A novel spinocerebellar ataxia type 15 family with involuntary movements and cognitive decline.

BACKGROUND AND PURPOSE: SCA15 is a recently identified spinocerebellar ataxia with pure cerebellar involvement. Here, we report a novel SCA15 Italian family with atypical clinical features. METHODS: Three affected members from a three-generation family segregating an autosomal dominant cerebellar ataxia underwent clinical examination and genetic tests for hereditary ataxia. RESULTS: All affected members present with cognitive impairment and two of them with mild intermittent involuntary movements in association with the clinical hallmarks of SCA15 (gait ataxia, balance impairment, and dysarthria). Genetic tests detected a large deletion spanning ITPR1 and SUMF1 genes in affected members. CONCLUSION: Our findings help enlarging the clinical spectrum of SCA15.

Prenatal diagnosis and molecular characterization of an interstitial 1q24.3-31.3 deletion: case report and review.

We describe a foetus with an interstitial deletion of 1q detected in amniotic fluid cells and we review the literature of similar pre- and postnatal cases, in order to identify prognostic factors useful for prenatal counselling. Foetal/parents karyotyping and FISH with whole chromosome 1 paint and BAC clone specific for 1q23-32 region were performed. Further 100 Kb resolution array-CGH analysis was executed after pregnancy termination on DNA extracted from foetal skin fibroblasts. Cytogenetic analyses revealed a de novo interstitial deletion involving the long arm of chromosome 1. FISH analysis confirmed that the deletion involves the intermediate 1q31.2 region. Foetal ultrasound (US), performed at 21 weeks of gestation, showed intrauterine growth restriction, shortening of the long bones, echogenic intracardiac focus and mild cerebral ventriculomegaly. Array-CGH localized the deletion in a DNA sequence of about 21 Mb in the 1q24.3-q31.3 region. Our findings, together with available data on patients with 1q deletion, suggest that the most severe phenotypes are not simply associated with larger deletion, and that the results of prenatal US assessment, rather than a fine molecular characterization of the deletion, should be taken into account for prognostic evaluation.

Familial pericentric inversion of chromosome 18: intrafamilial variability of the recombinant dup(18q).

A pericentric inversion of chromosome 18 [inv(18)(p11.32q22)] and its recombinants has been studied in a three-generation family. A mother/son couple, carrying the rec dup(18q), showed dysmorphisms and short stature but only the son had mild mental retardation and speech delay. Karyotype, FISH analysis with subtelomeric probes and a 0.8 Mb array-CGH investigations were used to analyze this recombinant, demonstrating no genomic differences between the two relatives. This is the first observation of familial transmission of a rec dup(18q), showing that this recombinant is associated with a mild phenotype with variable clinical picture.

The Italian external quality assessment scheme in classical cytogenetics: four years of activity.

BACKGROUND: The Italian external quality assessment scheme in classical cytogenetics was started in 2001 as an activity funded by the National Health System and coordinated by the Italian Public Institute of Health. OBJECTIVES: The aim of our work is to present data from the first 4 years of activity, 2001-2004. METHODS: Italian cytogenetics public laboratories were enrolled on a voluntary basis, and this nationwide program covered prenatal, postnatal and oncological diagnosis. The scheme is annual and retrospective; a panel of experts reviewed the quality of images and reports in order to assess technical, analytical and interpretative performance. RESULTS: Over the 4-year period, the number of participating laboratories increased: from 36 in 2001, 46 in 2002, 49 in 2003 to 51 in 2004. The overall technical performance was satisfactory. Inadequacy or lack of information in reporting was the most frequent analytical inaccuracy identified in all parts of the scheme. However, the percentage of complete reports increased significantly during the period: by 36% in postnatal diagnosis between 2001 and 2004 (p < 0.001) and by 42% in oncological diagnosis between 2002 and 2004 (p = 0.003). CONCLUSIONS: Our experience reveals that participation in external quality assessment programs has significant advantages, helping to standardize and to assure quality in cytogenetic testing.

