Redefining the risks of prenatally ascertained supernumerary marker chromosomes: a collaborative study.

BACKGROUND: A marker chromosome is defined as a structurally abnormal chromosome that cannot be identified by routine cytogenetics. The risk for phenotypic abnormalities associated with a marker chromosome depends on several factors, including inheritance, mode of ascertainment, chromosomal origin, and the morphology, content, and structure of the marker. METHODS: to understand the karyotype-phenotype relationship of prenatally ascertained supernumerary de novo marker chromosomes, we combined data from prenatal cases obtained from 12 laboratories with those from studies in the literature. We were able to obtain cytogenetic and phenotypic data from 108 prenatally ascertained supernumerary de novo marker chromosomes to refine the phenotypic risk associated with these markers. Because of the growing number of cases and because more techniques are available to delineate marker morphology, we have been able to group risk estimates into subcategories, such as by marker type and whether there are ultrasound abnormalities. RESULTS: If a de novo supernumerary marker chromosome is found prenatally, our data suggest there is a 26% risk for phenotypic abnormality when there is no other information defining the marker (such as chromosomal origin or information about the existing phenotype). However, if high resolution ultrasound studies are normal, this risk reduces to 18%. CONCLUSIONS: Our findings strongly support the value of additional genetic studies for more precisely defining the risk in individual cases involving marker chromosomes.

Genomic characterization of high-count MBL cases indicates that early detection of driver mutations and subclonal expansion are predictors of adverse clinical outcome.

High-count monoclonal B-cell lymphocytosis (MBL) is an asymptomatic expansion of clonal B cells in the peripheral blood without other manifestations of chronic lymphocytic leukemia (CLL). Yearly, 1% of MBLs evolve to CLL requiring therapy; thus being critical to understand the biological events that determine which MBLs progress to intermediate/advanced CLL. In this study, we performed targeted deep sequencing on 48 high-count MBLs, 47 of them with 2-4 sequential samples analyzed, exploring the mutation status of 21 driver genes and evaluating clonal evolution. We found somatic non-synonymous mutations in 25 MBLs (52%) at the initial time point analyzed, including 12 (25%) with >1 mutated gene. In cases that subsequently progressed to CLL, mutations were detected 41 months (median) prior to progression. Excepting NOTCH1, TP53 and XPO1, which showed a lower incidence in MBL, genes were mutated with a similar prevalence to CLL, indicating the early origin of most driver mutations in the MBL/CLL continuum. MBLs with mutations at the initial time point analyzed were associated with shorter time-to-treatment (TTT). Furthermore, MBLs showing subclonal expansion of driver mutations on sequential evaluation had shorter progression time to CLL and shorter TTT. These findings support that clonal evolution has prognostic implications already at the pre-malignant MBL stage, anticipating which individuals will progress earlier to CLL.

Tumor behavior in transitional cell carcinoma of the bladder in relation to chromosomal markers and histopathology.

Tumor cells from direct harvests and short term cultures were karyotyped from 15 patients with transitional cell carcinoma of the bladder. There were two tumors with an apparently normal diploid karyotype, eight with counts up to 50 and with marker chromosomes, and five with counts of 60 or more and with markers. The median duration between recurrences was 3 months for the near-diploid, and 3 months for the near-polyploid tumors. One patient whose tumor was normal diploid had a recurrence at 5 months and the second patient whose tumor had normal diploid tumor had no recurrence over 15 months. Four tumors (27% of the series) had a rearrangement involving band 3p14: three had +der(5)t(3;5)(p14;p14) and one had +der(6)t(1;3;6)(q21;p14;p23). Duplication 3p14----3pter was observed in four tumors, and deletion 11p15----11pter in five. Three other abnormalities were observed in three cases each: deletion 5p14----5pter, duplication 1q23----1q32 and deletion 6q21----6qter. Trisomy 7 was observed as a sole clonal abnormality in one carcinoma in situ. Thirteen of 15 patients had recurrence of their tumor. Tumor progression (either in stage or grade) was evident in seven recurrent tumors. Among the seven with tumor progression, three had 11p deletion, two had 11p deletion plus 3p duplication, one had 3p duplication, and one had trisomy 7. Four of the five that had 11p deletion underwent cystectomy and three have died. Three of eight near-diploid tumors progressed and four of five near-tetraploid tumors progressed. It will be important to characterize any cytogenetic changes that are of prognostic value, since the categorization of bladder tumors by other methods has been problematic.

