A 343 Italian cohort of patients analysed with array-comparative genomic hybridization: unsolved problems and genetic counselling difficulties.

BACKGROUND: The recent introduction of microarrays for genetic analyses has allowed higher etiological diagnostic rates in patient with intellectual disability (ID), autism spectrum disorders (ASD), epilepsy and multiple congenital anomalies (MCA), because of its resolution. This approach still results of high complexity and some limitations have been reported. In fact, it discloses several variants of unknown significance (VOUS) or incidental findings. In all cases, a massive amount of data is generated, because of this, the analysis and the interpretation is very difficult and often without a definitive conclusion. METHOD: We analysed an Italian cohort of 343 patients with ID, MCA and ASD by array-comparative genomic hybridization. The purpose of this work was to consider the proportion of the chromosomal abnormalities in such cohort and to assess the distribution of the different type of the chromosomal abnormalities concerning their pathogenic significance, their origin and their correlation to these clinical phenotypes. RESULTS: Array-comparative genomic hybridization analysis revealed 76 positive results. Abnormalities were detected in 27.8% of patients with ID, 11.1% with ASD, 10.7% with epilepsy and 19.4% with multiple congenital anomalies. The anomalies were classified in three major groups: group 1 (27 patients) with pathogenic alterations (P group); group 2 (34 patients) with VOUS potentially pathogenic (PP group); and group 3 (13 patients) with VOUS potentially benign (PB group). As expected, comparing the diagnostic groups, we observed a greater number of deletions in the P group and that all the abnormalities of the PB group were inherited. CONCLUSIONS: Our retrospective study resulted in confirming the high detection rate of microarrays. CNV classification remains a complex procedure. The difficulty in CNV classification points out the importance of the patient selection, helping the interpretation of the molecular cytogenetic results.

A new case of pure partial 7q duplication.

We report on an 18-month-old boy conceived by assisted reproduction technology with developmental delay, hypotonia, microcephaly, frontal bossing, a mild convergent squint, malformed ears, and a short neck. Karyotype analysis revealed a de novo 7q21.1q22.3 duplication characterized by array comparative genomic hybridization (array-CGH) as a segment of 18.69 Mb. Duplications of the long arm of chromosome 7 are uncommon. There are 18 reported cases of different 7q segments with a pure duplication with no additional deletion of other chromosomes. As a consequence, duplications of chromosome 7q have been classified in 4 groups on the basis of the involved region. The present case is included in group 3 which involves interstitial duplications of different sizes. In the literature, only one case with an apparently smaller duplication of the same region has been described. Despite this, the phenotype is different. Moreover, the 2 patients share some phenotypic features, such as psychomotor delay, hypotonia, frontal bossing, short neck, and strabismus. However, the absence of physical characterization in most of the reported cases could justify the lacking phenotype-genotype correlation in patients with partial 7q duplication. Further studies using recent molecular approaches such as array-CGH might permit a more clinically useful grouping of 7q duplications.

Inositide-specific phospholipase c beta1 gene deletion in the progression of myelodysplastic syndrome to acute myeloid leukemia.

Myelodysplastic syndrome (MDS) is an adult hematological disease that evolves into acute myeloid leukemia (AML) in about 30% of the cases. The availability of a highly specific probe moved us to perform in patients affected with MDS/AML, associated with normal karyotype, painting and fluorescence in situ hybridization (FISH) analysis aimed to check the inositide-specific phospholipase C (PI-PLC) beta1 gene, a player in the control of some checkpoints of the cell cycle. Here we present a preliminary observation in which FISH analysis disclosed in a small group of MDS/AML patients with normal karyotype the monoallelic deletion of the PI-PLCbeta1 gene. On the contrary, PI-PLC beta4, another gene coding for a signaling molecule, located on 20p12.3 at a distance as far as less than 1Mb from PI-PLCbeta1, is unaffected in MDS patients with the deletion of PI-PLC beta1 gene, hinting at an interstitial deletion. The MDS patients, bearing the deletion, rapidly evolved to AML. The data suggest the possible involvement of PI-PLCbeta1 in the progression of the disease and pave the way for a larger investigation aimed at identifying a possible high-risk group among MDS patients with a normal karyotype.

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 < 3 cM 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.

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

Duane syndrome (MIM 126800) 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 < 3 cM smallest region of deletion overlap (SRO) by comparing interstitial deletions at band 8q13 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 < 1 cM interval between markers SHGC37325 and W14901. In addition, the identification and mapping of two PAC clones flanking the translocation breakpoint, allowed us to further narrow the critical region to about 40 kb. 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.

FISH analysis in detecting 9p duplication (p22p24).

Authors report on a case of partial 9p duplication, involving the 9p22-9p24 region. This represents the second case of such duplication in which the breakpoints were precisely defined using fluorescence in situ hybridisation (FISH) with chromosome 9 specific painting and YAC DNA probes, localised onto 9p22-9p24 region. FISH analysis pinpointed chromosome breakpoints in dup(9)(p22p24) and excluded an insertion or a translocation from other chromosomes. The present report supports the segment 9p22-9p24 as the critical region for the observed phenotype of the duplication 9p syndrome.

FISH detection of mixed chimerism in 33 patients submitted to bone marrow transplantation.

