Duplications in addition to terminal deletions are present in a proportion of ring chromosomes: clues to the mechanisms of formation.

BACKGROUND AND METHODS: Ring chromosomes are often associated with abnormal phenotypes because of loss of genomic material at one or both ends. In some cases no deletion has been detected and the abnormal phenotype has been attributed to mitotic ring instability. We investigated 33 different ring chromosomes in patients with phenotypic abnormalities by array based comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH). RESULTS: In seven cases we found not only the expected terminal deletion but also a contiguous duplication. FISH analysis in some of these cases demonstrated that the duplication was inverted. Thus these ring chromosomes derived through a classical inv dup del rearrangement consisting of a deletion and an inverted duplication. DISCUSSION: Inv dup del rearrangements have been reported for several chromosomes, but hardly ever in ring chromosomes. Our findings highlight a new mechanism for the formation of some ring chromosomes and show that inv dup del rearrangements may be stabilised not only through telomere healing and telomere capture but also through circularisation. This type of mechanism must be kept in mind when evaluating possible genotype-phenotype correlations in ring chromosomes since in these cases: (1) the deletion may be larger or smaller than first estimated based on the size of the ring, with a different impact on the phenotype; and (2) the associated duplication will in general cause further phenotypic anomalies and might confuse the genotype-phenotype correlation. Moreover, these findings explain some phenotypic peculiarities which previously were attributed to a wide phenotypic variation or hidden mosaicism related to the instability of the ring.

Clinical spectrum of immunodeficiency, centromeric instability and facial dysmorphism (ICF syndrome).

BACKGROUND: Immunodeficiency, centromeric instability and facial dysmorphism (ICF syndrome) is a rare autosomal recessive disease characterised by facial dysmorphism, immunoglobulin deficiency and branching of chromosomes 1, 9 and 16 after PHA stimulation of lymphocytes. Hypomethylation of DNA of a small fraction of the genome is an unusual feature of ICF patients which is explained by mutations in the DNA methyltransferase gene DNMT3B in some, but not all, ICF patients. OBJECTIVE: To obtain a comprehensive description of the clinical features of this syndrome as well as genotype-phenotype correlations in ICF patients. METHODS: Data on ICF patients were obtained by literature search and additional information by means of questionnaires to corresponding authors. RESULTS AND CONCLUSIONS: 45 patients all with proven centromeric instability were included in this study. Facial dysmorphism was found to be a common characteristic (n = 41/42), especially epicanthic folds, hypertelorism, flat nasal bridge and low set ears. Hypo- or agammaglobulinaemia was demonstrated in nearly all patients (n = 39/44). Opportunistic infections were seen in several patients, pointing to a T cell dysfunction. Haematological malignancy was documented in two patients. Life expectancy of ICF patients is poor, especially those with severe infections in infancy or chronic gastrointestinal problems and failure to thrive. Early diagnosis of ICF is important since early introduction of immunoglobulin supplementation can improve the course of the disease. Allogeneic stem cell transplantation should be considered as a therapeutic option in patients with severe infections or failure to thrive. Only 19 of 34 patients showed mutations in DNMT3B, suggesting genetic heterogeneity. No genotype-phenotype correlation was found between patients with and without DNMT3B mutations.

Cryptic deletions are a common finding in "balanced" reciprocal and complex chromosome rearrangements: a study of 59 patients.