Characterization of human laryngeal primary and metastatic squamous cell carcinoma cell lines UM-SCC-17A and UM-SCC-17B.

The squamous cell carcinoma (SCC) cell lines UM-SCC-17A and -17B were derived, respectively, from the primary laryngeal cancer and a metastatic neck tumor of a patient who failed to respond to radiation therapy but achieved long-term remission after surgery. The karyotypes of both cell lines and a subline of 17A were pseudodiploid and stable in multiple in vitro passages. Several karyotypic abnormalities were common to all three cell lines and therefore represent mutations present in the tumor before the divergence of the metastatic and subline populations whereas those rearrangements observed only in one cell are more likely to be secondary. The shared mutations include: duplication of the short and proximal long arm of chromosome 2, isochromosome 3q, duplication 7, inversion 8, duplication of the distal long arm of 18, and monosomy 21 or ring 21. Each line had different rearrangements involving chromosome 7 that resulted in three copies of most of the short arm being present in both cell lines, except for high passages of 17B, in which one structurally normal 7 was replaced by a dicentric isochromosome, dic(7)(q11.22), resulting in four copies of 7p. The dic(7) may represent an in vitro mutation. An isochromosome 13q was noted in both the stemline and subline of UM-SCC-17A but not in UM-SCC-17B. A del(11p) and an iso(21q) were present only in the 17A subline. The cell lines expressed the membrane antigen phenotype characteristic of squamous cancers although the UM-SCC-17A subline differed with respect to three markers. Of these, the A9 and blood group antigen changes are thought to be associated with progression. The subline, which carried the del(11)(p13-p15.1), also failed to express the E7 antigen mapped to the band 11p13. It is possible that the two apparently normal 11s in this subline carry a point mutation or microscopically undetected deletion involving the E7 antigen locus.

UM-EC-1, a new hypodiploid human cell line derived from a poorly differentiated endometrial cancer.

The University of Michigan endometrial carcinoma cell line UM-EC-1 was derived from a poorly differentiated endometrial adenocarcinoma of a 66-yr-old white female. Cell cultures were started using both tumor explants and a cell suspension obtained from collagenase-treated tumor tissue. The collagenase-derived cell suspension gave rise to monolayer cultures which grew rapidly from the outset. This subline of UM-EC-1 has now been subcultured more than 50 times. Cells derived from the tumor explants grew more slowly initially, but after a lag phase of 5 to 6 wk, this subline also exhibited rapid logarithmic growth and reached the same growth rate as that of the collagenase-treated cells. The explant subline has been subcultured more than 37 times. The doubling time of both sublines is 24 h under optimal growth conditions. The karyotype of both cell cultures is 43, XX, inv(1)(p32q42), -4, +der(8) t(8;12)(p23.1;q22), del(9)(q11), -13, -13, +t(13;13) (p13;p13), del(18)(q), -19, -22, -22, +t(22;22)(p11;p11). The net result of the chromosome losses and rearrangements was monosomy 4, duplication 8p23.1----qter, deletion 9q11----9qter, duplication 12q22----qter, deletion 18q, and monosomy 19. The t(13;13) and the t(22;22) were dicentric by C-banding. Virtually all of the chromosome changes were stable in multiple passages except that there was mosaicism for chromosome 13. Some cells contained a single copy of 13 and others had t(13;13). The available evidence indicates the t(13;13) is an isochromosome. UM-EC-1 cells produced tumors histologically similar to the original tumor in male, female, and ovariectomized female athymic mice. UM-EC-1 cells express human class I histocompatibility antigens as assessed by binding of antibodies to nonpolymorphic HLA and beta-2-microglobulin antigens. Blood group antigens A and H were absent although the patient is blood type A and these antigens are normally expressed in endometrial glands. A rearrangement involving the region of chromosome nine that carries the ABH locus may be related to the absence of blood group antigen expression by these cells. The E7 membrane antigen, the locus for which resides on the short arm of chromosome 11, was expressed strongly which is consistent with the presence of two intact copies of chromosome 11 in these cells.

Maternal effect on intelligence in fragile X males and females.

