Deletion of a 5-cM region at chromosome 8p23 is associated with a spectrum of congenital heart defects.

BACKGROUND: Cytogenetic evidence suggests that the haploinsufficiency of > or =1 gene located in 8p23 behaves as a dominant mutation, impairing heart differentiation and leading to a wide spectrum of congenital heart defects (CHDs), including conotruncal lesions, atrial septal defects, atrioventricular canal defects, and pulmonary valve stenosis. An 8p heart-defect-critical region was delineated, and the zinc finger transcription factor GATA4 was considered a likely candidate for these defects. We narrowed this region and excluded a major role of GATA4 in these CHDs. METHODS AND RESULTS: We studied 12 patients (7 had CHD and 5 did not) with distal 8p deletions from 9 families by defining their chromosome rearrangements at the molecular level by fluorescent in situ hybridization and short-tandem repeat analysis. Subjects with 8p deletions distal to D8S1706, at approximately 10 cM from the 8p telomere, did not have CHD, whereas subjects with a deletion that included the more proximal region suffered from the spectrum of heart defects reported in patients with 8p distal deletions. The 5-cM critical region is flanked distally by D8S1706 and WI-8327, both at approximately 10 cM, and proximally by D8S1825, at 15 cM. Neither GATA4 nor angiopoietin-2 (ANGPT2; a gene in 8p23 involved in blood vessel formation) were found to be deleted in some of the critical patients. We also found that CHDs are not related to the parental origin of deletion. CONCLUSIONS: Haploinsufficiency for a gene between WI-8327 and D8S1825 is critical for heart development. A causal relationship does not seem to exist between GATA4 and ANGPT2 haploinsufficiency and CHDs.

Early onset of gastric carcinoma and constitutional deletion of 18p.

We report on the association of a gastric carcinoma and a constitutional deletion of the short arm of chromosome 18 in a 14-year-old patient. The phenotype of the patient, including microcephaly, ptosis, micrognathia, tetralogy of Fallot, and mental retardation, fits well with previously reported cases of del(18p); she also showed a positive serology against Helicobacter pylori. The comparison of the alleles of polymorphic loci located on the short arm of chromosome 18 between the patient and her parents showed a maternal origin of the abnormal chromosome. Loss of heterozygosity (LOH) for loci located in the long arm of chromosome 18 is a frequent event in gastric carcinomas; it was observed in the tumoral mass of our patient and again, the alleles lost were of maternal origin. We postulate that the constitutional chromosomal abnormality may have favored the loss of the abnormal chromosome in some cells and that the loss of the deleted chromosome 18 (demonstrated by LOH for this chromosome in the tumoral mass) has been an early step in the pathogenesis of the gastric carcinoma of our patient with Helicobacter pylori infection acting as a cofactor.

The breakpoints of a constitutional inversion of chromosome 9 associated with haemophagocytic lymphohistiocytosis are not linked to the disease gene.

Haemophagocytic lymphohistiocytosis (HLH) is a severe disorder of early infancy consistent with an autosomal recessive inheritance. Neither the genetic locus nor the biochemical defect is known for the disease. A constitutional pericentric inversion of chromosome 9, with breakpoints in bands 9p23 and 9q31, has been reported in a case of HLH, suggesting a possible relationship between this chromosome abnormality and the disease. We investigated such an association, performing a genetic linkage analysis in a set of five consanguineous HLH families. 27 polymorphic markers on chromosome 9 were studied, excluding most of chromosome 9 as a putative site for the HLH gene.

Unbalanced X-chromosome inactivation in haemopoietic cells from normal women.

We studied X-chromosome inactivation patterns in blood cells from normal females in three age groups: neonates (umbilical cord blood), 25-32 years old (young women group) and >75 years old (elderly women). Using PCR, the differential allele methylation status was evaluated on active and inactive X chromosomes at the human androgen receptor (HUMARA) and phosphoglycerate kinase (PGK) loci. A cleavage ratio (CR) > or = 3.0 was adopted as a cut-off to discriminate between balanced and unbalanced X-chromosome inactivation. In adult women this analysis was also performed on hair bulbs. The frequency of skewed X-inactivation in polymorphonuclear (PMN) cells increased with age: CR > or = 3.0 was found in 3/36 cord blood samples, 5/30 young women and 14/31 elderly women. Mathematical analysis of patterns found in neonates indicated that X-chromosome inactivation probably occurs when the total number of haemopoietic stem cell precursors is 14-16. The inactivation patterns found in T lymphocytes were significantly related to those observed in PMNs in both young (P < 0.001) and elderly women (P < 0.01). However, the use of T lymphocytes as a control tissue for distinguishing between skewed inactivation and clonal proliferation proved to be reliable in young females, but not in elderly women, where overestimation of the frequency of clonal myelopoiesis may appear.

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.

Ph-positive CML in blastic phase with monosomy 7 in a Down syndrome patient. Monitoring by interphase cytogenetics and demonstration of maternal allelic loss.

We report a case of Ph-positive chronic myelocytic leukemia in blastic phase in an 11-year-old boy with Down syndrome. Monosomy 7 was the only additional chromosomal anomaly in the blastic clone. Fluorescence in situ hybridization analysis on interphase nuclei with a centromeric probe specific to chromosome 7 proved to be efficient in disease monitoring, and showed, together with the results of chromosome analysis on metaphases, that B-lymphocytes at the origin of an EBV-established line were not part of the leukemic clone. The study of DNA polymorphisms showed that the origin of the constitutional trisomy 21 was a maternal anaphase I nondisjunction, that the chromosome 7 lost in the blastic marrow clone was the maternal one, and led us to postulate that the mother's chromosomes are prone to impairment of normal disjunction. The study of allelic losses of chromosome 7 loci proved to be a further possibility for disease monitoring.

