A family with autosomal dominant leukodystrophy linked to 5q23.2-q23.3 without lamin B1 mutations.

BACKGROUND AND PURPOSE: Duplications of lamin B1 (LMNB1) at 5q23 are implicated in adult-onset autosomal dominant leukodystrophy (ADLD) having been described in six families with diverse ethnic background but with a homogeneous phenotype. In a large Italian family, we recently identified a variant form of ADLD characterized clinically by absence of the autonomic dysfunction at onset described in ADLD and, on MRI, by milder cerebellar involvement with sparing of hemispheric white matter. Aim of this study was to investigate the genetic basis of this variant form of ADLD. METHODS: We carried out a genome-wide linkage analysis using microsatellite markers, and the genes in the candidate region were screened for point mutations. LMNB1 was also screened for deletions/duplications by real-time PCR, multiplex ligation-dependent probe amplification and Southern blot. RESULTS: We mapped the variant ADLD locus to 5q23.2-q23.3, a genomic region containing 11 genes including LMNB1. Neither gene copy-number defects nor point mutations in the LMNB1 gene were found. We also excluded point mutations in the coding exons of the other ten genes in the candidate region. However, expression of lamin B1 evaluated in lymphoblastoid cells was higher in patients than in healthy controls, and was similar to the lamin B1 expression levels found in a patient with LMNB1 duplication. CONCLUSIONS: This observation suggests that a mutation in an LMNB1 regulatory sequence underlies the variant ADLD phenotype. Thus, adult forms of ADLD linked to 5q23 appear to be more heterogeneous clinically and genetically than previously thought.

Can GH induce chromosome breaks or microsatellite instability in GH-deficient children?

The role of GH in carcinogenesis is unclear. We studied the effect of recombinant human GH (rhGH) in vitro on chromosomal and genomic instability. Thirteen children, aged 9.57 +/- 1.08 yr (mean +/- SEM), with complete (no.=5) or partial (no.=8) GH deficiency were evaluated before and during GH treatment. We examined the incidence of chromosome and chromatid breaks, and microsatellite instability after in vitro addition of two different doses of rhGH to peripheral blood lymphocytes obtained from the patients. No differences were observed between the samples of GH-deficient children before and after GH therapy as regards the percentage of chromosome and chromatids breaks, and in microsatellite instability. Our data show that in vitro addition of rhGH does not induce chromosomal and/or genomic instability in cultured lymphocytes.

Isochromosome (7)(q10) in Shwachman syndrome without MDS/AML and role of chromosome 7 anomalies in myeloproliferative disorders.

Shwachman syndrome (SS) is an autosomal recessive disorder in which bone marrow dysfunction is observed, with development of myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML) in up to one third of the cases. Inconclusive data are available as to increased chromosome breakage in SS, while chromosome 7 anomalies, and often an isochromosome (7)(q10), are frequent in cases with MDS/AML. We report on the consistent presence of an i(7)(q10) in the bone marrow and blood lymphocytes in one of two sisters affected with SS without any clinical or cytological signs of MDS/AML. Thus, this patient was either a case of constitutional mosaicism for the i(7)(q10), or this had to be acquired in a nondysplastic and non-neoplastic marrow clone. DNA polymorphism analysis demonstrated the paternal origin of the i(7q). We postulate that the SS mutation acts as a mutator gene, and causes karyotype instability; abnormal clones would thus arise in the marrow, and chromosome 7 anomalies, i(7q) in particular, will in turn lead to MDS/AML. If this interpretation is correct, it would be also an indication to consider chromosome 7 anomalies in general, out of SS, as primary changes in MDS/AML pathogenesis.

Cytogenetic abnormalities in PHA-stimulated lymphocytes from patients with Langerhans cell histocytosis. AIEOP-Istiocitosi Group.

The aetiopathogenesis of Langerhans cell histiocytosis (LCH) is still undefined. Constitutional abnormalities in LCH have rarely been reported. One study showed chromosomal instability in lesional cells from three patients. No chromosomal studies are available on peripheral blood lymphocytes. Peripheral blood lymphocytes were analysed for the presence of chromatid and/or chromosomal breaks and structural rearrangements. A fluorescence in situ hybridization (FISH) painting technique was also applied in two cases. Sixteen patients with multisystem (MS, n = 11) or single system (SS, n = 5) LCH were studied. either at the diagnosis (n = 8), during treatment (n = 2) or during follow-up, when asymptomatic (n = 6). Thirteen patients had chromosomal abnormalities. Eleven patients (69%) had chromatid and chromosomal breaks in 7-45% of cells. Overall, chromosome and chromatid breaks were significantly more frequent in the 11 patients with MS disease than in the five patients with SS disease: the mean percentage of cells showing chromosome and chromatid breaks was 13.4% in MS patients vs. 6.2% in SS patients (P = 0.003). Chromosomal abnormalities may be found in phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes of LCH patients at diagnosis, during the disease course and even during long-term follow-up, more frequently in MS disease. Chromosome instability may be considered as either a basic genetic instability or as a landmark of reaction to an environmental agent (viral?) that, through genome alteration, may play a role in histiocyte proliferation and, in some cases, also in the increased risk of malignancy.