If the mother is the fragile X gene carrier, her daughters (and sons) with the mutation are at high risk of mental retardation. If the father is the (clinically unaffected) carrier, his daughters are normal. This is strong evidence for a maternal effect. The decreased penetrance and variable expressivity in fra(X) offspring of carriers could be related, at least in part, to variabile expression or availability of some maternal factor between pregnancies. We hypothesize a maternal effect in fra(X), with variability in intelligence of heterozygotes and hemizygotes mediated mainly by the maternal uterus or placenta by virtue of different patterns of lyonization in those tissues between pregnancies. If the mother is a carrier, the maternal placenta could develop with a skewed proportion of the normal or the fra(X) genetically active. Each female or male embryo could be exposed to very different environments with respect to genetic activity of the fra(X) chromosome, depending on the site of implantation within the uterus. If the father contributes the fra(X), the intrauterine environment is invariably normal and so are the daughters. Modifiers of the intrauterine effect could include lyonization patterns in tissues of the carrier fetus, and preferential inactivation of the paternal X in extra-embryonic tissues. The ultimate phenotype of the developing heterozygote and hemizygote may be determined by a threshold effect and interaction between the maternal genotype, the placental genotype, and the fetal genotype. The possibility of maternal effect is testable and has implications for treatment.

Characterization of a de novo 48,XX,+r(X),+r(17) by in situ hybridization in a patient with neurofibromatosis (NF1).

We describe a patient with familial neurofibromatosis (NF1), short stature, developmental delay, and a de novo chromosome abnormality. In situ hybridization was done using chromosome specific centromere probes to characterize the karyotype as 46,XX/47, XX,+r(X) (p11q11)/47,XX,+r(17) (p11q11)/48, XX,+r(X) (p11q11),+r(17) (p11q11). The NF1 mutation, as well as each supernumerary ring chromosome, may have played a role in perturbing the normal developmental process of this patient.

Dysgenetic male pseudohermaphroditism in a 45,X/46,X,del(Y)(q11.1) mosaic infant.

We describe an infant with dysgenetic male pseudohermaphroditism and the karyotype 45,X/46,X,del(Y)(q11.1). Histologic examination of the resected gonads showed cortical dysplasia indicative of incipient gonadoblastoma.

The origin of inverted tandem duplications, and phenotypic effects of tandem duplication of the X chromosome long arm.

Tandem repeats of chromosome material can arise as inverted or as direct duplications. Such duplications of the X chromosome are instructive regarding X-linked genetic determinants of phenotype. We describe a 40-year-old woman with a direct duplication Xq13.3 to Xq27.2, short stature, gonadal dysgenesis, and secondary amenorrhea. Comparison of her phenotype with that of two other women with a direct duplication of part of Xq confirms the existence of statural determinants within the region X13 to Xq21, determinants of ovarian function within X22 to X27, and the X inactivation center within or proximal to band Xq13.3. In humans, direct duplications are more frequent than inverted, but both forms are rare. The mean age of parents is normal in subjects with direct duplications, but is advanced in subjects with inverted duplications. An inverted duplication can arise from a three-break rearrangement that includes a U-type exchange; a similar origin (two breaks and a U-type exchange) and a parental age association can be postulated for dicentric inverted duplications including dicentric isochromosome X.

Malformation syndrome of duplication 12q24.1 leads to qter.

While duplication and deletion of the short arm of chromosome 12 cause well-recognized syndromes, duplication of the long arm chromosome 12 is rarely observe. We are reporting a duplication of chromosome 12 distal to band q24.1 in a five-month-old child. His chromosome constitution is 46,XY,-4+der(4),t(4:12)(p16;q24.1)mat. The balanced translocation is also carried by his maternal grandmother and two of the mother's brothers. The malformation syndrome consisted of unusual facial appearance and anomalies of the musculoskeletal, cardiovascular, genitourinary, and central nervous systems. Four previously reported patients had similar break points on chromosome 12 with similar malformations; therefore, phenotype-karyotype correlation suggests a definitive malformation syndrome associated with duplication of chromosome region 12q24.1 leads to qter.

Child with velocardiofacial syndrome and del (4)(q34.2): another critical region associated with a velocardiofacial syndrome-like phenotype.

We report on a child with congenital heart disease (atrial septal defect, ventricular septal defect, pulmonic stenosis), submucosal cleft palate, hypernasal speech, learning difficulties, and right fifth finger anomaly manifestations, consistent with velocardiofacial syndrome (VCFS); however, cytogenetic analysis demonstrated a small terminal deletion of the segment 4q34.2 to 4qter. Fluorescent in situ hybridization did not identify a deletion of the critical region associated with VCFS. In previously reported 4q deletions with a breakpoint distal to 4q34.2, no cardiac defects or cleft of palate were reported. Our patient has a deletion of 4q34.2 to 4qter and has palate and cardiac involvement and minor learning difficulties, which implies that genes involved in heart and palate development lie distal to 4q34.2, and that the critical region for more severe mental retardation on 4q may reside proximal to 4q34.2. These results suggest that a distal 4q deletion can lead to a phenotype similar to VCFS and emphasizes the importance of searching for other karyotype abnormalities when a VCFS-like phenotype is present and a 22q deletion is not identified.