Using array comparative genome hybridisation (CGH) 41 de novo reciprocal translocations and 18 de novo complex chromosome rearrangements (CCRs) were screened. All cases had been interpreted as "balanced" by conventional cytogenetics. In all, 27 cases of reciprocal translocations were detected in patients with an abnormal phenotype, and after array CGH analysis, 11 were found to be unbalanced. Thus 40% (11 of 27) of patients with a "chromosomal phenotype" and an apparently balanced translocation were in fact unbalanced, and 18% (5 of 27) of the reciprocal translocations were instead complex rearrangements with >3 breakpoints. Fourteen fetuses with de novo, apparently balanced translocations, all but two with normal ultrasound findings, were also analysed and all were found to be normal using array CGH. Thirteen CCRs were detected in patients with abnormal phenotypes, two in women who had experienced repeated spontaneous abortions and three in fetuses. Sixteen patients were found to have unbalanced mutations, with up to 4 deletions. These results suggest that genome-wide array CGH may be advisable in all carriers of "balanced" CCRs. The parental origin of the deletions was investigated in 5 reciprocal translocations and 11 CCRs; all were found to be paternal. Using customized platforms in seven cases of CCRs, the deletion breakpoints were narrowed down to regions of a few hundred base pairs in length. No susceptibility motifs were associated with the imbalances. These results show that the phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions and that spermatogenesis is more prone to generate multiple chaotic chromosome imbalances and reciprocal translocations than oogenesis.

DNMT3B mutations and DNA methylation defect define two types of ICF syndrome.

ICF syndrome is a rare autosomal recessive disease characterized by variable immunodeficiency, centromeric instability, and facial abnormalities. Mutations in the catalytic domain of DNMT3B, a gene encoding a de novo DNA methyltransferase, have been recognized in a subset of patients. ICF syndrome is a genetic disease directly related to a genomic methylation defect that mainly affects classical satellites 2 and 3, both components of constitutive heterochromatin. The variable incidence of DNMT3B mutations and the differential methylation defect of alpha satellites allow the identification of two types of patients, both showing an undermethylation of classical satellite DNA. This classification illustrates the specificity of the methylation process and raises questions about the genetic heterogeneity of the ICF syndrome.

Late diagnosis in severe and mild intellectual disability in adulthood.

BACKGROUND: The diagnosis of intellectual disability (ID) is highly dependent on a comprehensive personal and family medical history, a complete physical examination and a careful developmental assessment of the patient. Our study intended to: (1) classify the aetiology of mild and severe ID in an adult population of 140 Italian subjects; (2) evaluate the frequency of associated medical conditions; (3) evaluate the age of diagnosis in both groups; and (4) underline the importance of aetiological diagnosis for adult ID patients also. METHODS: The study involved 140 consecutive adult Italian ID inpatients and outpatients neurologically investigated at the Neurological Institute C. Mondino of Pavia Service for Mental Retardation. A total of 80 patients had mild ID (MID group) (39 females, 41 males), mean age 34 years (range 19-61 years), mean IQ = 64 (range 51-75), and 60 had severe ID (SID group) (32 females, 28 males), mean age 30 years (range 19-69 years). They underwent a complete diagnostic work-up that comprised prenatal, perinatal and postnatal history, physical examinations, laboratory investigations, genetic survey and neuroradiological investigations to determine the aetiology of ID and to evaluate the presence of associated medical conditions. RESULTS: ID aetiology was classified as prenatal in 34% of the MID and 28% of the SID group. Perinatal and postnatal events were found in 6% of the MID and in 5% of the SID group. Associated medical conditions were found in 97 patients (47% MID and 26% SID). A genetic diagnosis was possible in 6% of patients above 20 years of age and in 5% of patients above 40 years. A diagnosis of cerebral dysgenesis was possible in 5% of patients above 20 years and 4% of patients above 40 years. CONCLUSIONS: A long interval between the diagnosis of ID and the aetiological definition can be observed in a significant percentage (24%) of our population, leading to unfortunate consequences of late diagnosis: late onset of a specific therapeutic program, genetic counselling that is frequently no more useful, and ineffective prenatal diagnosis, leading to the birth of other affected subjects (for familiar ID).

Genetic heterogeneity for a Nijmegen breakage-like syndrome.

