Complex chromosome 17p rearrangements associated with low-copy repeats in two patients with congenital anomalies.

Recent molecular cytogenetic data have shown that the constitution of complex chromosome rearrangements (CCRs) may be more complicated than previously thought. The complicated nature of these rearrangements challenges the accurate delineation of the chromosomal breakpoints and mechanisms involved. Here, we report a molecular cytogenetic analysis of two patients with congenital anomalies and unbalanced de novo CCRs involving chromosome 17p using high-resolution array-based comparative genomic hybridization (array CGH) and fluorescent in situ hybridization (FISH). In the first patient, a 4-month-old boy with developmental delay, hypotonia, growth retardation, coronal synostosis, mild hypertelorism, and bilateral club feet, we found a duplication of the Charcot-Marie-Tooth disease type 1A and Smith-Magenis syndrome (SMS) chromosome regions, inverted insertion of the Miller-Dieker lissencephaly syndrome region into the SMS region, and two microdeletions including a terminal deletion of 17p. The latter, together with a duplication of 21q22.3-qter detected by array CGH, are likely the unbalanced product of a translocation t(17;21)(p13.3;q22.3). In the second patient, an 8-year-old girl with mental retardation, short stature, microcephaly and mild dysmorphic features, we identified four submicroscopic interspersed 17p duplications. All 17 breakpoints were examined in detail by FISH analysis. We found that four of the breakpoints mapped within known low-copy repeats (LCRs), including LCR17pA, middle SMS-REP/LCR17pB block, and LCR17pC. Our findings suggest that the LCR burden in proximal 17p may have stimulated the formation of these CCRs and, thus, that genome architectural features such as LCRs may have been instrumental in the generation of these CCRs.

Molecular and cytogenetic analysis of familial Xp deletions.

Five families in which an Xp deletion is segregating and two families in which an X chromosome rearrangement including a deletion of the short arm is segregating were ascertained for study. Normal fertility was seen in all families. Members from 5 of the 7 families manifested short stature (height <5th centile), while normal height was present in two families. Studies of both the FMR-1 and the androgen receptor loci using PCR based X-inactivation analysis demonstrated that in all families analyzed, there is preferential inactivation of one X chromosome. Molecular cytogenetic analysis showed that members of 3 of the 7 families share a common breakpoint in an approximate 2-3 Mb region at Xp22.12, suggesting a possible hotspot for chromatin breakage. Previous genotype-phenotype correlations and deletion mapping have indicated that a gene for stature resides within the pseudoautosomal region in Xp22.33. Our findings indicate that the loss of this region is not always associated with short stature, suggesting that other factors may be involved.

Random X inactivation in a girl with a balanced t(X;9) and an abnormal phenotype.

X inactivation is the process by which mammalian females achieve dosage compensation by transcriptionally silencing one X chromosome. In chromosomally normal females, this process is random. However, most females with one abnormal X chromosome demonstrate complete skewing of X inactivation, presumably as the result of cell selection. We present a mentally retarded girl with a 46,X,t(X;9)(q28;q12) karyotype. Analysis of this patient's lymphocytes, using late replication banding and methylation assays for the androgen receptor (AR) and fragile X mental retardation (FMR1) genes, did not show the predicted nonrandom X inactivation pattern. Thus, this patient is functionally disomic for Xq28-qter in a proportion of her cells, most likely resulting in her abnormal phenotype. This case demonstrates the utility of correlating X inactivation patterns with phenotype in females with one structurally abnormal X chromosome, and suggests that both cytogenetic and molecular X inactivation studies should be included in the routine study of these individuals.

Characterization of neo-centromeres in marker chromosomes lacking detectable alpha-satellite DNA.

Recent studies have implicated alpha-satellite DNA as an integral part of the centromere, important for the normal segregation of human chromosomes. To explore the relationship between the normal functioning centromere and alpha-satellite DNA, we have studied eight accessory marker chromosomes in which fluorescence in-situ hybridization could detect neither pancentromeric nor chromosome-specific alpha-satellite DNA. These accessory marker chromosomes were present in the majority of or all cells analyzed and appeared mitotically stable, thereby indicating the presence of a functional centromere. FISH analysis with both chromosome-specific libraries and single-copy YACs, together with microsatellite DNA studies, allowed unequivocal identification of both the origin and structure of these chromosomes. All but one of the marker chromosomes were linear mirror image duplications, and they were present along with either two additional normal chromosomes or with one normal and one deleted chromosome. Indirect immunofluorescence analysis revealed that the centromere protein CENP-B was not present on these markers; however, both CENP-C and CENP-E were present at a position defining a 'neo-centromere'. These studies provide insight into a newly defined class of marker chromosomes that lack detectable alpha-satellite DNA. At least for such marker chromosomes, alpha-satellite DNA at levels detectable by FISH appears unnecessary for chromosome segregation or for the association of CENP-C and CENP-E at a functional centromere.