Fluorescence in situ hybridization (FISH) and cytogenetic analysis were carried out in 33 transplanted patients suffering from different hematologic disease using probes for X and Y chromosomes and ABL and BCR genes. FISH showed that recipient cells were invariably present during post-transplant follow-up. Stable minimal residual disease was associated with clinical and hematologic remission, while a progressive increase of host cells was strictly related with disease relapse. Cytogenetic investigation on the same samples showed recipient cells only in few cases. It was concluded that FISH analysis is useful for: (1) characterizing cases in which standard cytogenetic analysis has failed; (2) detecting host cells in sex-mismatched transplanted patients; and (3) evaluating Ph-negative CML with the BCR/ABL rearrangement. The possibility of detecting chromosome rearrangements in interphase nuclei using FISH analysis improves diagnosis and prediction of disease evolution and prompts earlier therapeutic approaches.

Spectral karyotyping (SKY) refinement of a complex karyotype with t(20;21) in a Ph-positive CML patient submitted to peripheral blood stem cell transplantation.

A patient with a Ph-positive chronic myeloid leukaemia (CML) was submitted to allogeneic peripheral blood stem cell transplantation from an HLA-haploidentical related donor 7 years after the diagnosis. Six months later, he showed a disease relapse while cytogenetic analysis displayed a complex karyotype. To characterise the chromosomal rearrangements spectral karyotype (SKY) analysis was used. This redefined all chromosome rearrangements and revealed a t(20;21)(q11;q22). FISH analysis with a specific probe for the AML1 gene disclosed disruption of this gene which was partially translocated on to the long arm of chromosome 20. It is likely that this rearrangement, unusual for CML, was implicated in the disease evolution towards blastic crisis (BC).

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.

Retrospective investigation of hematopoietic chimerism after BMT by PCR amplification of hypervariable DNA regions.

We report on nine patients submitted to BMT with sex-matched donors and investigated by means of PCR amplification of the VNTRs ApoB, D1S80, DXS52, and D17S5. In all cases it was possible to detect a polymorphism able to distinguish between donor and patient cells, thus allowing us to recognize the presence of complete or mixed chimerism. In eight patients PCR analysis showed a complete chimerism during the entire follow-up. Only one of these patients relapsed, while the others are alive and without any sign of relapse 56.2 months (mean) after BMT. Mixed chimerism was detected in only one patient, who relapsed 3 months after this finding. These results confirm the usefulness of the study of PCR-amplified VNTRs in the assessment of marrow engraftment after BMT, mostly in sex-matched transplants where, in the absence of specific chromosome rearrangements, cytogenetic or FISH analysis cannot be used.

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.

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.

Prenatal diagnosis using the triple test.

METHODS: A screening study performed on 2,803 pregnant women using the "triple test" is reported. RESULTS: Nine hundred and twenty-one had a high prior risk, having > 35 years while, after the screening, only 201 women had a positive test at risk higher than 1:270, and underwent to amniocentesis. The detection rate (DR) for all abnormalities was 91% while for Down's syndrome (DS) it was 87.5% and for neural tube defects 85.5%. Foetal abnormalities were detected in 20 cases (1:10) while 181 were false positive cases (6.5%), of which 151 for DS (5.4%). False negative were observed only in 2 cases within 2,339 at term pregnancies. CONCLUSIONS: The authors retain that high DR is related to the exactness of determination of gestation age calculated by scan and to the homogeneity of the examined population.

Assignment of the beta-arrestin 1 gene (ARRB1) to human chromosome 11q13.

Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: beta-adrenergic receptor kinase (beta ARK), which phosphorylates the agonist-occupied receptor, and its functional cofactor, beta-arrestin. beta ARK is a member of a multigene family, consisting of six known subtypes, which have also been named G-protein-coupled receptor kinases (GRK 1 to 6) due to the apparently unique functional association of such kinases with this receptor family. The gene for beta ARK1 has been localized to human chromosome 11q13. The four members of the arrestin/beta-arrestin gene family identified so far are arrestin, X-arrestin, beta-arrestin 1, and beta-arrestin 2. Here we report the chromosome mapping of the human gene for beta-arrestin 1 (ARRB1) to chromosome 11q13 by fluorescence in situ hybridization (FISH). Two-color FISH confirmed that the two genes coding for the functionally related proteins beta ARK1 and beta-arrestin 1 both map to 11q13.

A woman with an apparent non-mosaic 45,X delivered a 46,X,der(X) liveborn female.

A liveborn female with a phenotype suggestive of Down syndrome is reported. Cytogenetic lymphocyte analysis showed a 46,X der(X) karyotype. Fluorescence in situ hybridization (FISH) with a biotinylated probe specific for chromosome 21 showed no signal on the der(X). This marker was homogeneously painted using a specific probe for X chromosome. In addition, FISH analysis detected telomeres on the rearranged X. Therefore, the proband's karyotype was reevaluated as 46,X,del(X) (pter-->p22.2::p11.3-->qter). Cytogenetic analysis of 150 lymphocytes in the mother disclosed a homogeneous 45,X karyotype. FISH analysis of interphase nuclei using the X chromosome painting probe showed two domains of different sizes in 0.8% of cells. This led us to study further metaphases in the mother. In one out of 450 metaphases scored, after FISH with the X chromosome painting probe, the del(X) was observed, confirming that the rearranged X chromosome found in the newborn had segregated from a 45,X/46,X,del(X) mother.