Submicroscopic Deletions Research Articles

Extraskeletal chondrosarcoma with a allele loss in chromosome 22 and chromosome 10: Shen WV, Walter B, Cain MJ, Young R, Katz J, and Smith M. University of California, Irvine, CA, U.S.A.

Abstracts *B2 ANGIOSARCOMA OF THE SCALP WiTH A COMPLEX HYPO- TETRAPLOID KARYOTYPE. A Molina, CD Bangs and T Donlon. Stanford University Medical Center, Depadments of Medicne (Oncology) and Pathology (Clinical Cytogenetics), Stanford, Ca 94305 Angiosarcoma is a rare (less than 2% of all sott tissue sarcomas), aggressive malignant neoPlasm of vascular endothelial cell origin which can arise in any tissue of the body: Cutaneous angiosarcoma almost exclusively affects the elderly and primarily involves the head and neck region in the area of the scalp. We performed a ~togenetic study of a resected cutaneous angiosarcoma in a 72 year old man. The patient underwent wide surgica ! excision of several erythematous ulcerated:scalp noduJes present in the dermis and suboutis. Histological examination re~ealed diffuse infiltrative cells with pleomorphic hyperchromatic nuclei~ and vascular neoformat!ons: Numerous mitotic figures were seen. Tumor tissue was disaggregated with coitagenease and cultured. Chromosomes were analyzed by the G-band method. Twenty cells were examined; ieveaiing a hy~!tetraploid Cell line with a chromosome number ranging from 85-91 anda modal number of 87. A repfesentat ve karyotype was dentil ed 86i XY, %XI +YI ÷1, ~2 +2q?i ÷2q?, -3, ~3, +!(3p). +i(3p), +del(3)(p2t3), +del(3) (p21.3)i +6, +7i +7. ÷8. +der(8)t(1;8)(p13;plL2), +9, +10, +10, +11, +11 ,+12. +t3, ÷13p+. +14i +14. +15, +16; +16, +17; +18, +19, +19, +20, +20,+21. +22. +22, ÷Stoat. Consistent structural abnormalities were two chromosome #2% with abnormal long arms:, two short arm de!clad copies of #3i an abnorma! #8 derived from a translocation with #1; a #13 with extra material on the short arm and multiple imarker Chromosomes. NUmeric Variation between Ceils was seen_To our knowledge: this: isthe tirst cytogenetic Study on angiosarooma of tha scalp. Despite surgical resection and adjuvant radiation therapy, the Patient develapedwidespread metastatic disease: Cytogenetic aria!yeas pe~ormed at an ea!lier stage in the evolution of this malignancy might offer insights into the mechanisms underlying tum0r progression and the generation of aneuploi~< ,B3~fI~%SKELSTAL : ~ : •- WITS A A L L ~ E LOSS IN CBRQMOSCME 22 ~ C~RQMOSQME i0. Shen WV, Walter B, Cain MJ, Young R, Katz J, and Smith M. University of California, Irvine, CA, U.SoA. Extraskeletal Myxoid Chondrosarcoma is a rare tumor of the adulthood. Non-random reciprocal translocation between chromosome 9 and 22 has been reported associated with this tumor. We have performed cytogenetic and molecular genetic investigation of a myxoid chondrosarcoma from right frontal-temporal reglon in a 27 year old female. A portion of this tumor was snapped frozen for DNA analysis and the other portion was mechanically dissociated and put in the tissue culture medium. Twenty-five analyzable metaphases from a lO day short term culture of the tumor revealed 46 XX karyotype. When the tumor DNA was compared with lymphocyte DNA from the same person with the probe D22S9 (mapped to chromosome 22 qll region), control DNA was homozygous for this probe (5.8Kb), tumor DNA has extra lower bend at 5.2Kb in addition to the 5.8Kb bend. This finding is suggestive of tumor DNA had possible small internal deletion of one allele in the reglon of 22qli. Analysis of WBC DNA revealed homozygosity for the D22S] probe which is mapped to chromosome 22qli- q13. Markedly decreased hybridization signal was noted in the tumor DNA indicating loss of chromosome 22 sequence in the tumor cells. The tumor DNA and control DNA were also compared with 9q RFLP probes including ASSP3, Aldo B, no difference were observed. Hox I~ NGF-B and DIOSI loci were not informative. Allele loss was noted in the tumor DNA with DIOS4 probe. This study reveals s defec~ of extraskeletal chondrosarcoma may reside in the region of 22qli region which is consistent with the previous cyto- genetic observation. In our case, no translocation was observed, but submicroscopic deletion may be present. Allele loss in chromosome i0 warrants further study.