Sex chromosome markers: characterization using fluorescence in situ hybridization and review of the literature.

Fluorescence in situ hybridization (FISH) using biotin labeled X- and Y-chromosome DNA probes was utilized in the analysis of 23 sex chromosome-derived markers. Specimens were obtained through prenatal diagnosis, because of a presumptive diagnosis of Ullrich-Turner syndrome, mental retardation, and minor anomalies or ambiguous genitalia; three were spontaneous abortuses. Twelve markers were derived from the X chromosome and eleven from the Y chromosome; this demonstrates successfully the value and necessity of FISH utilizing DNA probes in the identification of sex chromosome markers. Both fresh and older slides, some of which had been previously G-banded, were used in these determinations. We have also reviewed the literature on sex chromosome markers identified using FISH.

Non-mosaic tetrasomy 9p in a liveborn infant with multiple congenital anomalies: case report and comparison with trisomy 9p.

Tetrasomy of the short(p) arm of chromosome 9 has been reported in few cases. Most of these children present with microbrachycephaly, wide forehead, hypertelorism, lowset, malformed ears, beaked noses, and micrognathia. Additional anomalies include short neck, congenital heart disease, genital abnormalities, multiple limb defects, hypotonia, and early death.

Reproductive outcome in 3 families with a satellited chromosome 4 with review of the literature.

We describe 3 families segregating for a translocation of the nucleolus organizer region (NOR) onto chromosome 4. Review of previously reported cases of translocations involving NOR and chromosome 4 shows that these translocations may be associated with variable reproductive outcomes. We provide evidence that imprinting is not the mechanism responsible for the variable reproductive outcomes in the case of satellited 4p chromosomes; this may offer indirect support for a ribosomal gene position effect. Translocated ribosomal genes may influence the expression of neighboring genes and could explain the variable phenotypes in individuals with satellited nonacrocentric chromosomes. We recommend that prenatal counseling of individuals with satellited nonacrocentric chromosomes should be cautious.

Molecular cytogenetic analysis of inv dup(15) chromosomes, using probes specific for the Prader-Willi/Angelman syndrome region: clinical implications.

Twenty-seven cases of inverted duplications of chromosome 15 (inv dup [15]) were investigated by FISH with two DNA probes specific for the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region on proximal 15q. Sixteen of the marker chromosomes displayed two copies of each probe, while in the remaining 11 markers no hybridization was observed. A significant association was found between the presence of this region and an abnormal phenotype (P < .01). This is the largest study to date of inv dup(15) chromosomes, that uses molecular cytogenetic methods and is the first to report a significant association between the presence of a specific chromosomal region in such markers and an abnormal phenotype.

Clinical, cytogenetic, and molecular evidence for an infant with Smith-Magenis syndrome born from a mother having a mosaic 17p11.2p12 deletion.

We describe an infant with del(17) (p11.2p12) whose deleted chromosome was inherited from a mosaic mother. The child had manifestations consistent with Smith-Magenis syndrome. The mother appeared to be of normal intelligence and she had minimal findings of Smith-Magenis syndrome. Separation of chromosome 17 homologues in somatic cell hybrids and molecular studies confirmed the cytogenetic diagnoses and the fact that the mother was mosaic. Furthermore, molecular analysis demonstrated novel breakpoints in this family, with the deletion extending into and completely encompassing the markers duplicated in Charcot-Marie-Tooth (CMT) disease. Although this Smith-Magenis syndrome patient is completely deleted for the CMT region, her electrophysiological findings are different from those found in CMT. This is the only reported case of Smith-Magenis syndrome with transmission from a partially affected mosaic mother. Transmission of interstitial deletions from mosaic parents may be more common than thought; therefore, parental chromosomes should be examined when interstitial deletions are identified.

Preaxial acrofacial dysostosis (Nager syndrome) associated with an inherited and apparently balanced X;9 translocation: prenatal and postnatal late replication studies.

We report on an infant with preaxial acrofacial dysostosis (Nager syndrome) who was diagnosed prenatally as having an apparently balanced X/autosome translocation [46,X,t(X;9)(p22.1;q32)mat] inherited from a previously diagnosed mosaic translocation carrier mother [46,XX/46,X,t(X;9)(p22.1;q32)]. Replication studies on amniocytes showed the normal X chromosome to be late replicating while the same studies repeated on the infant's lymphocytes showed the translocated X chromosome to be late replicating in most cells. Late replication studies of the mother's lymphocytes demonstrated that the normal X chromosome was late replicating in most cells. The presence of Nager syndrome in this infant may be the result of critical breakpoints and/or position effects on chromosome 9, inducing expression of a gene responsible for the syndrome.

Cytogenetic and molecular analysis in Angelman syndrome.

