Chromosome number and secondary constriction variation in 51 accessions of a citrus germplasm bank

The mitotic chromosomes of 51 citrus accessions from the Centro Nacional de Pesquisa em Mandioca e Fruticultura Tropical, Cruz das Almas, BA, Brazil, were analyzed. The sample included representatives of 20 Citrus species, one of Poncirus and seven hybrids. All accessions showed 2n = 18 without any evidence of numerical variation. The most clearly variable karyotype feature was the number and position of secondary constrictions (SECs). In 19 accessions the SECs were not identified, mainly due to the degree of chromatin condensation. In the remainder they varied in number from one to three per karyotype. They were found in the proximal region of one of the three largest chromosome pairs, in the terminal/subterminal region of a smaller chromosome or, more seldom, terminally in a larger chromosome. Only in a few cases were such constrictions observed simultaneously in both homologues of the same chromosome pair. The high variability of this karyotype feature may be due to the activation of this region in the previous interphase but may also indicate a high structural variability and heterozygosity of citrus germplasms

Deletion/inversion in the X-chromosome and increased telomeric associations in a female with primary amenorrhea

We describe a new case of a partial interstitial deletion and inversion of the long arm of the X-chromosome associated with a high incidence of telomeric associations in an 18-year old female who showed underdeveloped secondary sex characteristics, including small breasts and primary amenorrhea. Her karyotype was considered to be 46,X,del(Xq13 -> q22)inv(X)(q23-q27). The buccal mucosal cells showed absence of a typical Barr body, and the 5’-bromo-2-deoxyuridine incorporation studies revealed that neither the normal X-nor the abnormal X-chromosome was late replicating. The case is being presented for its extreme rarity

Evaluation of techniques for C and ASG banding of the mitotic chromosomes of Anastrepha species (Diptera, Tephritidae)

Methods previously described by Canovai et al. (Caryologia 47: 241-247, 1994) which produced C and ASG bands in mitotic chromosomes of Ceratitis capitata were applied to the chromosomes of several Anastrepha species. Metaphase plate yield was substantially increased by use of imaginal disks together with cerebral ganglia. The C-bands were quite prominent allowing the resolution of tiny blocks of heterochromatin. The ASG method produced G-like banded chromosomes, which permitted recognition of each individual chromosome. These simple techniques do not require special equipment and may be valuable for karyotype variability studies in fruit flies and other Diptera

Acute promyelocytic leukemia: the study of t(15;17) translocation by fluorescent in situ hybridization, reverse transcriptase-polymerase chain reaction and cytogenetic techniques

Acute promyelocytic leukemia (AML M3) is a well-defined subtype of leukemia with specific and peculiar characteristics. Immediate identification of t(15;17) or the PML/RARA gene rearrangement is fundamental for treatment. The objective of the present study was to compare fluorescent in situ hybridization (FISH), reverse transcriptase-polymerase chain reaction (RT-PCR) and karyotyping in 18 samples (12 at diagnosis and 6 after treatment) from 13 AML M3 patients. Bone marrow samples were submitted to karyotype G-banding, FISH and RT-PCR. At diagnosis, cytogenetics was successful in 10 of 12 samples, 8 with t(15;17) and 2 without. FISH was positive in 11/12 cases (one had no cells for analysis) and positivity varied from 25 to 93% (mean: 56%). RT-PCR was done in 6/12 cases and all were positive. Four of 8 patients with t(15;17) presented positive RT-PCR as well as 2 without metaphases. The lack of RT-PCR results in the other samples was due to poor quality RNA. When the three tests were compared at diagnosis, karyotyping presented the translocation in 80% of the tested samples while FISH and RT-PCR showed the PML/RARA rearrangement in 100% of them. Of 6 samples evaluated after treatment, 3 showed a normal karyotype, 1 persistence of an abnormal clone and 2 no metaphases. FISH was negative in 4 samples studied and 2 had no material for analysis. RT-PCR was positive in 4 (2 of which showed negative FISH, indicating residual disease) and negative in 2. When the three tests were compared after treatment, they showed concordance in 2 of 6 samples or, when there were not enough cells for all tests, concordance between karyotype and RT-PCR in one. At remission, RT-PCR was the most sensitive test in detecting residual disease, as expected (positive in 4/6 samples). An incidence of about 40% of 5' breaks and 60% of 3' breaks, i.e., bcr3 and bcr1/bcr2, respectively, was observed.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Acute myeloid leukemia in elderly patients: experience of a single center