Molecular dissection of a contiguous gene syndrome: frequent submicroscopic deletions, evolutionarily conserved sequences, and a hypomethylated "island" in the Miller-Dieker chromosome region.

The Miller-Dieker syndrome (MDS), composed of characteristic facial abnormalities and a severe neuronal migration disorder affecting the cerebral cortex, is caused by visible or submicroscopic deletions of chromosome band 17p13. Twelve anonymous DNA markers were tested against a panel of somatic cell hybrids containing 17p deletions from seven MDS patients. All patients, including three with normal karyotypes, are deleted for a variable set of 5-12 markers. Two highly polymorphic VNTR (variable number of tandem repeats) probes, YNZ22 and YNH37, are codeleted in all patients tested and make molecular diagnosis for this disorder feasible. By pulsed-field gel electrophoresis, YNZ22 and YNH37 were shown to be within 30 kilobases (kb) of each other. Cosmid clones containing both VNTR sequences were identified, and restriction mapping showed them to be less than 15 kb apart. Three overlapping cosmids spanning greater than 100 kb were completely deleted in all patients, providing a minimum estimate of the size of the MDS critical region. A hypomethylated island and evolutionarily conserved sequences were identified within this 100-kb region, indications of the presence of one or more expressed sequences potentially involved in the pathophysiology of this disorder. The conserved sequences were mapped to mouse chromosome 11 by using mouse-rat somatic cell hybrids, extending the remarkable homology between human chromosome 17 and mouse chromosome 11 by 30 centimorgans, into the 17p telomere region.

Prometaphase chromosomes in the tricho‐rhino‐phalangeal syndrome type I

Prometaphase chromosome analysis was undertaken in a patient with familial tricho-rhino-phalangeal syndrome type I (TRPS-I). The patient had apparently normal chromosomes and produced counterevidence to part of Bühler's hypothesis. Thus, although some cases of typical TRPS-I may be derived from a deletion of 8q24.12, others might be caused by gene mutation or submicroscopic deletion involving the corresponding locus within the band 8q24.11----24.13.

Unraveling the mysteries of Duchenne and Becker muscular dystrophy.

Through a process that has come to be known as reverse genetics, the gene and gene product involved in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) have been identified. The DMD/BMD gene is over 2 million base pairs in size and over 50% of DMD/BMD patients harbor submicroscopic deletions for portions of the gene. The gene product, named dystrophin, is 400 Kd in size. Dystrophin is present in skeletal, cardiac, and smooth muscles, as well as brain. The protein is absent or altered in DMD/BMD patient muscle. The normal function of dystrophin and the reasons why its alteration results in the DMD/BMD phenotypes are presently unknown. The discoveries to date, however, provide a starting point for investigating the fundamental pathogenetic mechanisms involved in DMD/BMD.

Monoamine oxidase deficiency in males with an X chromosome deletion

Mapping of the human MAOA gene to chromosomal region Xp21-p11 prompted our study of two affected males in a family previously reported to have Norrie disease resulting from a submicroscopic deletion in this chromosomal region. In this investigation we demonstrate in these cousins deletion of the MAOA gene, undetectable levels of MAO-A and MAO-B activities in their fibroblasts and platelets, respectively, loss of mRNA for MAO-A in fibroblasts, and substantial alterations in urinary catecholamine metabolites. The present study documents that a marked deficiency of MAO activity is compatible with life and that genes for MAO-A and MAO-B are near each other in this Xp chromosomal region. Some of the clinical features of these MAO deletion patients may help to identify X-linked MAO deficiency diseases in humans.

Gene diagnosis in X-linked ichthyosis

Three families segregating for X-linked ichthyosis (XLI) were analysed using the full-length STS cDNA probe and an anonymous polymorphic DNA sequence closely linked to the STS gene. In patients from two of the families, submicroscopic chromosomal deletions could be detected using both the STS and the GMGX9 (DXS237 locus) probes. Patients in the third family showed the same hybridization pattern as healthy males following molecular hybridization with either of the probes. The results of DNA analysis (indirect genotype diagnosis) agree well with those based on the arysulfatase C/beta-gal determination and prove the reliability of the biochemical test. Both methods are discussed for carrier detection, prenatal diagnosis, and genetic counseling.

