The syndrome associated with the partial D-monosomy

Summary A female child with multiple malformations and 46,XX,Dq-/46,XX chromosomal mosaicism is described. The main data concerning the phenotypes of other partial D-monosomics mentioned in the literature are reviewed. Despite the variability of clinical picture in these patients it is possible to delineate at least one distnct syndrome associated with partial monosomy D. The completeness of clinical picture as well as of the symptoms vary from case to case. The most typical while non constant somatic symptoms of this syndrome are: absence of the thumb and of the 1st metacarpal bone combined with the fusion of the 4th and 5th metacarpal bones; skull/brain defects of the arrhinencephaly/trigonocephaly type; colobomata; anal atresia. Sometimes retinoblastoma occurs. It is likely that the chromosome involved in this syndrome is D1(13) chromosome.

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Zusammenfassung Ein Mädchen mit multiplen Mißbildungen und dem Mosaik 46,XX,Dq-/46,XX wird beschrieben. Bie dieser Gelegenheit werden die wesentlichen Daten über den Phänotyp anderer Fälle mit partieller D-Monosomie aus der Literatur zusammengefßt. Trotz der Variabilität des klinischen Bildes ist es möglich, wenigstens ein ausgeprägtes Monosomie D-Syndrom abzugrenzen. Die Vollständigkeit des klinischen Bildes und der Symptome variiert von Fall zu Fall. Die typischsten, wenn auch inkonstanten Symptome sind die folgenden: Fehlen des Daumens und des Os metacarpale 1; Verschmelzung des 4. und 5. Metacarpale; Schädel-Hirn-Defekte vom Arrhinencephalie-Trigonocephalie-Typ; Kolobome; Analatresie. In mehreren Fällen kommt auch ein Retinoblastom vor. Wahrscheinlich ist das betreffende Chromosome D1 (13).

     

Retinoblastoma. A model of hereditary fragile chromosomal regions.

Direct karyotyping of tumour cells from three familial and two sporadic cases of retinoblastoma revealed the existence of a Dq- marker chromosome. The hypothesis is launched that a specific region on the long arm of one of the D chromosomes is the site of a locus which is essential for the sustained differentiation of specialized retinal tissue and may be the site of other loci essential for the maturation of other embryonic tissues. Fragility of this region and its potentiality for breakage under the influence of various environmental insults could be the basic cytological event leading to the development of sporadic retinoblastoma. Mutants at these loci, including those of sustained differentiation, could be a less common operational event whereby some variants could enhance the fragility of their respective chromosomal region and thereby explain the genetic transmission of retinoblastoma in certain families. It is common for the critical functional disruption of the locus to precede the deletion which may then be considered the terminal event in the fragile region.     

Low incidence of deletion of the esterase D locus in retinoblastoma patients

Esterase D was quantitatively measured in the red blood cells from three patients from three separate kindreds who had abnormalities of chromosome 13. The esterase D activity was proportional to the number of copies of the q14 region of chromosome 13 present. These findings confirm published data localizing the esterase D gene to chromosome band 13q14, a region which is important in the etiology of retinoblastoma. Fifty-one additional retinoblastoma patients not known to have any chromosomal defect also underwent esterase D determination. In none of these patients did the esterase D measurement detect a 13q14 deletion. The normal esterase D levels in this series of 51 retinoblastoma patients suggest that deletion of an esterase D locus is infrequent in retinoblastoma patients. It must be noted that patients who are mosaics, with a 13q14 deletion in only a fraction of all somatic cells, could possibly have normal red blood cell esterase D levels. Further study is necessary to determine if esterase D determination of all retinoblastoma patients is a worthwhile clinical tool.     

Constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma.

