G. Calabrese

Microdeletions in interval 6 of the Y chromosome detected by STS-PCR in 6 of 33 patients with idiopathic oligo- or azoospermia

It has been proposed that interval 6 of the human Y chromosome contains the gene or genes that control spermatogenesis (AZF, azoospermia factor). We have studied this region in 33 patients with oligo- or azoospermia, using PCR amplification of the YRRM1 (RBM1) gene and of 13 sequence-tagged sites (STSs), all mapping within interval 6. Six of the 33 patients showed no amplification of specific STSs, whereas there was no failure of amplification in normal male controls. We deduce that these six patients had microdeletions in interval 6 of the Y chromosome that correlated with the oligo- or azoospermia of these individuals. On biopsy of the testis, two of these patients showed a low number of germ cells, and four showed arrest with spermatides. We conclude that PCR amplification of Y-specific regions is a powerful and very sensitive tool for screening infertile men.

Detection of an insertion deletion of region 8q13-q21.2 in a patient with Duane syndrome: implications for mapping and cloning a Duane gene

Duane syndrome (MIM126800) is an autosomal dominant disease responsible for 1% of all strabismus cases and has been related to a 8q12–13 contiguous gene syndrome. We report on an insertion of chromosome region 8q13–q21.2 on to band 6q25 in a patient presenting with Duane syndrome, mental retardation, and other dysmorphisms. FISH analysis using chromosome 8 radiation hybrid LIA2L indicated a concurrent deletion within the 8q rearranged region. These results were corroborated by STR-PCR analysis and FISH using YAC contig WC8.8 disclosed a deletion in 8q13. Comparison of the two known patients with Duane syndrome associated with deletion of 8q identifies a small region of overlap (SRO) of <3xa0cM extending from D8S533 and D8S1767 in which a Duane syndrome locus is assigned. In addition YAC analysis in our patient showed that 8q rearrangement was rather complex since 8q deletion and insertion occurred in two distinct segments separated by a region which maintained its location on 8q.

Complex translocations of the Ph chromosome and Ph negative CML arise from similar mechanisms, as evidenced by FISH analysis

The authors report on 13 patients with chronic myeloid leukemia (CML) studied by serial karyotyping and fluorescence in situ hybridization (FISH) of their bone marrow cells. Ten patients had complex translocations of the Ph chromosome while the remaining three were Ph negative. FISH analysis revealed in all 13 patients the translocation of the ABL protooncogene into chromosome 22 at band q11. Moreover, in all complex translocations but one, FISH with a chromosome 22 painting probe demonstrated on one chromosome 9 at band q34 the presence of material from chromosome 22, in addition to signals on the third chromosome involved in complex changes. Therefore, in this study complex translocations appeared as secondary changes resulting from two consecutive translocations with a total of at least four breaks. The first translocation gave rise to the standard t(9;22)(q34;q11). The second one included a break distal to the original breakpoint at band 9q34 and another one on a third chromosome. Furthermore FISH using S1 and S15 probes, mapped at band 22q11.2 or 22q12, gave evidence that in complex translocations the secondary breakpoint on der(9) was in the translocated segment 22q11-qter between bands q11 and q12. FISH analysis also disclosed the presence of material from chromosome 22 on one chromosome 9 in the three patients with Ph negative CML, demonstrating that in these cases a retranslocation between chromosomes 9q+ and 22q- had occurred. Consequently, the four-break mechanism could also be invoked for the three Ph negative CML patients.

p53 loss and point mutations are associated with suppression of apoptosis and progression of CML into myeloid blastic crisis

A longitudinal investigation using fluorescence in situ hybridization (FISH) analysis, PCR-SSCP, and in situ detection of apoptosis by the terminal deoxynucleotidyl Transferase (TdT) method was carried out on 13 chronic myelogenous leukemia (CML) patients to study the p53 gene behavior and the apoptotic process during the course of the disease. At diagnosis, FISH showed no loss of the p53 gene on interphase nuclei, and no point mutation was detected by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) and sequencing. During the disease course, FISH analysis showed a significative loss of allele (LOA) rate for the p53 gene in eight patients that in seven cases was associated with a suppression of apoptotic process and the progressive expansion of the p53+/p53- clone. DNA sequencing showed in two of these eight patients a point mutation on the other allele, consisting in the formation of a stop codon in one case, and in a frameshift mutation in the other. Six patients had a myeloid blastic crisis (BC), five a lymphoid BC, and the other two an erythroid and an undifferentiated BC, respectively. All patients with myeloid BC and the one with undifferentiated BC disclosed a progressive expansion of the clone with p53 loss that was associated with a significant reduction in apoptosis. On the contrary in the 5 patients with lymphoid BC no significant p53 LOA rate was observed during the course of the disease. In these patients apoptotic process also persisted in the acute phase although in a lower rate as compared to CP.