Norrie disease as part of a complex syndrome explained by a submicroscopic deletion of the X chromosome

A 15-year-old male patient with the typical ocular symptoms of Norrie disease is described. Additionally, he presents severe mental retardation, growth disturbances, hypogonadism, and increased susceptibility to infections. This complex syndrome is apparently segregating through three generations: four other male relatives of the patient were blind from birth and died from recurrent infections between the ages of three to 15 months. The DNA sequence of the DXS7 locus (L1.28 probe), known to be closely linked to the Norrie gene, was not found in the patient's DNA. This result suggests that the more complex clinical picture seen is the result of a deletion of the X chromosome spanning DXS7, the Norrie gene, and several neighbouring loci. A detailed clinical description of the patient is given and compared to that of similar cases.

Molecular characterization of chromosome 7 long arm deletions in myeloid disorders

Partial deletion of the long arm of chromosome 7 is a common abnormality in the bone marrow cells of patients with myelodysplastic syndrome (MDS) or acute nonlymphocytic leukemia (ANLL). This study was undertaken to characterize the chromosome breakpoints in molecular terms and to determine if hemizygosity or submicroscopic deletions occur in patients without any cytogenetically detectable abnormality of chromosome 7. We studied restriction fragment length polymorphisms with 10 chromosome 7-specific DNA probes in separated WBC fractions. No molecular abnormalities occurred in lymphocyte-derived DNA. Several probes located in band 7q22 or distally thereof detected deletion of one allele in granulocyte-derived DNA from all four patients with chromosome 7 long arm deletion. In the granulocytes of one patient heterozygosity for the T cell receptor beta chain gene (in band 7q35) indicated that the deletion was interstitial. NJ-3, a proalpha2(I)collagen gene probe (in band 7q21–22) detected heterozygosity in the granulocytes of one patient. No hemizygosity or deletions were found in four patients with two normal chromosomes 7. These results confirm that mature granulocytes but not lymphocytes are derived from the abnormal clone. Interstitial deletions exist, and the extent of deleted genomic material varies among patients.

Molecular Characterization of Chromosome 7 Long Arm Deletions in Myeloid Disorders

Partial deletion of the long arm of chromosome 7 is a common abnormality in the bone marrow cells of patients with myelodysplastic syndrome (MDS) or acute nonlymphocytic leukemia (ANLL). This study was undertaken to characterize the chromosome breakpoints in molecular terms and to determine if hemizygosity or submicroscopic deletions occur in patients without any cytogenetically detectable abnormality of chromosome 7. We studied restriction fragment length polymorphisms with 10 chromosome 7-specific DNA probes in separated WBC fractions. No molecular abnormalities occurred in lymphocyte-derived DNA. Several probes located in band 7q22 or distally thereof detected deletion of one allele in granulocyte-derived DNA from all four patients with chromosome 7 long arm deletion. In the granulocytes of one patient heterozygosity for the T cell receptor beta chain gene (in band 7q35) indicated that the deletion was interstitial. NJ-3, a proalpha2(I)collagen gene probe (in band 7q21-22) detected heterozygosity in the granulocytes of one patient. No hemizygosity or deletions were found in four patients with two normal chromosomes 7. These results confirm that mature granulocytes but not lymphocytes are derived from the abnormal clone. Interstitial deletions exist, and the extent of deleted genomic material varies among patients.

Isolation of anonymous DNA sequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation.

Choroideremia, an X-chromosome linked retinal dystrophy of unknown pathogenesis, causes progressive nightblindness and eventual central blindness in affected males by the third to fourth decade of life. Choroideremia has been mapped to Xq13-21 by tight linkage to restriction fragment length polymorphism loci. We have recently identified two families in which choroideremia is inherited with mental retardation and deafness. In family XL-62, an interstitial deletion in Xq21 is visible by cytogenetic analysis and two linked anonymous DNA markers, DXYS1 and DXS72, are deleted. In the second family, XL-45, an interstitial deletion was suspected on phenotypic grounds but could not be confirmed by high-resolution cytogenetic analysis. We used phenol-enhanced reassociation of 48,XXXX DNA in competition with excess XL-45 DNA to generate a library of cloned DNA enriched for sequences that might be deleted in XL-45. Two of the first 83 sequences characterized from the library were found to be deleted in probands from family XL-45 as well as from family XL-62. Isolation of these sequences proves that XL-45 does contain a submicroscopic deletion and provides a starting point for identifying overlapping genomic sequences that span the XL-45 deletion. Each overlapping sequence will be studied to identify exons from the choroideremia locus.

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

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.