In most patients with isolated unilateral retinoblastoma, tumor development is initiated by somatic inactivation of both alleles of the RB1 gene. However, some of these patients can transmit retinoblastoma predisposition to their offspring. To determine the frequency and nature of constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma, we analyzed DNA from peripheral blood and from tumor tissue. The analysis of tumors from 54 (71%) of 76 informative patients showed loss of constitutional heterozygosity (LOH) at intragenic loci. Three of 13 uninformative patients had constitutional deletions. For 39 randomly selected tumors, SSCP, hetero-duplex analysis, sequencing, and Southern blot analysis were used to identify mutations. Mutations were detected in 21 (91%) of 23 tumors with LOH. In 6 (38%) of 16 tumors without LOH, one mutation was detected, and in 9 (56%) of the tumors without LOH, both mutations were found. Thus, a total of 45 mutations were identified in tumors of 36 patients. Thirty-nine of the mutations-including 34 small mutations, 2 large structural alterations, and hypermethylation in 3 tumors-were not detected in the corresponding peripheral blood DNA. In 6 (17%) of the 36 patients, a mutation was detected in constitutional DNA, and 1 of these mutations is known to be associated with reduced expressivity. The presence of a constitutional mutation was not associated with an early age at treatment. In 1 patient, somatic mosaicism was demonstrated by molecular analysis of DNA and RNA from peripheral blood. In 2 patients without a detectable mutation in peripheral blood, mosaicism was suggested because 1 of the patients showed multifocal tumors and the other later developed bilateral retinoblastoma. In conclusion, our results emphasize that the manifestation and transmissibility of retinoblastoma depend on the nature of the first mutation, its time in development, and the number and types of cells that are affected.     

Frequency of somatic and germ-line mosaicism in retinoblastoma: implications for genetic counseling.

Although mosaicism can have important implications for genetic counseling of families with hereditary disorders, information regarding the incidence of mosaicism is available for only a few genetic diseases. Here we describe an evaluation of 156 families with retinoblastoma; the initial oncogenic mutation in the retinoblastoma gene had been identified in these families. In 15 ( approximately 10%) families, we were able to document mosaicism for the initial mutation in the retinoblastoma gene, either in the proband or in one of the proband's parents. The true incidence of mosaicism in this group of 156 families is probably higher than our findings indicate; in some additional families beyond the 15 we identified, mosaicism was likely but could not be proven, because somatic or germ-line DNA from key family members was unavailable. Germ-line DNA from two mosaic fathers was analyzed: in one of these, the mutation was detected in both sperm and leukocyte DNA; in the other, the mutation was detected only in sperm DNA. Our data suggest that mosaicism is more common than is generally appreciated, especially in disorders such as retinoblastoma, in which a high proportion of cases represent new mutations. The possibility of mosaicism should always be considered during the genetic counseling of newly identified families with retinoblastoma. As demonstrated here, genetic tests of germ-line DNA can provide valuable information that is not available through analysis of somatic (leukocyte) DNA.     

Isochromosome 6p,a unique chromosomal abnormality in retinoblastoma: Verification by standard staining techniques,new densitometric methods,and somatic cell hybridization

Summary Study of chromosome rearrangements in retinoblastoma tumors revealed that all tumors contained either an unusual isochromosome and/or extra copies of chromosome 1q. Extra copies of chromosome 1q occur in many malignancies. The pattern of G-bands suggested that the isochromosome was derived from either the short arm of chromosome 6, i(6p), or the long arm of chromosome 17, i(17q). Standard staining techniques using G-, C-, Q-, and R-banding; high resolution G-banding; and density profile analysis were consistent with the characteristic isochromosome of retinoblastoma being i(6p), rather than i(17q). This conclusion was substantiated by the analysis of segregants derived from retinoblastoma X mouse hybrid cells which had been grown in bromodeoxyuridine to select for loss of chromosome 17. The unique isochromosome was not lost under these conditions confirming that it is an i(6p) rather than an i(17q). The i(6p) abnormality has not been observed frequently in other tumors, but occurs in 60% of retinoblastoma tumors. Thus, although the mutation predisposing to retinoblastoma is known to map at 13q14, somatic amplification of genes on 1q and 6p may play a role in the pathogenesis of this tumor.     

Erbliche Disposition zu Tumoren des Auges

Ocular neoplasms are associated with several hereditary cancer predisposition syndromes. Of these, hereditary retinoblastoma is the most important. Historically, hereditary retinoblastoma has been the prototype dominant cancer predisposition, and Knudson’s study of this tumour has resulted in the two-step mutation hypothesis, which has been seminal for developing a further understanding of hereditary cancers. Although ocular manifestations are of minor relevance for the prognosis in tumour syndromes other than retinoblastoma, they may be important for the recognition of the underlying cancer predisposition. There is no monogenic predisposition for the development of uveal melanoma, which is the most frequent malignant ocular neoplasm in humans. However, epidemiological data suggest that genetic factors may play a role. The study of somatic alterations in these tumours has shown that the loss of one chromosome 3 is closely associated with an adverse prognosis. Such a somatic genotype-phenotype association is highly exceptional among solid tumours.     