An intermediate phenotype between Hay-Wells and Rapp-Hodgkin syndromes in a patient with a novel P63 mutation: confirmation of a variable phenotypic spectrum with a common aetiology.

The ankyloblepharon-ectodermal defects-cleft lip and palate (Hay-Wells or AEC) and the Rapp-Hodgkin syndrome (RHS) are rare autosomal dominant ectodermal dysplasias due to mutations in the transcription factor gene P63. Both are caused by mutations affecting SAM or TID domains of TP63 protein. The two disorders share common features and may represent different phenotypic expressions of the same clinical entity. To date more than 20 P63 mutations have been described associated with AEC and RHS, the majority of which are missense or nonsense mutations. Molecular heterogeneity cannot account for the clinical heterogeneity, because the same mutations were observed both in patient with RHS and with AEC syndrome. Here we report on a novel P63 mutation (the first repeat variation described in the gene) in a patient showing overlapping phenotype of AEC and RH syndromes.

A novel mutation in the SDHD gene responsible for familial paraganglioma. Medical and psychological implications.

Familial paragangliomas/pheochromocytomas are dominantly inherited disorders characterized by the development of highly vascularized tumors of the head and neck, derived from non-chromaffin cells of the extra-adrenal paraganglia, and tumors with endocrine activity, derived from chromaffin cells, usually located in the adrenal medulla and pre- and para-vertebral thoracoabdominal regions. Germline inactivating heterozygous mutations in one of the genes encoding for succinate dehydrogenase subunits B, C or D (SDHB, SDHC or SDHD) are responsible for hereditary paragangliomas (PGLs), accounting for nearly 70% of familial cases. Particularly in the SDHD gene, different types of mutations have been found, nevertheless, alterations other than point mutations and deletion leading to missense/nonsense/splicing mutations are extremely rare. Here we report a family with multiple cases of PGL which co-segregates with a novel SDHD gene mutation predictable to give rise to an abnormal gene product (CybS). The identification of the molecular event responsible for PGL in our family made genetic counseling particularly useful for younger first degree relatives at risk to develop this late-onset disease.

Prenatal diagnosis of a de novo satellited chromosome 18 (18ps) associated with 18p deletion.

De novo satellited non-acrocentric chromosomes are very rare findings in prenatal diagnosis. Here we report the first case of a de novo 18ps, associated with del(18p), detected at prenatal diagnosis. A 37 years old woman underwent Chorionic Villus Sampling (CVS) for advanced maternal age. Cytogenetic analysis on direct CVS preparation (CVSc) revealed a male karyotype with a nonfamilial satellited 18ps and a reciprocal translocation t(17;19)(P11.1;q11) of maternal origin. The mesenchimal CVS culture (CVSm) showed a mosaic of cell lines with various involvement of chromosome 18: 18ps [36/70]/ r(18) [25/70]/ del(18p) [3/70]/ -18 [6/70]. Amniotic fluid cells (AFC) confirmed the homogeneous karyotype found at CVSc. The molecular cytogenetic characterization, performed on AFC, allowed the following diagnosis: 46,XY, +15, dic(15;18)(p11.1;p11.2), t(17;19)(p11.1;q11)mat. ish dic(15;18)(tel 18p-, D15Z1+, wcp18-, wcp 18+, D18Z1+, tel 18q+). The foetal autopsy disclosed subtle facial dysmorphisms and corpus callosum hypoplasia. In case of prenatal detection of de novo terminal ectopic NORs an accurate cytogenetic and molecular analysis should be performed in order to rule out subtle unbalancements.

Presence of chromosomal abnormalities and lack of AIDS retrovirus DNA sequences in AIDS-associated Kaposi's sarcoma.

