Aicardi syndrome associated with autosomal genomic imbalance: coincidence or evidence for autosomal inheritance with sex-limited expression?

Aicardi syndrome (AIS), a rare neurodevelopmental disorder thought to be caused by an X-linked dominant mutation, is characterized by 3 main features: agenesis of corpus callosum, infantile spams and chorioretinal lacunae. A genome-wide study of a girl with AIS lead us to identify a 6q deletion;12q duplication, derived from a maternal 6q;12q translocation. The two intellectually impaired brothers of the proband showed the same genomic anomalies, but not the constellation of features characterizing the AIS. This could be either a coincidental observation of 2 rare conditions, but can also suggest an alternative hypothesis for the genetic etiology of AIS, indicating the existence of a subset of autosomal genes whose mutation could act in a sex-confined manner.

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.

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.

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.

MASA syndrome: ultrasonographic evidence in a male fetus.

The recent identification of a common etiology among MASA syndrome (McKusick 303300), X-linked hydrocephalus (HSAS) (McKusick 307000) and other related neurological disorders, which had previously been considered distinct nosological entities, allowed us to diagnose MASA syndrome in a male fetus in a primigravida at the 29th week of gestation by sonographic signs of the MASA spectrum such as hydrocephalus and hypoplasia of corpus callosum. Indeed, the evidence of an X-linked neurological disease in the brother and the maternal uncle of the pregnant women enabled us to estimate a 25% risk of a male fetus being an affected hemizygote. The way in which a prenatal diagnosis, based on instrumental procedures, was reached is described since the authors were unable to perform, at the time of the observation, a molecular confirmation which was carried out only after birth.

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.

Glycosaminoglycan metabolism and cytokine release in normal and otosclerotic human bone cells interleukin-1 treated.

Glycosaminoglycans (GAGs), normal components of the extracellular matrix (ECM), and the glycosidases, that degrade them, play a key role in the bone remodelling process. The effects of interleukin-1 alpha (IL-1 alpha) on GAG metabolism in normal and otosclerotic human bone cells as well as its capacity to modulate IL-1 alpha, IL-1 beta and IL-6 secretion in both populations was analyzed. The amount of radiolabeled GAGs was lower in otosclerotic than in normal bone cells. IL-1 alpha reduced newly synthesized cellular and extracellular GAGs in normal cells, but only those of the cellular compartment in otosclerotic bone cells. It depressed heparan sulphate (HS) more in normal cells and chondroitin sulphate (CS) more in otosclerotic bone cells. The HA/total sulphated GAG ratio was shifted in favour of the latter in otosclerotic cells, whereas the opposite effect was seen after IL-1 alpha treatment. There was little difference in the beta-D-glucuronidase levels of the normal and pathological cells, while beta-N-acetyl-D-glucosaminidase was significantly increased in otosclerotic bone cells. As the activity of neither enzyme was modified by treatment with IL-1 alpha, the cytokine seems to exert its influences on GAG synthesis rather than on the degradation process. IL-1 alpha, IL-1 beta and IL-6 secretion was markedly higher in otosclerotic cells. IL-1 alpha modulated the secretion of each interleukin differently, thus resulting in a cytokine cascade that may act in autocrine/paracrine manner on target cells. The authors suggest that changes in the cytokine network may have a specific, yet still unknown, role during normal and pathological osteogenesis.

Centralized cytogenetic analysis of pediatric acute leukemia: results of an Italian collaborative experience.

BACKGROUND AND OBJECTIVE: Cytogenetic analysis of acute leukemia yields important information which has been demonstrated to be correlated to patient survival. A reference laboratory was created in order to perform karyotype analysis on all cases of acute leukemia enrolled in the AIEOP (Associazione Italiana Emato-Oncologia Pediatrica) protocols. METHODS: From January 1990 to December 1995, 1115 samples of children with ALL or AML were sent in for cytogenetic analysis. The results of cell cultures were screened in the Reference Laboratory and then the fixed metaphases were sent to one of the six cytogenetic laboratories for analysis. RESULTS: The leukemic karyotypes of 556 patients were successfully analyzed. An abnormal clone was detected in 49% of cases of ALL and in 66% of AML. In ALL the most frequent abnormality was 9p rearrangement. Other recurrent abnormalities were t(9;22), t(4;11) and t(1;19). In AML t(8;21), t(15;17) and 11q23 rearrangement were the most frequent structural abnormalities. These findings are similar to the results obtained in other multicenter studies using a similar approach. INTERPRETATION AND CONCLUSIONS: Our data confirm the feasibility of performing cytogenetic analysis in a centralized laboratory on mailed samples of bone marrow and/or peripheral blood; this is very important considering that cytogenetic analysis of neoplastic tissue requires a special laboratory and expert staff.