Analysis of banding patterns and mosaic configurations in a case of ring chromosome 15

Summary Cytogenetic studies on lymphocytes from a 14-year-old mentally retarded girl with somatic anomalies suggestive of a chromosomal abnormality revealed a ring chromosome 15. The long arm of the defective chromosome is broken at band q24 or q25. The silver staining technique for nucleolus organizer regions showed that the ring had lost the achromatic stalk and the satellite. The chromosomal mosaicism resulting from the structural instability of the ring chromosome was analyzed and compared with 6 cases reported in the literature. It is proposed that the clinical manifestations in the different patients with ring chromosome 15 result from both the deficiency in the long arm and the mosaic configurations.     

High rate of detection of 13q14 deletion mosaicism among retinoblastoma patients (using more extensive methods)

Summary Using a cell population with a high proportion of early mitotic cells and by examining more cells derived from peripheral lymphocytes, we found three cases with a 13q14 deletion mosaicism among fifteen retinoblastoma patients; one with a de novo 13/18 balanced translocation, and another with a monosomy 13(q13»q21.2 or 21.3). The three patients with a 13q14 deletion mosaicism had sporadic retinoblastoma (two had bilateral and one unilateral retinoblastoma). The results indicate that 13q14 deletion mosaicism plays a major role in the etiology of this tumor.     

Fibroblasts from Retinoblastoma patients: Enhanced growth in fetal calf serum and a normal response to ionizing radiation

Retinoblastoma is a rare malignant eye tumor found almost exclusively in young children. In 30% of cases, the tumor is bilateral and is inherited as an autosomal dominant trait. In such patients, all of the cells in the body must carry the mutation predisposing to retinoblastoma. To search for the expression of the gene in cells outside the retina, we have studied several in vitro properties of skin fibroblasts from patients with bilateral retinoblastoma. Measurement in low concentrations of fetal calf serum of the initial growth rate and the plating efficiency show that fibroblasts from retinoblastoma donors grow significantly better than those from normal donors. However, we were unable to confirm the results of other investigators that fibroblasts from donors with bilateral retinoblastoma are unusually sensitive to ionizing radiation. In family studies, skin fibroblasts from normal siblings had the same radiation sensitivity as fibroblasts from sibling with retinoblastoma.     

Detection of mutations of the RB1 gene in retinoblastoma patients by using exon-by-exon PCR-SSCP analysis.

Most sporadic cases of retinoblastoma, malignant eye tumor of children, may require the identification of a mutation of the retinoblastoma gene (RB1 gene) for precise genetic counseling. We established a mutation detection system of and screened for the RB1 gene mutation in 24 patients with retinoblastoma--12 bilateral patients and 12 unilateral patients. Mutation analysis was performed by PCR-mediated SSCP analysis in the entire coding region and promoter region, as an initial screening method, followed by direct genomic sequencing. Possible oncogenic mutations were identified in 14 (58%) of 24 tumors, of which 6 were single base substitutions, 4 were small deletions, 3 were small insertions, and 1 was a complex alteration due to deletion-insertion. A constitutional somatic mosaicism was suggested in one bilateral patient. A majority (57%) of mutations were found in E1A binding domains, and all were presumed to truncate the normal gene products. The mutation analysis presented here may provide a basis for the screening system of RB1 gene mutations in retinoblastoma patients.     

Quantification of the paternal allele bias for new germline mutations in the retinoblastoma gene

New germline mutations in the human retinoblastoma gene are known to arise preferentially on paternally derived chromosomes, but the magnitude of that bias has not been measured. We evaluated 49 cases with a new germline mutation and found that in 40 cases (82%) the mutation arose on the paternally derived allele. We also evaluated 48 cases likely to have a somatic initial mutation; in this group the initial mutation arose on paternal or maternal chromosomes with approximately equal frequency. There was no statistically significant difference in the average age of fathers of children with new paternal germline mutations from the average age of fathers of children with new maternal germline mutations or somatic initial mutations. Combining the data with that from previous reports from other groups, the proportion of new germline mutations arising on a paternally derived allele is 85% (based on 72 cases; 95% confidence interval = 76–93%). This number can be useful in the genetic counseling of some families with retinoblastoma. Received: 18 December 1996 / Accepted: 30 April 1997     

Two-hit model for sporadic congenital anomalies in mice with the disorganization mutation.