Acute myeloid leukemia (AML) is a disease predominantly of older adults. Treatment of AML in the elderly is complicated not only by comorbidities but also by the high prevalence of poor prognosis markers. Thirty-one consecutive unselected patients with AML older than 60 years (representing 33% of all AML cases diagnosed at our institution during the same period) were followed over a period of 5 years (1997-2002). A high incidence of AML with multilineage dysplasia (45%) and no favorable cytogenetic abnormalities but 62% intermediate and 38% unfavorable karyotypes were found. Sixteen patients (52%) were selected for induction of intensive cytotoxic treatment and complete remission was achieved only by some of these intensively treated patients (7 of 16). Of these, 3 remained alive without disease (median: 11 months), 1 patient died shortly after complete remission, and 3 patients relapsed and died from refractory disease. Only 1 patient that was refractory to intensive cytotoxic treatment remained alive with disease under supportive care. Fifteen patients (48%) were managed with palliative/supportive care: 7 received palliative treatment and supportive care, 8 received supportive care only, and 4 patients remained alive with disease under supportive care (median: 9 months). Mortality rate was 74% and overall survival at two years was 12%. To the best of our knowledge, there is no previous report regarding elderly patients with AML in Brazilian subsets. The present data are similar to previously reported studies showing that elderly AML patients are not only older but also biologically distinct from younger AML patients, particularly in terms of the high incidence of poor prognostic karyotypes and resistance to therapy.

Prevalence of Y chromosome deletions in a Brazilian population of nonobstructive azoospermic and severely oligozoospermic men

We determined the prevalence of Y chromosome deletions in a population of 60 Brazilian nonobstructive azoospermic and severely oligozoospermic men. PCR-based screening of microdeletions was performed on lymphocyte DNA for the presence of 14 sequence-tagged sites (STS) located in the azoospermic factor (AZF) on the Yq chromosome. All STS were amplified efficiently in samples from 12 fertile men tested, but failed to be amplified in samples from fertile women, indicating the specificity of PCR conditions for Yq screening. Overall, 4 of the 60 infertile patients tested (6.7%) exhibited deletion of the Y chromosome, 2 of them being severely oligozoospermic patients (P10 and P32) and 2 azoospermic men (patients P47 and P57). Patients P47 and P57 presented larger deletions in the AZFa, AZFb and AZFc subregions, with apparent loss of Yq material evidenced by karyotype analysis. Patients P10 and P32 presented deletions confined to the AZFc region, involving the DAZ locus. Male relatives of patients P10 and P32 had no Y chromosome deletions and presented a normal karyotype, suggesting a de novo status of the deletions found. Our data add to the growing literature showing that microdeletions of the Y chromosome can be the cause of male idiopathic infertility.

Somatic cytogenetic and azoospermia factor gene microdeletion studies in infertile men

The objective of the present study was to determine the frequency of somatic chromosomal anomalies and Y chromosomal microdeletions (azoospermia factor genes, AZF) in infertile males who seek assisted reproduction. These studies are very important because the assisted reproduction techniques (mainly intracytoplasmic sperm injection) bypass the natural selection process and some classical chromosomal abnormalities, microdeletions of AZF genes or some deleterious genic mutations could pass through generations. These genetic abnormalities can cause in the offspring of these patients male infertility, ambiguous external genitalia, mental retardation, and other birth defects. We studied 165 infertile men whose infertility was attributable to testicular problems (60 were azoospermic, 100 were oligospermic and 5 were asthenospermic). We studied 100 metaphases per patient with GTG banding obtained from temporary lymphocyte culture for chromosomal abnormality detection and performed a genomic DNA analysis using 28 Y chromosome-specific sequence-tagged sites for Y AZF microdeletion detection. Karyotyping revealed somatic anomalies in 16 subjects (16/165 = 9.6%). Of these 16, 12 were in the azoospermic group (12/60 = 20%) and 4 were in the oligospermic group (4/100 = 4%). The most common chromosomal anomaly was Klinefelter syndrome (10/165 = 6%). Microdeletions of AZF genes were detected in 12 subjects (12/160 = 7.5%). The frequencies detected are similar to those described previously. These results show the importance of genetic evaluation of infertile males prior to assisted reproduction. Such evaluation can lead to genetic counseling and, consequently, to primary and secondary prevention of mental retardation and birth defects.