Concurrent cytogenetic and molecular investigations in uterine and ovarian neoplasms.

We studied a group of 24 uterine and ovarian neoplasms with the purpose to verify if any correlation could be established between chromosomal abnormalities, loss of heterozigosity (LOH) and microsatellite instability (MIN). Tumor specimens obtained from 24 women (12 affected by ovarian and 12 by uterine neoplasms) were split in two parts, one was used for short term cultures for cytogenetic investigation while from the second DNA was extracted for molecular studies. We studied 22 polymorphic loci from 19 chromosomes and compared the alleles observed in the tumor with those observed in the DNA obtained from peripheral blood. Extensive loss of heterozigosity was observed when total or partial chromosomal loss was observed in at least 50% of the examined cells; MIN did not correlate with any particular cytogenetic abnormality nor with LOH.

Does growth hormone treatment increase chromosomal abnormalities?

OBJECTIVE: The relationship between growth hormone (GH) therapy and malignancy, including leukaemias, remains controversial. In order to study this possible relationship further, we have investigated whether GH treatment induces chromosomal abnormalities in peripheral blood lymphocytes. DESIGN: Open, prospective study in a University Hospital to examine peripheral blood mononuclear cells in subjects with GH-deficiency (GHD) before and during GH treatment. SUBJECTS: Twelve idiopathic GHD patients, aged 1.8-12.5 years, were evaluated before and after 3, 6 and 12 months of GH therapy (0.6 IU/kg per week subcutaneously). Two additional GHD patients, aged 16.6 and 18 years, were studied 1 year after long-term GH therapy had been discontinued, and 12 age-matched healthy subjects were evaluated as controls. METHODS: We examined the incidence of chromosome and chromatid breaks, fragments, structural rearrangements and aneuploidies in 100 metaphases for each blood sample. A total of 5300 cells was analysed in the 14 patients. RESULTS: The proportion of cells with chromatid and chromosome breaks ranged from 0% to 6% in patients before treatment and from 1% to 5% in controls. During GH therapy the incidence of aneuploid metaphases ranged from 0% to 7% and was comparable with values observed in controls. Chromosomal loss and gain was random. CONCLUSIONS: We observed no increase in chromosomal abnormalities in GH-treated patients when compared with age-matched healthy controls.

Homozygous and compound heterozygous mutations at the Werner syndrome locus.

The Werner syndrome (WS) is a rare autosomal recessive progeroid disorder. The Werner syndrome gene (WRN) has recently been identified as a member of the helicase family. Four distinct mutations were previously reported in three Japanese and one Syrian WS pedigrees. The latter mutation was originally described as a 4 bp deletion spanning a spliced junction. It is now shown that this mutation results in a 4 bp deletion at the beginning of an exon. Nine new WRN mutations in 10 additional WS patients, both Japanese and Caucasian, are described. These include three compound heterozygotes (one Japanese and two Caucasian). The new mutations are located all across the coding region.

A search for double minute chromosomes in cultured lymphocytes from different types of tumors.

Double minutes are considered the products of DNA amplification and are rare in normal human cells. They have been observed in cultured lymphocytes in selected samples of human populations as one of the characteristics of the so-called rogue cells. We scored 9500 metaphases of cultured lymphocytes from 65 subjects with a variety of heredity and sporadic tumors and from 30 healthy subjects. The 15 cells with double minutes were found in subjects with multiple endocrine neoplasia type 1 (14 cases) and with familial adenomatous polyposis (1 case). Only one rogue cell was found among the 15 cells with double minutes.

Cytogenetic studies in venous tissue from patients with varicose veins.

Cytogenetic investigation of primary cell cultures from fragments of varicose veins of seven patients with familial varicosity and seven patients with the sporadic type revealed the presence of metaphases with structural abnormalities, clonal trisomies of chromosomes 7, 12, and 18, and monosomy of chromosome 14 only in cases with the familial type, while the sporadic cases had no similar chromosome aberrations. The immunophenotypical results are consistent with fibroblast lineage of the cultured cells. These results suggest that karyotypic variations in familial varicose vein tissue cultures could in some way be associated either with the genotypic constitution responsible for the familial type or a longer duration of disease on average than those with sporadic varicosities.

Stimulation of DNA synthesis by endothelin-1 in primary cultures of human dental pulp.