The frequent occurrence of Kaposi's sarcoma (KS) in association with the acquired immune deficiency syndrome (AIDS) could be due to the fact that the etiological agent of this tumor is the same retrovirus causing AIDS, to another oncogenic virus frequently found in AIDS patients, or to the unmasking of the tumorigenic potential of KS cells by immunosuppression. We have therefore investigated the presence of DNA sequences homologous to the AIDS retrovirus, cytomegalovirus (CMV), and hepatitis B virus in 13 KS necropsies and biopsies from AIDS patients. All KS DNA samples were negative for AIDS retrovirus or hepatitis B DNA sequences. Two DNAs from necropsies contained CMV DNA, but the data suggested the presence of replicating CMV DNA due to generalized infection. We have also studied cell cultures derived from KS skin biopsies of AIDS patients. These cultures had a short lifetime in vitro and expressed some markers of endothelial cells. The cells were not tumorigenic in nude mice but contained a number of chromosomal rearrangements which were often monoclonal within the same culture. However, these abnormalities were different from culture to culture and even in cultures from the same biopsy. The presence of these chromosomal abnormalities seemed to correlate with the cell positivity for endothelial markers. Taken together these results indicate that neither the AIDS retrovirus, CMV, or hepatitis B virus is directly responsible for the altered growth of KS cells, that KS may be polyclonal even within the same lesion, and that KS cells have a tendency to karyotypic rearrangements.

Mapping of chromosome 17 breakpoints in acute myeloid leukemias.

The 17q11-21 chromosomal region is frequently involved in non-random structural rearrangements associated with the M1 and M2 subtypes of acute myeloid leukemias (AML), as well as with the 15;17 translocation typical of the promyelocytic subtype. A number of genes have been localized in this region including the c-erbA-1 and c-erbB-2 proto-oncogenes, the genes coding for the granulocyte-colony stimulating factor (G-CSF), the retinoic acid receptor alpha (RAR alpha) and the myeloperoxidase enzyme (MPO). However, the precise location of these genes in relationship to the 17q11-21 breakpoint(s) has not been determined. Using in situ hybridization on metaphase chromosomes, we established the position of the breakpoints in relationship to the c-erbA-1, c-erbB-2, G-CSF, RAR alpha and MPO loci in a series of AML cases bearing 17q11-21 rearrangements. We report: (i) that the respective position of the five genes is centromere - c-erbA-1 - G-CSF - c-erbB-2 - RAR alpha - MPO - telomere; (ii) that the breakpoints of the various AML subtypes are variably located between the centromere and c-erbB-2 in M1 and M2; (iii) that the breakpoints are consistently located between c-erbB-2 and RAR alpha/MPO in M3; and (iv) that the breakpoint on chromosome 17 in the 15;17 translocation is located on 17q21 and not on 17q11-12 as previously reported.

Immunological and molecular studies in a case of follicular lymphoma with an extra chromosome 12 and t(2;8) translocation.

Two sequential lymph node biopsies taken from a non-Hodgkin lymphoma patient revealed two karyotype abnormalities peculiar to B cell neoplasias: trisomy 12 and t(2;8)(p12;q24) translocation. The first was documented in all cells analyzed, while the second was present in 20% of the metaphases from the first biopsy and in 100% from the second. This suggests that the t(2;8) translocation arose as a secondary karyotypic change. In addition, although immunological characterization of the neoplastic cells disclosed a monoclonal B cell population that expressed immunoglobulin kappa light chains, as usually found in Burkitt's lymphoma with t(2;8) translocation, Southern blot analysis provided evidence of rearrangement in only one kappa chain allele.

Prenatal diagnosis in Italy.