Cryptic mosaicism involving a second chromosome X in patients with Turner syndrome

The high abortion rate of 45,X embryos indicates that patients with Turner syndrome and 45,X karyotype could be mosaics, in at least one phase of embryo development or cellular lineage, due to the need for the other sex chromosome presence for conceptus to be compatible with life. In cases of structural chromosomal aberrations or hidden mosaicism, conventional cytogenetic techniques can be ineffective and molecular investigation is indicated. Two hundred and fifty patients with Turner syndrome stigmata were studied and 36 who had female genitalia and had been cytogenetically diagnosed as having "pure" 45,X karyotype were selected after 100 metaphases were analyzed in order to exclude mosaicism and the presence of genomic Y-specific sequences (SRY, TSPY, and DAZ) was excluded by PCR. Genomic DNA was extracted from peripheral blood and screened by the human androgen receptor (HUMARA) assay. The HUMARA gene has a polymorphic CAG repeat and, in the presence of a second chromosome with a different HUMARA allele, a second band will be amplified by PCR. Additionally, the CAG repeats contain two methylation-sensitive HpaII enzyme restriction sites, which can be used to verify skewed inactivation. Twenty-five percent (9/36) of the cases showed a cryptic mosaicism involving a second X and approximately 14% (5/36), or 55% (5/9) of the patients with cryptic mosaicism, also presented skewed inactivation. The laboratory identification of the second X chromosome and its inactivation pattern are important for the clinical management (hormone replacement therapy, and inclusion in an oocyte donation program) and prognostic counseling of patients with Turner syndrome.

Association of loss of heterozygosity with cytogenetic abnormalities in acute myeloid leukemia and myelodysplastic syndrome

Deletions on chromosomes 5 and 7 are frequently seen in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). It is assumed that these deletions indicate loss of tumor suppressor genes on these chromosomes and until these tumor suppressor genes are identified, the functional consequences of these deletions and the molecular basis of these myeloid disorders cannot be completely understood. We evaluated loss of heterozygosity (LOH) in 44 patients (18 MDS and 26 AML, diagnosed according to WHO classification criteria) at diagnosis, using a four-microsatellite marker panel: an intragenic marker on the 7th intron of gene IRF-1 of the 5q31.1 region and three markers located inside the 7q31.1 region and correlated the LOH with karyotype abnormalities. The microsatellites chosen corresponded to chromosome regions frequently deleted in MDS/AML. The samples with Q (peak area) less than or equal to 0.50 were indicative of LOH. The percent of informative samples (i.e., heterozygous) for the intragenic microsatellite in gene IRF-1 and in loci D7S486, D7S515 and D7S522 were 66.6, 73.7, 75.5, and 48.8%, respectively. Cytogenetic abnormalities by G-banding were found in 36% (16/44) of the patients (2 of 18 MDS and 14 of 26 AML patients). We found a significantly positive association of the occurrence of LOH with abnormal karyotype (P < 0.05; chi-square test) and there were cases with LOH but the karyotype was normal (by G-banding). These data indicate that LOH in different microsatellite markers is possibly an event previous to chromosomal abnormalities in these myeloid neoplasias.

Comparison of I-FISH and G-banding for the detection of chromosomal abnormalities during the evolution of myelodysplastic syndrome

Myelodysplastic syndrome (MDS) patients with a normal karyotype constitute a heterogeneous group from a biological standpoint and their outcome is often unpredictable. Interphase fluorescence in situ hybridization (I-FISH) studies could increase the rate of detection of abnormalities, but previous reports in the literature have been contradictory. We performed I-FISH and conventional karyotyping (G-banding) on 50 MDS patients at diagnosis, after 6 and 12 months or at any time if a transformation to acute myeloid leukemia (AML) was detected. Applying a probe-panel targeting the centromere of chromosomes 7 and 8, 5q31, 5p15.2 and 7q31, we observed one case with 5q deletion not identified by G-banding. I-FISH at 6 and 12 months confirmed the karyotype results. Eight cases transformed to AML during follow-up, but no hidden clone was detected by I-FISH in any of them. The inclusion of I-FISH during follow-up of MDS resulted in a small improvement in abnormality detection when compared with conventional G-banding.

The novel p.E89K mutation in the SRY gene inhibits DNA binding and causes the 46,XY disorder of sex development

Male sex determination in humans is controlled by the SRY gene, which encodes a transcriptional regulator containing a conserved high mobility group box domain (HMG-box) required for DNA binding. Mutations in the SRY HMG-box affect protein function, causing sex reversal phenotypes. In the present study, we describe a 19-year-old female presenting 46,XY karyotype with hypogonadism and primary amenorrhea that led to the diagnosis of 46,XY complete gonadal dysgenesis. The novel p.E89K missense mutation in the SRY HMG-box was identified as a de novo mutation. Electrophoretic mobility shift assays showed that p.E89K almost completely abolished SRY DNA-binding activity, suggesting that it is the cause of SRY function impairment. In addition, we report the occurrence of the p.G95R mutation in a 46,XY female with complete gonadal dysgenesis. According to the three-dimensional structure of the human SRY HMG-box, the substitution of the conserved glutamic acid residue by the basic lysine at position 89 introduces an extra positive charge adjacent to and between the positively charged residues R86 and K92, important for stabilizing the HMG-box helix 2 with DNA. Thus, we propose that an electrostatic repulsion caused by the proximity of these positive charges could destabilize the tip of helix 2, abrogating DNA interaction.