Nijmegen breakage syndrome (NBS) is a rare, autosomal-recessive chromosome instability disorder characterized by growth and developmental defects, immunodeficiency, high susceptibility to lymphoid malignancies, hypersensitivity to ionizing radiation and aberrant cell-cycle checkpoint control. The disease is caused by mutations in the NBS1 gene, which encodes nibrin, a component of the hMre11-Rad50-p95 complex involved in cellular response to DNA double-strand breaks. Genetic heterogeneity has been suggested in at least two patients with the NBS phenotype, but no mutation in the NBS1 gene; recently, mutations in the gene encoding the enzyme ligase IV have been identified in patients with signs of NBS. We describe a boy with an NBS clinical phenotype but no mutation in either the NBS1 or the LIG4 genes. The analysis of his cellular phenotype reveals chromosome instability and radiosensitivity, but normal cell-cycle checkpoint control. In addition, a literature review was carried out to summarize and compare data of all NBS-like patients reported to date. This case confirms genetic heterogeneity for NBS. We believe that dissecting the clinical and cellular phenotypes of this and other NBS-like patients will provide useful information for the research of new genes involved in cellular response to DNA damage and the assessment of cancer risk in NBS-like syndrome.

G2-phase radiation response in lymphoblastoid cell lines from Nijmegen breakage syndrome.

The relationship between G2-phase checkpoint activation, cytoplasmic cyclin-B1 accumulation and nuclear phosphorylation of p34CDC2 was studied in Nijmegen breakage syndrome cells treated with DNA damaging agents. Experiments were performed on lymphoblastoid cell lines from four Nijmegen breakage syndrome patients with different mutations, as well as on cells from an ataxia telangiectasia patient. Lymphoblastoid cell lines were irradiated with 0.50-2 Gy X-rays and the percentage of G2-phase accumulated cells was evaluated by means of flow cytometry in samples that were harvested 24 h later. The G2-checkpoint activation was analysed by scoring the mitotic index at 2 and 4 h after treatment with 0.5 and 1 Gy X-rays and treatment with the DNA double-strand break inducer calicheamicin-gamma1. Cytoplasmic accumulation of cyclin-B1 was evaluated by means of fluorescence immunostaining or Western blotting, in cells harvested shortly after irradiation with 1 and 2 Gy. The extent of tyrosine 15-phosphorylated p34CDC2 was assessed in the nuclear fractions. Nijmegen breakage syndrome cells showed suboptimal G2-phase checkpoint activation respect to normal cells and were greatly different from ataxia telangiectasia cells. Increased cytoplasmic cyclin-B1 accumulation was detected by both immunofluorescence and immunoblot in normal as well as in Nijmegen breakage syndrome cells. Furthermore, nuclear p34CDC2. phosphorylation was detected at a higher level in Nijmegen breakage syndrome than in ataxia telangiectasia cells. In conclusion, our data do not suggest that failure to activate checkpoints plays a major role in the radiosensitivity of Nijmegen breakage syndrome cells.

Ring chromosome 9 with a 9p22.3-p24.3 duplication.

UNLABELLED: A ring chromosome 9 containing an inverted 9p22.3-p24.3 duplication was found in a girl presenting with some of the phenotypic characteristics of ring 9 syndrome such as trigonocephaly, microcephaly, hypotelorism, micrognathia, single palmar crease, and bilateral clinodactyly. The typical facial dysmorphic features of 9p duplication, ascribed to trisomy of the band p22, were not present in this patient. Cytogenetic and molecular studies indicated that the duplicated region of band p22 in the ring is confined to the sub-band 22.3. CONCLUSION: The chromosome region responsible for the 9p duplication syndrome appears to be restricted to sub-bands p22. 1-22.2.

Abnormal methylation does not prevent X inactivation in ICF patients.

DNA undermethylation is a characteristic feature of ICF syndrome and has been implicated in the formation of the juxtacentromeric chromosomal abnormalities of this rare syndrome. We have previously shown that in female ICF patients the inactive X chromosome (Xi) is also undermethylated. This result was unexpected since female ICF patients are not more severely affected than male patients. Here we show that CpG island methylation is abnormal in some ICF patients but in other ICF patients, the difference in methylation pattern between Xi and Xa (active X) is maintained. The consequences of Xi undermethylation on gene expression were investigated by enzyme assays. They showed that significant gene expression did not correlate with CpG island methylation status. The widespread Xi undermethylation does not affect overall Xi replication timing and does not prevent Barr body formation suggesting that a normal methylation pattern is not required for normal chromatin organization of Xi. Molecular investigation of some X-chromosome intron regions showed that the methylation changes in ICF female patients extend to non CpG islands sequences. Our results suggest that the genetic alteration of DNA methylation in ICF syndrome has little consequence on X chromosome gene expression and chromatin organization.