X-radiation-induced chromosome breakage in retinoblastoma lymphocytes

We have examined the spontaneous and X-radiation-induced chromosomal damage in normal humans and in patients with retinoblastoma using the BudR-Giemsa technique in lymphocytes cultured for 48 h.9 sporadic unilateral non-hereditary cases, 11 hereditary cases (8 bilateral sporadic and 3 unilateral hereditary cases) and 20 healthy individuals were studied simultaneously. No difference in the spontaneous frequency of chromatid and chromosome aberrations was observed between patients and controls. After treatment with 150 rad the frequency of chromosome exchange aberrations was higher in unilateral hereditary cases than the controls (42.0% ± 5.3 and 22.3 ± 2.6 respectively; p = 0.05). In bilateral sporadic retinoblastoma 2 different groups were observed. A hypersensitive group showed a significant increment in radiation-induced chromosomal exchange aberrations over the control group (46.2% ± 5.4 and 24.2% ± 2.1 respectively; p = 0.01). The other group had a chromosomal exchange frequency similar individuals (26/5% ± 2.0 and 24.2% ± 0.4 respectively; p = 0.10). Sporadic unilateral non-hereditary retinoblastoma had an exchange chromosomal aberration frequency similar to control individuals (26.1% ± 2.8 and 24.6% ± 2.7 respectively; p > 0.10). These results suggest that: 1. (a) There is no relationship between spontaneous chromosome fragility and retinoblastoma. 2. (b) Sporadi unilateral non-hereditary retinoblastoma has normal chromosome sensitivity to X-irradiation. 3. (c) Some hereditary cases of retinoblastoma are sensitive to X-rays while other behave like normals. A mutation or a submicroscopic deletion at a DNA repair locus which is independent of the retinoblastoma gene may cause this radiosensitivity.

Lip pits and deletion 1q32 → 41

A patient with an interstitial deletion of chromosome 1q[del(1q32----41)] was found to have, among other anomalies, congenital lower-lip pits. Lip pits are rare and are found mainly in association with the van der Woude syndrome and the popliteal pterygium syndrome; we cannot find a report of their association with a chromosome anomaly. To our knowledge, interstitial deletion of the segment 1q32----41 has not been reported. This observation raises the possibility that the van der Woude syndrome may be due to a submicroscopic deletion of chromosome 1q.

The screening of Duchenne muscular dystrophy patients for submicroscopic deletions.

We have probed the DNA of 156 Duchenne muscular dystrophy (DMD) patients, representing 140 kindreds, with cloned DNA sequences derived from Xp21 and known to show deletions in some DMD patients. Sixteen cases showed a deletion, as defined by lack of hybridisation to one or more of the four probes used. However, two of these cases were brothers, so 15 independent deletions (10.7%) are represented. The deletion map is compatible with the suggested order for the sites of the probes used in the study, that is, telomere----pERT87.15----pERT87.8----pERT87.1----pX J1.1----754----centromere. Further mapping of these deletions and characterisation of the deletion breakpoints should facilitate more accurate molecular localisation of the gene or genes which, when mutated, are responsible for causing DMD.

Prader-Willi Syndrome-Reply

In Reply .—The comments of Drs Wu and Hasen concerning our recent article 1 are appreciated. Three things mentioned by them deserve further comment. First, we of course concur that not all persons clinically thought to have Prader-Willi syndrome have demonstrable deletions of chromosome 15. While this could reflect true causal heterogeneity, we suspect that in many instances lack of rigorous clinical diagnostic criteria may be equally important in explaining this apparent heterogeneity. Second, those instances in which there is no cytogenetically detectable deletion may mean that less than (rather than more than) a simple absence of q11-q12 is sometimes sufficient. This hypothesis—that submicroscopic deletions or even point mutations within this region may sometimes yield the same phenotype—will probably eventually be proved or disproved using DNA probe technology (in conjunction with more rigorous clinical assessment, as already alluded to). Finally, we certainly agree that more needs to be learned about possible

Loss of a Harvey ras allele in sporadic Wilms' tumour

Genomic changes within chromosome band 11p13 appear to have a role in the initiation of Wilms' tumour. The human Harvey ras oncogene, c-Ha-ras 1, has been located by Jhanwar et al. immediately adjacent to this region at band 11p14 .1, although several groups have assigned the gene more distally at band 11p15 . We have examined tumour DNA from two cases of sporadic Wilms' tumour, and report here that in both cases one of the two constitutional c-Ha-ras 1 alleles was absent. One tumour had a reciprocal translocation between the short arm of chromosome 11 (at band 11p13), and the long arm of chromosome 12, with no visible loss of chromosomal material. The loss of a c-Ha-ras 1 allele in association with this translocation indicates that a submicroscopic deletion had occurred. The resulting hemizygosity may have had a role in tumour initiation. Our results indicate that the c-Ha-ras 1 gene and the 'Wilms' tumour locus' may be in close proximity. It would, therefore, be premature to exclude the possibility that these two sites are functionally related.