Cytogenetic studies of Brazilian pediatric myelodysplastic syndrome cases: challenges and difficulties in a large and emerging country

Myelodysplastic syndromes (MDS) and juvenile myelomonocytic leukemia (JMML) are rare hematopoietic stem cell diseases affecting children. Cytogenetics plays an important role in the diagnosis of these diseases. We report here the experience of the Cytogenetic Subcommittee of the Brazilian Cooperative Group on Pediatric Myelodysplastic Syndromes (BCG-MDS-PED). We analyzed 168 cytogenetic studies performed in 23 different cytogenetic centers; 84 of these studies were performed in patients with confirmed MDS (primary MDS, secondary MDS, JMML, and acute myeloid leukemia/MDS+Down syndrome). Clonal abnormalities were found in 36.9% of the MDS cases and cytogenetic studies were important for the detection of constitutional diseases and for differential diagnosis with other myeloid neoplasms. These data show the importance of the Cooperative Group for continuing education in order to avoid a late or wrong diagnosis.

Prenatal management, pregnancy and pediatric outcomes in fetuses with septated cystic hygroma

It has been reported that, compared with simple increased nuchal translucency, fetal cases with septated cystic hygroma (CH) are more likely to face perinatal handicaps. However, pediatric outcomes and proper prenatal counseling for this anomaly have not yet been truly defined. We performed this study to determine pregnancy and pediatric outcomes of fetuses with septated CH. We searched records for cases with septated CH and collected data for structural abnormalities, karyotype analysis, and pregnancy outcomes. Fetuses born with septated CH were also evaluated for their pediatric outcomes. Sixty-nine fetuses with septated CH were enrolled in the study. Results showed that chromosomal abnormalities were present in 28 fetuses (40.6%), and the most common aneuploidy was Turner syndrome (n=14, 20.3%); 16 (23.2%) of the remaining cases, in which aneuploidy was not found, had coexistent structural malformations; 25 (36.2%) cases had normal karyotype and morphology. The total number of live births and infants with unfavorable neurologic follow-up were 13 (18.8%) and 2 (2.9%), respectively. Septated CH is associated with poor perinatal outcomes; therefore, karyotype analysis and ultrasonographic anomaly screening should be performed as initial steps, and expectant management should be offered to couples with euploid fetuses that have normal morphology.

Possible benefit of consolidation therapy with high-dose cytarabine on overall survival of adults with non-promyelocytic acute myeloid leukemia

In adults with non-promyelocytic acute myeloid leukemia (AML), high-dose cytarabine consolidation therapy has been shown to influence survival in selected patients, although the appropriate doses and schemes have not been defined. We evaluated survival after calculating the actual dose of cytarabine that patients received for consolidation therapy and divided them into 3 groups according to dose. We conducted a single-center, retrospective study involving 311 non-promyelocytic AML patients with a median age of 36 years (16-79 years) who received curative treatment between 1978 and 2007. The 131 patients who received cytarabine consolidation were assigned to study groups by their cytarabine dose protocol. Group 1 (n=69) received <1.5 g/m2 every 12 h on 3 alternate days for up to 4 cycles. The remaining patients received high-dose cytarabine (≥1.5 g/m2 every 12 h on 3 alternate days for up to 4 cycles). The actual dose received during the entire consolidation period in these patients was calculated, allowing us to divide these patients into 2 additional groups. Group 2 (n=27) received an intermediate-high-dose (<27 g/m2), and group 3 (n=35) received a very-high-dose (≥27 g/m2). Among the 311 patients receiving curative treatment, the 5-year survival rate was 20.2% (63 patients). The cytarabine consolidation dose was an independent determinant of survival in multivariate analysis; age, karyotype, induction protocol, French-American-British classification, and de novo leukemia were not. Comparisons showed that the risk of death was higher in the intermediate-high-dose group 2 (hazard ratio [HR]=4.51; 95% confidence interval [CI]: 1.81-11.21) and the low-dose group 1 (HR=4.43; 95% CI: 1.97-9.96) than in the very-high-dose group 3, with no significant difference between those two groups. Our findings indicated that very-high-dose cytarabine during consolidation in adults with non-promyelocytic AML may improve survival.