Congenital anomalies have complex etiologies involving both genetic and nongenetic components. Many are sporadic, without obvious evidence for heritability. An important model for these anomalies is a mutation in laboratory mice that is called "disorganization" (Ds), which functions as a variable autosomal dominant and leads to a wide variety of congenital anomalies involving many developmental processes and systems. Variable expressivity, asymmetrical manifestations, and low penetrance suggest that somatic events determine the location and nature of these anomalies. A statistical analysis suggests that occurrence of anomalies in mice with the Ds mutation follows a Poisson distribution. These results suggest that congenital anomalies in mice with the Ds mutation occur independently of each other. We propose that Ds causes a heritable predisposition to congenital anomalies and that Ds and appropriate somatic events combine to compromise normal development. We also propose that some sporadic, nonheritable congenital anomalies involve somatic mutations at Ds-like loci. Ds may therefore serve not only as a model for developmental anomalies in cell fate and pattern formation but also for complex developmental traits showing variable expressivity, low penetrance, and sporadic occurrence in mice and humans.     

Somatic mosaicism in a patient with bilateral retinoblastoma.

We describe two cell lines with different deletions of the retinoblastoma gene in a patient with bilateral retinoblastoma. This patient has transmitted the mutation less frequent in his lymphocytes to two affected children. We cloned, mapped, and sequenced the junction fragments of the two deletions and found that they share one breakpoint but extend into opposite directions. An insertion of 4 bp of unknown origin is present between the breakpoints in one of the deletions. The second deletion shows a more complex rearrangement, including an inversion at the 5' end. Short regions of homology were found at the breakpoints and flanking the inversion. These results support the notion that bilateral retinoblastoma may not only be due to a germ-line mutation but also to a postzygotic mutation leading to somatic mosaicism.     

Detection of submicroscopic deletions and a DNA polymorphism at the retinoblastoma locus

Summary DNA samples from 60 unrelated patients with retinoblastoma were screened by Southern blot hybridization using two probes that are closely linked to the retinoblastoma locus within human chromosome band 13q14. Seven of 44 patients with bilateral or multifocal unilateral retinoblastoma and one patient with unifocal unilateral retinoblastoma were found to have a heterozygous deletion for the anonymous DNA sequence H3-8. Three of the eight deletions did not include the esterase D locus and were undetectable by conventional cytogenetic analysis. The findings are compatible with the deletions being the cause of retinoblastoma in these cases and provide a basis for DNA diagnosis in nearly 20% of patients with bilateral and multifocal unilateral retinoblastoma. The H3-8 probe also detects a restriction fragment length polymorphism that is a useful genetic marker in some families.     

Somatic mosaic activating mutations in PIK3CA cause CLOVES syndrome

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies (CLOVES) is a sporadically occurring, nonhereditary disorder characterized by asymmetric somatic hypertrophy and anomalies in multiple organs. We hypothesized that CLOVES syndrome would be caused by a somatic mutation arising during early embryonic development. Therefore, we employed massively parallel sequencing to search for somatic mosaic mutations in fresh, frozen, or fixed archival tissue from six affected individuals. We identified mutations in PIK3CA in all six individuals, and mutant allele frequencies ranged from 3% to 30% in affected tissue from multiple embryonic lineages. Interestingly, these same mutations have been identified in cancer cells, in which they increase phosphoinositide-3-kinase activity. We conclude that CLOVES is caused by postzygotic activating mutations in PIK3CA. The application of similar sequencing strategies will probably identify additional genetic causes for sporadically occurring, nonheritable malformations.     

International Commission for Protection Against Environmental Mutagens and Carcinogens. ICPEMC working paper no. 4. Mutation surveillance of sentinel anomalies in Hungary, 1980-1984

Sentinel phenotypes are indicators of germinal dominant gene mutations. 23 sentinel abnormalities and 2 sentinel childhood tumours (bilateral retinoblastoma and Wilms' tumour), i.e., 25 sentinel anomalies of autosomal dominant origin were selected from the material of the Hungarian Congenital Malformation Registry, 1980-1984. Furthermore, cases of sentinel childhood tumours from the Hungarian Childhood Tumour Registry and some extra notifications of sentinel abnormalities were also included. Experts examined index patients and their parents in order to confirm or exclude nosological diagnosis, to separate sporadic and familial cases, to obtain environmental history and to give genetic counselling. The revised total observed prevalence of 25 sentinel anomalies was 3.80 per 10,000 livebirths. Only 12% of cases examined were familial. According to the statistical power calculation, 47,500 livebirths are needed to detect a doubling of mutation rate with probabilities of type I and II errors of 0.05 level. In Hungary the average number of yearly births was 135,548 in the study period.