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.

Glycosaminoglycan metabolism in otosclerotic bone cells.

Normal and otosclerotic bone cells were cultured in vitro in serum-free medium to evaluate single glycosaminoglycan (GAG) class synthesis and secretion. Moreover, the degradative process was studied by inhibiting the lysosomal functions through the addition of ammonium chloride to the cultures, an ammine known to inhibit lysosomal degradation by neutralizing organelle activity. Otosclerotic bone cells accumulated a lower amount of GAG both in the cellular and extracellular pool compared to normal ones. The decrease was markedly higher for secreted GAG. Moreover a different pattern of single GAG class distribution was observed in the two cell types considered. In the medium of otosclerotic cells a percentage increase of hyaluronic acid (HA) and dermatan sulphate (DS) and a percentage decrease of heparan sulfate (HS) and chondroitin sulfate (CS) were observed compared to normal bone cells. Ammonium chloride had a lower effect on pathologic than on normal cells, indicating a decrease in the degradative process in otosclerotic bone cells. These results were also confirmed by the experiments on GAG uptake and degradation and by the dosage of enzymatic activity of two exoglycosidases. Since extracellular GAG composition influences bone deposition and mineralization, these data support the hypothesis that otosclerosis is the result of an error in the connective tissue matrix structure.

Phenotype of in vitro human otosclerotic cells and its modulation by TGF beta.

A study was carried out to obtain a more detailed picture of the phenotypes of human otosclerotic and normal bone cells and to analyse the response of both populations to treatment with TGF beta 1. Total collagen synthesis was found to be decreased, but fibronectin secretion increased in otosclerotic with respect to normal cells. Although overall glycosaminoglycan (GAG) synthesis was lower in otosclerotic cells, the sulphated GAG to hyaluronic acid (HA) ratio was higher, in particular there was greater expression of chondroitin (CS) and dermatan sulphates (DS). TGF beta 1 induced a more marked increase in collagen and fibronectin release and greater production of sulphated GAGs as DS and heparan sulphate (HS) in the otosclerotic cells. The fact that the phenotype of the otosclerotic cells differed from that of the normal cells and could be modified by TGF beta 1 treatment, suggests that TGF beta 1 is implicated in the pathogenesis of otosclerosis.

Interferon-alpha plus low-dose cytosine arabinoside in advanced phase chronic myelogenous leukaemia patients.

Twenty-nine late chronic and accelerated phase chronic myelogenous leukaemia (CML) patients were entered in a pilot study designed to test the therapeutic efficacy of treatment with interferon-alpha (IFN-alpha) and low-dose cytosine arabinoside (ARA-C). IFN-alpha was administered at a dose of 2-10 x 10(6) IU/day and ARA-C at 15 mg/m2/day for 14 days each month. The treatment was well tolerated by 73% of the patients. Side effects were mainly asthenia, anorexia, anaemia and piastrinopenia. Haematological and cytogenetic responses were evaluated in the 19 patients who received more than 6 cycles. Four complete haematological response, 7 partial haematological response, 6 minor haematological response, 2 stable disease were obtained in this patient group. Two complete cytogenetic responses and 2 minor cytogenetic responses were detected in these patients. Suppression of secondary Ph' positive clones which appeared during the previous IFN-alpha treatment was documented in 3 accelerated phase patients after ARA-C was added to their IFN-alpha treatment. It would therefore seem that late chronic and accelerated phase CML patients benefit from combined IFN-alpha/ARA-C treatment and achieve haematological and cytogenetic responses not obtained during previous treatment without being exposed to undue toxicity. However, we cannot judge whether it offers any advantage in terms of survival.

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.

Repeated PCR in CML during IFN-alpha therapy.

Repeated PCR analysis was performed on bone marrow and/or peripheral blood samples from 4 CML patients in complete cytogenetic remission during treatment with IFN-alpha. Two patients became PCR-negative. One was negative for the analyses carried out from the 9th to the 30th months, but reverted to PCR positivity 10 months after IFN was reduced from 1.5 x 10(6) IU/day to 1 x 10(6) IU and given on alternate days. Although the dose was again raised to 3 x 10(6) IU/day, 8 months later her peripheral blood cells were still PCR-positive, but remained persistently Ph'-negative. Another patient became PCR-negative at the 42nd month and remained so at the last analysis performed 3 months later. Two patients were persistently PCR-positive. Cytogenetic relapse was documented in both, in one while still on full therapy. Ph'-positive metaphases reappeared in the other patient 7 months after discontinuing IFN-alpha therapy.