence was observed for mantle cell lymphomas (32%) and lymphoplasmacytic lymphoma (21%) among B cell lymphomas, and for angioimmunoblastic lymphoma (AILT) (46%) and Sézary syndrome (SS) (50%) among T cell lymphomas. To determine whether the dual genotype corresponds to the presence of two distinct monoclonal populations or to the presence of both rearrangements within the same lymphoma cells, single-cell microdissection was used after immunohistochemistry and a single-cell combined IgH and TCRgamma gene analysis was designed after a whole-genome amplification step. This protocol was applied to the study of two nodal B cell lymphomas (one diffuse large B cell lymphoma and one mantle cell lymphoma) and two cutaneous T cell lymphomas (one AILT and one SS). Two cases (SS and mantle cell lymphoma) were true bigenotypic lymphomas, as both IgH and TCRgamma monoclonal rearrangements were detected in the same cells. Conversely, in the diffuse large B cell lymphoma and AILT cases, large CD22+ single cells exhibited only the monoclonal IgH rearrangement but not the TCRgamma gene that was detected in CD3+ single cells. Such an approach allows the identification of true bigenotypic lymphoma among dual genotypic lymphoma. Specific genetic alterations may be further amplified from microdissected cryopreserved material, such as the t(11;14) breakpoint detected in bigenotypic B cells of the mantle cell lymphoma case.     

DNA analysis of two patients with a non-fluorescent y chromosome

Summary Results of DNA study on two patients of gonadal dysgenesis with a 45,X/46,X,Ynf (non-fluorescent Y chromosome) karyotype are described. In one patient who developed gonadoblastoma, all 12 loci on the non-fluorescent part of Yq were detected. Another patient did not have gonadoblastoma at 20 years, and only the proximal 6 loci out of 12 were detected.     

Townes-Brocks syndrome

Townes-Brocks syndrome (TBS) is an autosomal dominant disorder with multiple malformations and variable expression. Major findings include external ear anomalies, hearing loss, preaxial polydactyly and triphalangeal thumbs, imperforate anus, and renal malformations. Most patients with Townes-Brocks syndrome have normal intelligence, although mental retardation has been noted in a few.     

Supernumerary marker chromosomes (SMCs) in Turner syndrome are mostly derived from the Y chromosome

DNA and FISH (fluorescence in situ hybridization) analysis were carried out in 12 patients with stigmata of Turner syndrome to determine whether the Supernumerary M arker C hromosome (SMC) found cytogenetically in each of these patients was derived from the Y chromosome. The presence of a Y chromosome in these patients may predispose them to develop gonadoblastoma. PCR-Southern blot analysis, followed by FISH, was used to detect the presence of Y chromosome material. The S ex determining R egion Y (SRY), T estis S pecific P rotein Y -encoded (TSPY) and Y -chromosome R NA R ecognition M otif (YRRM) genes, which map at Yp11.31, Yp11.1–11.2 and Yp11.2/Yq11.21–11.23, respectively, were selected as markers, because they span the whole Y chromosome, and more importantly, they are considered to be involved in the development of gonadoblastoma. It was shown that in 12 patients, all of whom had an SMC, the SMC of 11 was derived from the Y chromosome. Furthermore, the presence of the SRY, TSPY and YRRM gene sequences was determined and FISH analysis confirmed the Y origin of the SMCs. The methodology described in this report is a rapid, reliable and sensitive approach which may be easily applied to determine the Y origin of an SMC carried in Turner syndrome. The identification of an SMC is important for the clinical management and prognostic counseling of these patients with Turner syndrome.     

Partial gonadal dysgenesis in a patient with a marker Y chromosome

We evaluated a patient with partial gonadal dysgenesis including a right dysgenetic testis and a left streak gonad with rudimentary fallopian tube and uterus. She had ambiguous external genitalia and was raised female. Although her height is normal (25th centile at age 12 years), she has some findings of Ullrich–Turner syndrome. Her karyotype was reported to be 46, X, + marker; subsequent molecular investigations showed the marker to be the short arm of the Y chromosome. Genomic DNA, isolated from leukocytes of the patient and her father, was digested with a variety of restriction endonucleases and subjected to Southern blot analysis. A positive hybridization signal was obtained with probes for the short arm of the Y chromosome (pRsY0.55, SRY, ZFY, 47Z, pY-190, and YC-2) in DNA from the patient, indicating the presence of most if not all of the short arm, while long arm probes (HinfA and pY3.4) indicated that at least 75% of the long arm of the Y chromosome was missing. The gene responsible for testicular determination (TDF) is on the distal portion of the short arm of the Y chromosome; Yq has no known influence on sex determination. Hence, the deletion of the long arm of the Y chromosome cannot explain the gonadal dysgenesis in this patient. One explanation for the gonadal dysgenesis and Ullrich–Turner phenotype in the patient could be undetected 45, X/46,X, + marY mosaicism but no such mosaicism was observed in peripheral lymphocytes. Several investigators have suggested the presence of an “anti-Turner” gene near TDF. Hence it is possible that the clinical phenotype in our patient results from a Y chromosomal defect in sequences flanking TDF, which reduces the function of both TDF and the “anti-Turner” genes.     

Duplication 3q(q21 → qter) without limb anomalies

We report on a 2.5-month-old boy with hypertelorism, hypertrichosis, anteverted nostrils, malformed ears, thin lips, downturned corners of the mouth, micrognathia, short neck, cryptorchidism, and bilateral simian creases without limb anomalies. Cytogenetic studies showed a duplication 3q → qter 46,XY,der(6),t(3;6)(q21;p25)pat. The absence of limb anomalies is noteworthy; all 12 previously reported patients with the same duplication had limb anomalies. The uniqueness of this report provokes speculations regarding limb morphogenesis in embryos with chromosome anomalies. The concepts of chronogenetics, heterochrony, and developmental field defects appear relevant to yet another set of patients with chromosome anomalies.     

Deletion of 20p 11.23→pter with normal growth hormone-releasing hormone genes

Using a molecular analysis of the DNA from a patient with a deletion of chromosome 20 [46,XX,del(20)(p11.23)], we have excluded the growth hormone-releasing hormone (GHRH) gene from the region 20p11.23→pter. The patient had minor facial anomalies, Rieger eye anomaly, a congenital heart defect, severe failure to thrive, and a neurosecretory problem in growth hormone (GH) secretion. Since the GHRH gene was previously mapped to chromosome 20, we used molecular genetic methods to determine whether the growth abnormalities were due to the deletion of this gene. DNAs of the patient and 2 normal control subjects were analyzed by quantitative Southern blotting using a DNA probe for the GHRH gene and 2 reference DNA probes mapping to chromosome 21. The GHRH gene was found to be present in 2 copies in the patient. This indicates that the gene for GHRH maps to the region outside the patient's deletion, in 20p11.23→qter. Furthermore, our results suggest that genes other than GHRH on 20p are important for developmental steps leading to normal neurosecretory function of GH and may also be involved in generating Rieger eye anomaly. Finally, GH deficiency and Rieger eye anomaly should be sought in other patients with deletions of 20p.