Ring chromosome 13 with loss of the region D13S317-D13S285: phenotypic overlap with XK syndrome.

We report on a patient with a multiple congenital abnormalities/mental retardation (MCA/MR) syndrome including facial abnormalities, agenesis of the corpus callosum, heart defect, 1st ray anomalies of the upper limb, and ambiguous genitalia, whose phenotype overlaps a previous description of XK syndrome. The patient has a ring chromosome (13) with deletion 13q32-qter. Molecular analysis demonstrated loss of the region from D13S317 to D13S285 and a paternal origin of the anomaly.

Ataxic gait and mental retardation with absence of the paternal chromosome 8 and an idic(8)(p23.3): imprinting effect or nullisomy for distal 8p genes?

A female child with mild dysmorphisms, motor and mental retardation had a 45,XX,-8,-8,+psu dic(8)(p23.3) karyotype in blood lymphocytes, skin fibroblasts and in a lymphoblastoid cell line. DNA analysis showed that the proposita was nullisomic for the 8pter region distal to D8S264, at less than 1 cM from the telomere. Analysis of DNA polymorphisms of 38 loci spread along the entire chromosome 8 revealed that only maternal alleles were present, distributed in four heterozygous and four homozygous regions. This finding indicated that the rearrangement occurred during maternal meiosis in a chromosome recombinant with a minimum of seven crossovers. To our knowledge this is the first case of uniparental maternal disomy for chromosome 8 and of nullisomy for the distal 1-cM portion of the short arm. The available data are in favour of the assumption that no imprinted genes are present on chromosome 8. Thus, dysmorphisms, motor and mental retardation of the proposita are likely to be caused by the nullisomy for the region distal to D8S264, a region in which a recessive gene for epilepsy with progressive mental retardation is known to be located.

Constitutional trisomy 8 as first mutation in multistep carcinogenesis: clinical, cytogenetic, and molecular data on three cases.

Three patients, with constitutional trisomy 8 mosaicism (CT8M), who developed a malignancy are reported. The diagnoses were refractory anaemia, acute lymphoblastic leukaemia, and idiopathic myelofibrosis. In the child with acute leukaemia, the CT8M was diagnosed at birth due to severe dysmorphisms and malformations; the other two patients showed a milder phenotype, and the CT8M was diagnosed only after the finding of trisomy 8 in neoplastic cells. The review of eight similar, previously reported cases and the clinical, cytogenetic, and molecular studies performed in our patients led us to make the following observations: (I) CT8M predisposes to neoplasms, preferentially to myelo- or lymphoproliferative diseases; (2) a gene dosage effect for glutathione reductase in red blood cells was seen in two of our patients; (3) the wide phenotypic variation of CT8M was confirmed: trisomy 8 in neoplastic cells of phenotypically near-normal cases may be misinterpreted as acquired; and (4) molecular studies suggested a postzygotic origin of the trisomy in our three cases, with the supernumerary chromosome being of paternal origin in one case and of maternal origin in the other two. We postulate that the trisomy 8 in neoplasms may often occur by mitotic nondisjunction in an early embryonic multipotent cell and that what is usually interpreted as an acquired trisomy 8 may in fact be CT8M. The constitutional trisomy 8 would act as a pathogenetically important first mutation in multistep carcinogenesis. Whenever trisomy 8 is found in malignancies, the patient should be reevaluated clinically to exclude CT8M, and CT8M patients should be monitored for the possible development of malignancies.

Hereditary haemorrhagic telangiectasia with extensive liver involvement is not caused by either HHT1 or HHT2.

Hereditary haemorrhagic telangiectasia (HHT) is a genetically heterogeneous dominant disorder. Two disease loci have been mapped to chromosomes 9q3 and 12q. In a large pedigree, with an unusually high number of patients with liver vascular malformations, both previously mapped loci have been excluded. The loci for two other inherited vascular malformation diseases, cerebral cavernous malformations and multiple cutaneous and mucosal venous malformations, have also been excluded. Thus we conclude that at least a third, as yet unmapped, HHT locus does exist, possibly associated with high frequency of liver involvement.

The same molecular mechanism at the maternal meiosis I produces mono- and dicentric 8p duplications.

We studied 16 cases of 8p duplications, with a karyotype 46,XX or XY,dup(8p), associated with mental retardation, facial dysmorphisms, and brain defects. We demonstrate that these 8p rearrangements can be either dicentric (6 cases) with the second centromere at the tip of the short arm or monocentric (10 cases). The distal 8p23 region, from D8S349 to the telomere, including the defensin 1 locus, is deleted in all the cases. The region spanning from D8S252 to D8S265, at the proximal 8p23 region, is present in single copy, and the remaining part of the abnormal 8 short arm is duplicated in the dicentric cases and partially duplicated in the monocentric ones. The distal edge of the duplication always spans up to D8S552 (8p23.1), while its proximal edge includes the centromere in the dicentric cases and varies from case to case in the monocentric ones. The analysis of DNA polymorphisms indicates that the rearrangement is consistently of maternal origin. In the deleted region, only paternal alleles were present in the patient. In the duplicated region, besides one paternal allele, some loci showed two different maternal alleles, while others, which were duplicated by FISH analysis, showed only one maternal allele. We hypothesize that, at maternal meiosis I, there was abnormal pairing of chromosomes 8 followed by anomalous crossover at the regions delimited by D8S552 and D8S35 and by D8S252 and D8S349, which presumably contain inverted repeated sequences. The resulting dicentric chromosome, 8qter-8p23.1(D8S552)::8p23.1-(D8S35)-8q ter, due to the presence of two centromeres, breaks at anaphase I, generating an inverted duplicated 8p, dicentric if the breakage occurs at the centromere or monocentric if it occurs between centromeres.