Prader-Willi-like syndrome in a patient with an Xq23q25 duplication.

We report on a 24-year old woman with an Xq duplication and findings suggestive of Prader-Willi syndrome (PWS). Her birth weight was at the 3rd centile and her birth length was less than the 3rd centile. She was hypotonic and had a weak cry as an infant. There were no feeding difficulties, although her mother reports that as an infant, she was "small for her age." Excessive weight gain began between 3 and 4 years. The patient's development was delayed and she received special education. She has a history of hiding food. She has a sleep disturbance disorder and inappropriate social behavior. At the age of 24 years her height was below the 5th centile and weight >>95th centile. She has physical findings typical of PWS, skin picking, and speech articulation defects. Cytogenetic analysis showed a 46,X,dup(X)(q23q25) karyotype. Fluorescent in situ hybridization (FISH) studies using a chromosome X painting probe demonstrated that the rearrangement was intrachromosomal. The X-chromosome fold scoring technique was used to determine the X inactivation pattern and indicated that some cells expressed the abnormal X chromosome. Results of FISH studies using the SNRPN probe localized to 15q11q13 and DNA studies using the PW71B and SNRPN probes were normal. The duplicated X chromosome, random X inactivation pattern, and the negative molecular studies for PWS indicate that the abnormal X chromosome is the basis of this patient's phenotype. This patient emphasizes the importance of obtaining a karyotype even when a syndrome diagnosable by molecular methods is strongly suspected.

Sequential G-banding and fluorescent in situ hybridization on peripheral blood, bone marrow, and amniotic fluid samples.

We describe a method for fluorescent in situ hybridization (FISH) on previously G-banded slides and mounted coverslip preparations. The technique is successful on previously G-banded slides from peripheral blood and bone marrow specimens and on amniotic fluid samples from in situ harvests. Satellite sequence probes, whole chromosome paints, and unique sequence probes have all produced reliable results.

Analphoid 3qter markers.

Two cases of marker chromosomes derived from a non-centromeric location were studied to determine the characteristics of these markers with respect to the presence of functional centromeres and whether an associated phenotype could be described. The markers were characterized by fluorescence in situ hybridization and centromeric protein studies. Assessments were done to identify clinical features. Case 1 is a girl referred at age 1.5 years with swirly areas of hyperpigmentation, bilateral preauricular pits, hypotonia, developmental delay, and seizures. Case 2 is a male first evaluated as a newborn and then later during the first year of life. He had streaky hypopigmentation, right preauricular pit, accessory nipples, postaxial polydactyly, asymmetric cerebral ventricles, duplicated right kidney, a right pulmonary artery stenosis, and seizures. Mosaicism for an extra marker from the 3qter region was present in both cases. Both markers had a constriction near one end and were C-band negative. Centromeric protein studies indicated absence of CENP-B, presence of CENP-C (data for case 1 only), and presence of CENP-E. Marker chromosomes were thus identified with a chromosomal origin far from their usual centromeric region and yet appeared to have functional centromeres. These two cases did not permit a specific clinical phenotype to be ascribed to the presence of tetrasomy for 3q26.2 approximately 3q27.2-->3qter.

Inverted duplication of chromosome 5p14p15.3 confirmed with in situ hybridization.

Duplication of the short arm of chromosome 5 [dup(5)(p13.1p15.3)] has been associated with craniofacial malformations, cardiac defects, renal and intestinal malformations, limb abnormalities, and mental retardation. We report a 2-year-old white girl with a de novo 46,XX,inv dup(5)(p14p15.3) chromosome constitution, who presented with motor and language delays, bilateral strabismus, small posteriorly angulated ears, a high-arched palate, mild hypotonia, and an atrial septal defect. A CT scan of the head was normal. In situ hybridization with a cosmid probe specific for sub-band 5p15.3 (Oncor, Inc., Gaithersburg, MD) was used to identify the origin and orientation of the extra material. The milder manifestations in our patient are consistent with the hypothesis that significant phenotypic effects are associated with duplication of material proximal to band 5p14. This study demonstrates the usefulness of in situ probes in identifying the origin and orientation of duplicated genetic material.