A new case of Yq microdeletion transmitted from a normal father to two infertile sons

During the last few years, microdeletions of the long arm of the Y chromosome, involving loci AZFa, AZFb, and AZFc, have been identified as a major cause of infertility, leading to the disruption of genes involved in spermatogenesis. These microdeletions are usually de novo mutations, but in six cases transmission from fertile fathers to infertile sons has been reported. In four cases, the transmission occurred to a single son, and in one of these a widening of the deletion was shown.1–4 In the remaining two cases, the microdeletion was transmitted to multiple sons, resulting in different defects of spermatogenesis.5,6 Here, we describe a third family with a Yq microdeletion transmitted by a father to his two infertile sons.

Chromosome changes in 19 patients with Waldenström's macroglobulinemia

We report on 19 patients with Waldenströms macroglobulinemia (WM) who were studied cytogenetically at the onset and during progression of the disease. We found a high frequency of chromosome changes confirming the claim of other authors that, during progression of the disease, a large number of residual neoplastic cells, insensitive to conventional chemotherapy, persist. In turn, this may be the cause of the difficulty of inducing remission (21% of cases) and of the short survival (mean, 35 months). In our experience it is difficult to identify the primary chromosome abnormalities because of the late clinical stage at which the chromosomes were examined. However, changes involving chromosomes #10, #11, and #12 may be unfavorable events in patients with WM.

Narrowing the Duane syndrome critical region at chromosome 8q13 down to 40 kb

Duane syndrome (MIMxa0126800) is an autosomal dominant disorder characterised by primary strabismus and other ocular anomalies, associated with variable deficiency of binocular sight. We have recently identified a <3cM smallest region of deletion overlap (SRO) by comparing interstitial deletions at bandxa08q13 in two patients (one described by Vincent et al, 1994, and the other by Calabrese et al, 1998). Here we report on another patient with Duane syndrome carrying a reciprocal translation t(6;8)(q26;q13). FISH and PCR analyses using a YAC contig spanning the SRO narrowed the Duane region to a <1cM interval between markers SHGC37325 and WI4901. In addition, the identification and mapping of two PAC clones flanking the translocation breakpoint, allowed us to further narrow the critical region to about 40xa0kb. As part of these mapping studies, we have also refined the map position of AMYB, a putative candidate gene, to 8q13, centromeric to Duane locus. AMYB is expressed in brain cortex and genital crests and has been previously mapped to 8q22.

Clustering of Y chromosome deletions in subinterval E of interval 6 supports the existence of an oligozoospermia critical region outside the DAZ gene.

Y chromosome molecular analysis was performed using the STS-PCR technique in 50 patients with oligozoospermia. Microdeletions of interval 6 of the Y chromosome were detected in seven patients, in six of whom subinterval E was affected. All patients retained the RBM1 and DAZ genes, while in one deletion involved the SPGY gene. The size of the deletion was not apparently related to the severity of the disease. These results suggest the presence of an oligozoospermia critical region on the Y chromosome within subinterval E of interval 6.

Cytogenetics and bone marrow transplantation

The authors report hematologic and cytogenetic data on 19 patients treated with allogeneic bone marrow transplantation (BMT) for severe hematologic disorders: 8 patients with chronic myelogenous leukemia, 6 with acute leukemia, 3 with severe aplastic anemia, 1 with refractory anemia, and 1 with beta-thalassemia major. Cytogenetic assays were performed on marrow cells before conditioning, 30 days after BMT, and at subsequent times. The authors discuss the role of cytogenetic studies in the evaluation of bone marrow engraftment, leukemic transformation of the graft, and disease relapse.

Complex chromosome translocations of standard t(8;21) and t(15;17) arise from a two-step mechanism as evidenced by fluorescence in situ hybridization analysis

The authors report the results of cytogenetic and fluorescence in situ hybridization (FISH) analysis performed on complex chromosome translocations (CCTs) of t(8;21) and t(15;17) standard translocations associated with two M2 subtypes of acute myeloid leukemia (AML-M2) and four acute promyelocytic leukemia (APL), respectively. In one of two AML-M2 patients FISH analysis showed part of chromosome 21 on the der(8) and material from this chromosome on the der(21) and on chromosome 1 at band p32, suggesting that the t(8;21) occurred as the primary step. In the second AML-M2 patient. FISH displayed part of chromosome 21 on the der(8) and material from this chromosome on the der(21) but not on the third rearranged chromosome. Therefore, it is unclear whether chromosome 2 was rearranged secondary to the standard t(8;21). In four APL patients, FISH analysis showed material derived from chromosome 17 on the der(15). Moreover, in two patients with an i(17q) FISH disclosed material from chromosome 15 at the ends of both arms of the i(17q), suggesting that it occurred after the standard t(15;17). In the remaining two APL patients, FISH showed material from chromosome 15 on the der(17) and on chromosome 21 at band q22 in one case, and material of the p arm of chromosome 17 on chromosome 4 at band q11 in the other, demonstrating that in these two cases the first mutation also had been the t(15;17). Therefore, FISH analysis revealed that CCTs in five patients were secondary changes which occurred after standard t(8;21) and t(15;17), thus clarifying the hierarchy of the cytogenetic events, their role in the pathogenesis of the disease, and the associated clinic-hematologic findings.