Impaired p53-mediated DNA damage response, cell-cycle disturbance and chromosome aberrations in Nijmegen breakage syndrome lymphoblastoid cell lines.

PURPOSE: To investigate the p53 ionizing radiation-induced response, G1/S cell-cycle block and cytogenetic damage in Nijmegen breakage syndrome (NBS) cells characterized by different haplotypes. MATERIAL AND METHODS: Lymphoblastoid cell lines derived from three normals, five NBS and two ataxia telangiectasia (AT) individuals were treated with moderate doses of X-rays and changes in the p53 response were studied by dose-response and time-course experiments. Multiparametric flow cytometry analysis of bromodesoxyuridine-incorporated cells was carried out to analyse G1/S checkpoint alterations. Cytogenetic damage induced by 2 Gy radiation was assessed in cells harvested 28 h later. RESULTS: Comparison of mean values of p53 accumulation in NBS, AT and control cells indicated that protein induction in NBS cells was between normal and AT cells. Cell-cycle experiments showed a markedly reduced S-phase fraction in irradiated samples of normal cell lines, while NBS, and particularly AT cells, showed less reduction in S-phase fraction. Irrespective of differences in p53 induction and G1/S block, chromatid-type aberrations were induced at a comparable level in both syndromes, while being almost absent in normal cells. CONCLUSIONS: The data suggested that failure of NBS cells to initiate cell-cycle delay cannot account alone for their extreme sensitivity to radiation.

Cerebellar dysgenesis and mental retardation associated with a complex chromosome rearrangement.

Cerebellar hypoplasia, mild mental retardation, skeletal abnormalities, and ataxia were present in a 40 years old patient with a complex chromosome rearrangement (CCR). Chromosomes 2, 5, 16, and 17 were involved in the CCR. For the definition of the eight breakpoints leading to the rearrangement FISH with whole chromosomes paintings and specific telomeric probes was employed. Gene disruption, positional effect variegation, and sub-microscopic deletions are all possible causes for the abnormal phenotype observed in the patient.

Identical de novo mutation at the D4F104S1 locus in monozygotic male twins affected by facioscapulohumeral muscular dystrophy (FSHD) with different clinical expression.

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive hereditary neuromuscular disorder, transmitted in an autosomal dominant fashion. Its clinical expression is highly variable, ranging from almost asymptomatic subjects to wheelchair dependent patients. The molecular defect has been linked to chromosome 4q35 markers and has been related to deletions of tandemly repeated sequences located in the subtelomeric region detected by probe p13E-11 (D4F104S1). We describe a pair of monozygotic male twins affected by FSHD, carrying an identical de novo p13E-11 EcoRI fragment of paternal origin and showing great variability in the clinical expression of the disease, one being almost asymptomatic and the other severely affected. Their medical history was the same, with the exception of an anti-rabies vaccination performed at the age of 5 in the more severely affected twin. We hypothesise that the vaccination might have triggered an inflammatory immune reaction contributing to the more severe phenotype.

A variant of the Nijmegen breakage syndrome with unusual cytogenetic features and intermediate cellular radiosensitivity.