In Italy, there are about 560,000 births per year. The number of prenatal diagnoses (PND) performed is estimated at 80,000 examinations per year, but no official data are available regarding the distribution of the different procedures. There are no official registers, either at a national or at a regional level, concerning PND and particularly the invasive procedures (as amniocentesis, chorionic villus sampling, chordocentesis). Thanks to the direct interest of some scientific societies, such as the Italian Association of Medical Cytogenetics, the Italian Association of Medical Genetics, the Italian Society for the Study of Metabolic Hereditary Diseases and the Italian Society of Gynaecology and Obstetrics, it has been possible to identify the number and distribution of public and private structures interested in genetic counselling and prenatal diagnosis during the last decades. As to the congenital malformations, there is a regional epidemiological system of surveys (started in 1982) co-ordinated by the Epidemiological and Biostatistical Laboratory of the 'Istituto Superiore di Sanità' (National Board of Health). This institution has published data for the period between 1986 and 1990 concerning the trend of incidence for the most important malformations at birth. Cytogenetic PND in public services is allowed for the following indications: maternal age 35 and over, previous child with a chromosomal anomaly, parent with a constitutional chromosomal abnormality and abnormal findings at the ultrasound examinations. Current methods in use consist in echography, amniocentesis, chorionic villus sampling, fetal blood sampling and maternal serum screening. At the moment, new approaches to the fetal tissue sampling are, as follows: amniotic fluid filtration, transcervical cell sampling and isolation of fetal cells from maternal blood. Furthermore, areas under development are 3D sonography and first-trimester anatomic survey sonography. PND is financed by regional laws, the National Health Service and private funds. There is a current legislation on termination of pregnancy (Law 194/1978). This law permits voluntary interruption of pregnancy within the first 90 days, while it is permitted between 90 and 180 days only in cases of severe fetal anomalies and over 180 days for serious risks for the woman's life: but it is necessary to do everything to save the fetal life. No law has been issued yet on pre-implantation diagnosis. Presently, the major problems are: the unbalanced distribution of financial resources among the different regions, the irrational number and distribution of centres for PND, inadequate prenatal counselling, especially in central/southern Italy, where counselling is somehow lacking. Therefore, guidelines for appropriate prenatal counselling should be established. For the future, we believe that the best results in this field are probably related to the advances of research (there is a target programme of the Italian National Research Council called 'Genetic Engineering', which is in its fourth year of financing). This programme will hopefully be cost-effective and improve the quality of PND so that more congenital anomalies can be detected at lower expenses in the future.

Evolving modalities of treatment with interferon alfa-2b for Ph1-positive chronic myelogenous leukaemia.

We have administered interferon alfa-2b, alone or in combination with chemotherapy, to 126 Ph1-positive chronic myelogenous leukaemia patients. Of 71 early chronic phase (CP) patients (less than 12 months from diagnosis), 41 (58%) obtained a complete haematological response (CHR). Daily interferon was more effective than intermittent administration. In previously untreated patients, the response was significantly influenced by risk status at diagnosis. Thirty-four out of 71 (48%) patients improved cytogenetically, the median of Ph1+ mitoses declining from 100% to 66% with complete Ph1-suppression in one case. Of 46 late CP patients (greater than 12 months from diagnosis), 32 (70%) achieved CHR with interferon alone or combined with chemotherapy. All 10 patients with disease well controlled by chemotherapy obtained stable CHR with interferon alone. Of 36 partial responders to conventional chemotherapy, 22 (61%) obtained CHR on interferon plus low-dose hydroxyurea. Ph1 mosaicism was reached by 16 (35%) late CP patients (median Ph1+ cells 75%). Of nine accelerated phase patients on interferon plus chemotherapy, one attained CHR, and two responded partially. At a median follow up of 36 months, of 41 CHR patients in early CP, 15 are controlled on interferon, 12 have had autologous bone marrow transplantation (BMT), and two allogeneic BMT. Blastic transformation (BT) has occurred in eight of 41 CHR patients (19%) versus 17 of 30 (57%) non-responders and partial responders to interferon. At a median follow up of 22 months, of 32 late CP patients obtaining CHR, 26 remain on interferon, one had allogeneic BMT, one had autologous BMT, and one developed BT (versus five out of 14 with less than CHR). These studies confirm the haematological and cytogenetic efficacy of interferon in CML and indicate that the disease status at the start of treatment is critical in determining the success of therapy.

Altered adenylate cyclase activity in human otosclerotic bone cell cultures.

Adenylate cyclase (AC) activity was studied in whole homogenates of normal and otosclerotic bone cell cultures. When Mn2+ or Ca2+ was added to the medium there was a similar increase in AC activity in both cell types. F- provoked a greater rise in normal than in pathological cells, whereas 0.01 mM guanosine triphosphate (GTP) significantly raised cAMP synthesis in otosclerotic cells only. Mn2+ + calcitonin (Ct) increased AC activity in both cell preparations. With Ca2+ as cofactor there was no significant rise in either normal or pathological cells. However, while the combination Ca2+ + Ct + GTP had little effect on normal cells, it markedly increased cAMP synthesis in the pathological cells. 1 microgram/ml of the beta-blocker propranolol inhibited the effect Ct exerts on AC in normal cells, but enhanced it in otosclerotic cells. It would, therefore, seem that the pathogenesis of otosclerosis could be associated with an alteration in the AC system associated with Ct receptors.