Endothelin (ET), besides being a powerful vasoactive agent, acts as a mitogen in some cell types. ET-like immunoreactivity has been recently detected by immunocytochemistry in the vascular endothelium of human tooth germ and dental pulp, thus providing evidence for local ET production in these tissues. The effects of ET-1 on DNA synthesis in primary cultures of human dental pulp were now investigated. DNA synthesis was evaluated by flow cytometric assay and by 5-bromo,2'deoxyuridine incorporation as detected by immunocytochemistry. Cultured cells were morphologically similar to dental pulp cells and displayed vimentin immunoreactivity. Incubation of cultures with ET-1 resulted in a dose-dependent increase in the number of S-phase-traversing cells over control (unstimulated) cultures. Control skin fibroblasts were also responsive to ET. This finding raises the possibility that the multifunctional peptide ET-1 might subserve growth-promoting activity in the human tooth. It is tentatively suggested that such as an activity might be important during tooth development and in pulp inflammation and healing.

Homozygotes for the autosomal dominant neoplasia syndrome (MEN1).

Families in which both parents are heterozygotes for the same autosomal dominant neoplasia syndrome are extremely unusual. Recently, we had the unique opportunity to evaluate three symptomatic siblings from the union between two unrelated individuals affected by multiple endocrine neoplasia type 1 (MEN1). When the three siblings and their parents and relatives were genotyped for 12 markers tightly linked to the MEN1 locus, at 11q13, two of the siblings were found to be homozygotes, and one a heterozygote, for MEN1. With regard to the MEN1 syndrome, no phenotypic differences were observed between the two homozygotes and the heterozygotes. However, the two homozygotes showed unexplained infertility, which was not the case for any of the heterozygotes. Thus, MEN1 appears to be a disease with complete dominance, and the presence of two MEN1 alleles with mutations of the type that occur constitutionally may be insufficient for tumor development.

Cytogenetics of multiple endocrine neoplasia syndromes. III. Analysis of an insulinoma from a subject with MEN 1 by chromosome painting.

The cytogenetics of an insulinoma from a subject with MEN 1 characterized by the consistent presence of double minute chromosomes (dmins) and by five characteristic marker chromosomes was investigated with fluorescence in situ hybridization after labeling with a chromosome 11 library. The dmins were consistently negative for 11q material, with the exception of one metaphase which had two positive dmins. This indicated that the dmins are not derived massively from chromosome 11 and that they can be heterogeneous in their origin. One of the marker chromosomes, tentatively identified as a del (7), turned out to be the product of a 7;11 translocation.

Chromosome abnormalities in a case of pituitary adenoma.

Chromosome analysis of primary cultures from an ACTH-secreting pituitary adenoma revealed the presence of one clone of cells with deletion of 18p and some random structural and numerical abnormalities.

Cytogenetics of multiple endocrine neoplasia syndrome. II. Chromosome abnormalities in an insulinoma and a glucagonoma from two subjects with MEN1.

Cytogenetic analysis of two pancreatic islet tumors, an insulinoma and a glucagonoma was ascertained in two subjects with multiple endocrine neoplasia type 1 (MEN1). The insulinoma had a modal peak at 84 chromosomes. Most cells were pseudotetraploid, and in all cells the normal chromosomes were represented in varied numbers, i.e., from 1 to 7 copies. The tumor had 5 characteristic and consistent marker chromosomes which were identified as deletions of chromosomes 1, 2, 7, 16, and 17. All metaphases had several double minute chromosomes (dmin) of variable size and possible intermediate structures between dmin and homogeneously staining chromosomal regions. The glucagonoma had a nearly equal proportion of normal metaphases and metaphases with structural and numerical abnormalities with no consistent trend.

Cytogenetics of multiple endocrine neoplasia syndromes. I. Two different, unique clonal chromosome changes in a medullary thyroid carcinoma and in a C-cell thyroid hyperplasia.

In short-term cultures of tumor tissue from a medullary thyroid carcinoma (MTC), we found a large clone of cells with a balanced translocation t(9;12)(p24;q22). A large clone with a balanced translocation t(10;16)(p11;q24) was also found in cultures from a C-cell thyroid hyperplasia. No clearcut evidence for chromosome instability was observed in the lymphocytes of the two patients. The mother of the first patient died of MTC; two relatives of the second patient had MTC and one of them had pheochromocytoma. These findings classify the two subjects as MEN 2A patients with different phenotypic expression but with the same type of chromosomal abnormality.

Chromosome abnormalities in lymphocytes and fibroblasts of subjects with multiple endocrine neoplasia type 1.

A consistent degree of chromosome instability was found in cultured lymphocytes and in fibroblasts derived from skin biopsies of three patients with multiple endocrine neoplasia type 1 (MEN1). In both tissues, there was a significant increase in chromosomal breakage. Dicentrics and tricentrics, rings, translocations, deletions, acentric fragments, and presumptive double minutes were the most frequent abnormalities in lymphocytes. The fibroblasts of two patients had large clones consisting of trisomy 7 and trisomy 18 cells, respectively.