We report the first Italian case of Nijmegen breakage syndrome (NBS). The proband is an immunodeficient, microcephalic, 11 year old boy with a "bird-like" face. He developed a T cell rich B cell lymphoma. Spontaneous chromosomal instability was detected in T and B lymphocytes and fibroblasts; chromosomes 7 and 14 were only sporadically involved in the rearrangements and no clonal abnormality was present. The patient appeared to be sensitive both to ionising radiation and to bleomycin, although his sensitivity did not reach the level of AT reference cells. After bleomycin treatment, inhibition of DNA synthesis was low when compared with normal cells, but higher than observed in an AT reference strain. Moreover, cell cycle analysis, after drug exposure, showed a progressive reduction in the percentage of S phase cells, but the G1 arrest, found in normal cells, was not observed. On clinical evaluation our patient shares features with NBS subjects, but cytogenetic and cell biological data do not completely overlap with those reported in Nijmegen breakage syndrome. The ethnic origin of our patient might account for these differences, as expression of different allelic forms at the NBS locus.

Chromosomal sensitivity to clastogenic agents and cell cycle perturbations in Nijmegen breakage syndrome lymphoblastoid cell lines.

The relationship between chromosomal breakage and perturbations of cell cycle progression was investigated in lymphoblastoid cell lines established from a healthy donor, two subjects affected by Nijmegen Breakage Syndrome (NBS) and an ataxia-telangiectasia (AT) patient. The cytogenetic analysis revealed a similar chromosomal hypersensitivity in both NBS and AT cells exposed in the G1 phase to 200 cGy X-rays or in G2 to 15-30 cGy. Similarly, no differences were observed in the frequency of chromatid-type aberrations induced in G2 by 1-2 pg/ml calicheamicin gamma 1I, a DNA double-strand break inducer. In addition, as observed in AT cells, the rate of G2 radiation-induced chromosomal damage was less enhanced in NBS than in control cells following 3-h incubation with inhibitors of DNA synthesis/repair (cytosine arabinoside, aphidicolin, DMSO, hydroxyurea, caffeine). This is suggestive of an altered DNA lesion-processing pathway common to both syndromes. Despite the close resemblance of cellular phenotypes in the two syndromes, the analysis of mitotic indices carried out at 2 and 4 h postirradiation indicated that NBS sustained a G2-delay greater than that observed in AT cells, Furthermore, the flow cytometric analysis of 50-300 cGy irradiated cells at 10 and 20 h before harvesting showed that NBS cells sustained a G2/M phase arrest markedly lower than AT cells. Our data indicate that NBS and AT gene products are involved in a common pathway of radiation-induced chromosomal damage, but in a different one for cell cycle control after irradiation.

Monosomy of distal 4q does not cause facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary neuromuscular disorder transmitted in an autosomal dominant fashion. FSHD has been located by linkage analysis in the most distal part of chromosome 4q. The disease is associated with deletions within a 3.2 kb tandem repeat sequence, D4Z4. We have studied a family in which an abnormal chromosome 4 segregates through three generations in phenotypically normal subjects. This chromosome is the derivative of a (4;D or G) (q35;p12) translocation. Molecular analysis of the region 4q35 showed the absence of the segment ranging from the telomere to locus D4F104S1. Probe p13E-11 (D4F104S1), which detects polymorphic EcoRI fragments containing D4Z4, in Southern blot analysis showed only one allele in the carriers of the abnormal chromosome 4. Probe p13E-11 EcoRI fragments are contained in the subtelomeric region of 4q and their rearrangements associated with FSHD suggested that the gene responsible for the muscular dystrophy could be subject to a position effect variegation (PEV) because of its proximity to subtelomeric heterochromatin. The absence of the 4q telomeric region in our phenotypically normal cases indicates that haploinsufficiency of the region containing D4Z4 does not cause FSHD.

An analysis of Xq deletions.

We characterized by fluorescence in situ hybridization and Southern blotting 14 partial Xq monosomies, 11 due to terminal deletions and 3 secondary to X/autosome translocations. Three cases were mosaics with a XO cell line. In view of the possible role played by telomeres in chromosome segregation, we hypothesize a relationship between the loss of telomeric sequences in terminal deletions and the presence of 45,X cells. A correlation between phenotype and extent of deletion reveal that there is no correspondence between the size of the deletion and impairment of gonadal function. Turner stigmata are absent in patients without an XO cell line, when the breakpoint is distal to Xq24. A low birthweight is present whenever the breakpoint is at q22 or more proximal.