Variant Philadelphia translocation in an immunologically and clinically typical case of chronic myeloid leukaemia.

Cytogenetic analysis of bone marrow from a chronic myeloid leukemia patient in chronic phase revealed a classical Philadelphia chromosome from a complex translocation t(2;9;22). The break points on 9 and 22 were, apparently, the same as for the standard translocation (9;22). However, whereas the terminal band of 9 (9q34) was translocated in the usual site, that is on 22q-, the tract deleted from 22 was present on band p13 of chromosome 2. The finding of this rare 22 translocation in classical CML would seem to support the hypothesis that the crucial event in the pathogenesis of CML is the translocation of band 9q34, that contains the c-abl oncogene, onto the Ph' chromosome, rather than the translocation of the tract deleted from 22 to some other chromosome site.

Partial trisomy 1q in idiopathic myelofibrosis.

Three cases of idiopathic myelofibrosis with partial trisomy of the long arm of chromosome 1 are described. Partial trisomy 1q was the only karyotypic change detectable in unstimulated peripheral blood cell cultures of one and bone-marrow cultures of two patients at diagnosis. The extra segment from chromosome 1 was located on different karyotype sites, i.e. 1qter, 1p34 and 6p22-23; 1q21-32 was the shortest overlapping region and the only trisomic segment in one of the three patients. These findings suggest that partial trisomy 1q is a primary chromosome aberration in myelofibrosis relevant in the pathogenesis of this hematologic disorder.

t(4;21) (p16;q22) in blastic crisis of a chronic myeloid leukemia with variant Philadelphia translocation.

A chronic myeloid leukemia (CML) patient who had presented a t(2;9;22) translocation during the chronic phase developed an unusual t(4;21) (p16;q22) translocation during the M2 type FAB classification blastic crisis. The role of these two recombinant chromosomes in the genesis of the terminal phase is discussed, particularly as the breakpoint on chromosome 21 near to the ets-2 oncogene locus, seems to be the same as that described in the t(8;21) (q22;q22) translocation specific of type M2 AML.

5q- syndrome terminating in acute myeloid leukemia. Karyotype evolution and immunologic characterization of blast cells.

We report a case of 5q- syndrome that progressed to acute nonlymphocytic leukemia after 9 years of clinically and morphologically stable disease. The transition from the chronic to the leukemic phase was characterized by the appearance of an additional cytogenetic anomaly [inv(2)] in the cell carrying the 5q-, together with the expansion of a clone showing an apparently normal karyotype.

Cytogenetic and molecular study of two human neuroblastoma cell lines.

Long-term cell cultures (GI-LA-N and GI-ME-N) were established from the metastases of two disseminated neuroblastomas (NB). The first was obtained from a lymph node biopsy of a stage III NB after 7 months of chemotherapy, and the second from a bone marrow specimen of a stage IV NB after 6 months of chemotherapy. Cytogenetic investigation revealed several structural and numerical alterations in both cell cultures, but the only common chromosomal aberration was partial monosomy of 1p. Moreover, at the 5th in vitro passage, GI-LA-N displayed a high number of double minutes, not seen in GI-ME-N even after 33 subcultures. Molecular analysis revealed N-myc oncogene amplification in GI-LA-N cells, whereas, only one copy was found in GI-ME-N. No structural N-myc rearrangement was detected in either cell culture.

Clinical and cytogenetic findings in monocyte--macrophage system malignancies with initial spontaneous regression.

Clinical and cytogenetic features are described in two patients with rare monocyte-macrophage malignancy. Their conditions clinically were different, but initial spontaneous regression was observed, and in both patients a No. 15 anomaly was involved.