We report on cytogenetic and molecular analyses of 29 Angelman syndrome (AS) individuals ascertained in 1990 through the first National Angelman Syndrome Conference. High resolution GTG- and GBG-banded chromosomes were studied. Standard molecular analysis with six 15q11q13 DNA sequences was used to analyze copy number and parental origin of 15q11q13. Concordance between molecular and cytogenetic data was excellent. The combined data showed that 23 of the 27 probands (85%) on whom we had definitive results have deletions of the chromosome 15q11q13 region. Two classes of deletion were detected molecularly: most patients were deleted for the 5 more proximal probes, but in 2 cases the deletion extended distally to include in sixth probe. In the 13 cases where the parental origin of the deleted chromosome 15 could be established, it was maternal. There were no cases of uniparental disomy. Cytological observations of the relative sizes of the heterochromatic regions of the short arm of chromosome 15 suggested that chromosomes with large heterochromatic blocks may be more prone to de novo deletion.

Proximal interstitial deletion of 7q: a case report and review of the literature.

A male infant with multiple congenital anomalies was found to have a deletion of 7q [46,XY,del(7)(pter----q11.2::q22----qter)]. The father had a balanced rearrangement involving chromosomes 7 and 9, interpreted as 46,XY,dir ins(9;7), (9pter----9p12::7q22----7q11.2::9p12----++ +9qter;7pter---- 7q11.2::7q22----7qter). C-banding showed that the rearrangement occurred as a new event in the paternal grandfather's germ-line. Including the present patient, 16 cases of proximal 7q deletion (q11----q21/q22) have been described to date. This is a sufficient number of cases to permit comparison of manifestations to attempt delineation of karyotype-phenotype relationships in different proximal interstitial deletions of 7q.

A paternal balanced translocation [t(7;22)(q32;q13.3)] leading to reciprocal unbalanced karyotypes in two consecutive pregnancies.

A family is reported in which a man with a balanced reciprocal translocation [46,XY,t(7;22)(q32;q13.3)] fathered a daughter who was trisomic for the region 7q32----7qter and monosomic for 22q13.3----22qter, and a male fetus who was monosomic for 7q32----qter and trisomic for 22q13.3----22qter. The meiotic segregation of this translocation, as well as the phenotypes of the unbalanced offspring, are discussed.

Segregation products of male mice doubly heterozygous for the RB(6.16) and RB (16.17) translocations: influence of sperm karyotype on fertilizing competence under varying mating frequencies.

The meiotic segregants of male mice heterozygous for Rb(6.16)24Lub and Rb(16.17)7Bnr were viewed, for the first time, at first cleavage metaphase. Chromosomes were analyzed after G-banding, C-banding, and karyotyping. To study sperm aging effects, chromosomes of 202 one-cell zygotes derived from males mating at intervals of approximately 3, 14, and 21 days were examined. At least 89.6% of sperm-derived complements were products of 2:2 segregation; at most, a possible 6.4% were 3:1 segregants. The six expected types of 2:2 segregants, both balanced and unbalanced, were equifrequent in the total zygote population derived from sperm of all ages. When the data were analyzed according to mating frequency, the 3-day sperm population considered most likely to be fresh showed a deficiency of the segregant nullisomic for chromosome 6 and disomic for chromosome 17, when compared with the reciprocal segregant (P less than 0.025) as well as to all other 2:2 segregants (P less than 0.05). However, these sperm fertilized in greater numbers (P less than 0.01) than their reciprocal segregant (disomic for 6 and nullisomic for 17) in the 14-day sperm population. While sperm with chromosomal abnormalities are capable of fertilization, the competence of segregants nullisomic for 6 and disomic for 17 apparently depends on the prior storage period in the male. Further, the results suggest that the effect of aneuploidy on sperm function is dependent on the specific chromosome(s) involved.

Second meiotic nondisjunction is not increased in postovulatory aged murine oocytes fertilized in vitro.

We used an in vitro fertilization system to examine the effects of postovulatory oocyte age on nondisjunction at the second meiotic division. After ovulatory-inducing injections of hormone, we recovered mouse oocytes either at the estimated time of ovulation (controls) or 2, 4, 5, 10, or 14 h later. Oocytes were subjected to an in vitro fertilization procedure, and chromosomal preparations were made from first cleavage metaphase eggs. The first cleavage assay reveals morphologically distinguishable paternal and maternal chromosomes. Many of the aged oocytes were activated rather than fertilized by the in vitro procedure, but could still be analyzed for nondisjunction. We found a tendency toward retention of the second polar body after 10 and 14 h aging. A total of 488 maternal genomes, 290 of which were in the control group, were analyzable for nondisjunction. Seven hyperhaploid genomes (2.4%) were observed in the controls and 6 (3.0%) in the combined aged group. The difference between these two frequencies is not significant (Gadj = 0.164, P greater than 0.50). In the aged group, one hyperhaploid genome was in the 2-h population, three in the 5-h population, and two in the 10-h population. We were unable to find any significant increase in the frequency of nondisjunction after postovulatory oocyte aging.