Segregation of recessive phenotypes in somatic cell hybrids: role of mitotic recombination, gene inactivation, and chromosome nondisjunction.

Somatic cell hybrids heterozygous at the emetine resistance locus (emtr/emt+) or the chromate resistance locus (chrr/chr+) are known to segregate the recessive drug resistance phenotype at high frequency. We have examined mechanisms of segregation in Chinese hamster cell hybrids heterozygous at these two loci, both of which map to the long arm of Chinese hamster chromosome 2. To follow the fate of chromosomal arms through the segregation process, our hybrids were also heterozygous at the mtx (methotrexate resistance) locus on the short arm of chromosome 2 and carried cytogenetically marked chromosomes with either a short-arm deletion (2p-) or a long-arm addition (2q+). Karyotype and phenotype analysis of emetine- or chromate-resistant segregants from such hybrids allowed us to distinguish four potential segregation mechanisms: (i) loss of the emt+- or chr+-bearing chromosome; (ii) mitotic recombination between the centromere and the emt or chr loci, giving rise to homozygous resistant segregants; (iii) inactivation of the emt+ or chr+ alleles; and (iv) loss of the emt+- or chr+-bearing chromosome with duplication of the homologous chromosome carrying the emtr or chrr allele. Of 48 independent segregants examined, only 9 (20%) arose by simple chromosome loss. Two segregants (4%) were consistent with a gene inactivation mechanism, but because of their rarity, other mechanisms such as mutation or submicroscopic deletion could not be excluded. Twenty-one segregants (44%) arose by either mitotic recombination or chromosome loss and duplication; the two mechanisms were not distinguishable in that experiment. Finally, in hybrids allowing these two mechanisms to be distinguished, 15 segregants (31%) arose by chromosome loss and duplication, and none arose by mitotic recombination.

Segregation of Recessive Phenotypes in Somatic Cell Hybrids: Role of Mitotic Recombination, Gene Inactivation, and Chromosome Nondisjunction

Somatic cell hybrids heterozygous at the emetine resistance locus (emtr/emt+) or the chromate resistance locus (chrr/chr+) are known to segregate the recessive drug resistance phenotype at high frequency. We have examined mechanisms of segregation in Chinese hamster cell hybrids heterozygous at these two loci, both of which map to the long arm of Chinese hamster chromosome 2. To follow the fate of chromosomal arms through the segregation process, our hybrids were also heterozygous at the mtx (methotrexate resistance) locus on the short arm of chromosome 2 and carried cytogenetically marked chromosomes with either a short-arm deletion (2p-) or a long-arm addition (2q+). Karyotype and phenotype analysis of emetine- or chromate-resistant segregants from such hybrids allowed us to distinguish four potential segregation mechanisms: (i) loss of the emt+- or chr+-bearing chromosome; (ii) mitotic recombination between the centromere and the emt or chr loci, giving rise to homozygous resistant segregants; (iii) inactivation of the emt+ or chr+ alleles; and (iv) loss of the emt+- or chr+-bearing chromosome with duplication of the homologous chromosome carrying the emtr or chrr allele. Of 48 independent segregants examined, only 9 (20%) arose by simple chromosome loss. Two segregants (4%) were consistent with a gene inactivation mechanism, but because of their rarity, other mechanisms such as mutation or submicroscopic deletion could not be excluded. Twenty-one segregants (44%) arose by either mitotic recombination or chromosome loss and duplication; the two mechanisms were not distinguishable in that experiment. Finally, in hybrids allowing these two mechanisms to be distinguished, 15 segregants (31%) arose by chromosome loss and duplication, and none arose by mitotic recombination.

46,XX Turner’s syndrome

A 25-year-old woman with gonadal dysgenesis, as manifested by estrogen deficiency, elevated urinary gonadotropins, primary amenorrhea, absence of ovarian masses by pelvic pneumography, and characteristic somatic features of Turner's syndrome, showed no demonstrable chromosome abnormality upon karyotypic analysis of cultured blood cells and skin fibroblasts. Buccal smears showed normal-sized Barr bodies and a normal sex chromatin index. Chromosome autoradiography disclosed allocyclic condensation of the late-replicating X chromosome during stages of metaphase. Although the cytologic and cytogenetic data do not exclude submicroscopic deletion or undetected translocation of the X chromosome, the possibility of intrachromosomal changes resulting in a genetically deficient X chromosome and Turner's syndrome is suggested as a probable etiologic mechanism.