Interstitial deletion of chromosome 10, del(10) (q11.2q22.1) in a boy with developmental delay and multiple congenital anomalies.

We describe a 4-year-old boy with an interstitial deletion of the long arm of chromosome 10: del(10) (q11.2q22.1). Frontal bossing, hypertelorism, bright blue iris color, up-slanting palpebral fissures, a flat nasal bridge, a broad nose, apparently low-set ears, micrognathia, deep philtrum, and hypotonia were noted neonatally. A murmur was noted at age 5 1/2 months and surgical repair of subaortic stenosis was required at 4 years. At 4 years micrognathia was no longer evident, but the palate was high-arched. The pattern of abnormalities included postnatal-onset slow growth, short stature, mental retardation, and cardiac anomalies.

Mother and son with deletion of 3p25-pter.

We report on a mother and son with a 3p25-pter deletion. Both have postnatal growth retardation, mental retardation, apparently low-set or malformed ears, and telecanthus. The mother also has ptosis and multiple joint pains, while the son has a long philtrum and anteverted nares. These phenotypes are compared to those of other 3p- patients. Both patients have many manifestations previously described. The son appears to be more severely affected than the mother.

Duplication 3p21----3pter and cyclopia.

We report on a patient with an interchromosomal duplication of 3p, from 3p21 to 3pter, which apparently arose de novo. The infant had multiple malformations including holoprosencephaly and cyclopia. It is possible that duplication 3p has a generalized effect on the holoprosencephalon or the cleavage of the embryonic forebrain. Fibroblasts from the patient are available from the NIGMS Human Genetic Mutant Cell Repository (GM 7216).

Chromosome 20 deletion in multiple endocrine neoplasia type 2: expanded double-blind studies.

Multiple endocrine neoplasia (MEN) type 2A and 2B are autosomal dominant syndromes in which medullary thyroid cancers are associated with adrenal pheochromocytomas. We have expanded our double-blind studies of high-resolution G-banded chromosomes from lymphocytes to a total of 12 MEN-2A families, 7 MEN-2B (mucosal neuroma phenotype) families and 23 non-MEN control subjects. Eighteen of 23 different control subjects were scored as having normal chromosomes 20, and 15 of 21 MEN-2A and 4 of 8 MEN-2B patients were scored as having an interstitial deletion: del(20) (p12.2p12.2). These findings suggest that the dominant mutation in many MEN-2A and MEN-2B families is a visible deletion within band 20p12.2. Combining the results of these double-blind studies with those of the only other comparable reported double-blind series provides a statistical probability of less than 1/1000 that the association between MEN-2A and the deletion was observed by chance alone. However, the occasional discrepancies in classification using presently available techniques preclude the use of high resolution chromosome studies for the diagnosis of MEN-2.

Familial papillary carcinoma of the thyroid.

Of 226 consecutive papillary carcinoma patients, 14 indicated that at least one other relative was similarly affected. Pathology confirmation was obtained in 8 of the 14 families. Of the eight families with documented familial papillary carcinoma, one had five members, another had four members, and yet another had three members affected. The remaining families had two members affected. In those families with two or more persons with confirmed papillary carcinomas of the thyroid, 20 first- and second-degree relatives were examined. Of those, one had a previously unidentified papillary carcinoma and 6 had a benign thyroid disease (4 primary hypothyroidism and 2 simple goiters). High-resolution chromosome studies of four patients from four different families were normal, and there was no increase in chromosome breakage in a fifth patient from yet another family. Autosomal dominant inheritance is possible. Although there was no family history of lipomas, osteomas, or intestinal polyposis to suggest Gardner syndrome, four parents of our familial papillary carcinoma patients had colon cancer. In addition, three other relatives died of unidentified intra-abdominal cancer. The apparently high frequency of colon cancer and other abdominal cancer in relatives was an additional concern. Based on our observations, three clinical recommendations can be made: obtain a family history of all patients with papillary carcinoma of the thyroid, since between 3.5 to 6.2% will have another affected relative; when two or more persons in a family have papillary carcinoma of the thyroid, all first- and second-degree relatives should have a neck palpation by an experienced examiner; and families with two or more persons with papillary carcinoma should be observed for possible colon cancer.