Segment duplications in subtelomeric regions of human chromosome 13

Owing to a great progress in studying the human genome, its euchromatic portion is almost completely sequenced; the complete sequence is still unknown only for pericentric and telomeric regions and short arms of acrocentric chromosomes. Extended satellite blocks and segment duplications located in these regions substantially hinder the joining of the sequenced fragments and construction of the full-length genome map. The sequence was established for a 1.5-kb human chromosome 13 subtelomeric region, which is about 10 kb away from the rDNA cluster, and deposited in GenBank under accession no. AF478540. The region showed 83-84% homology to the pericentric region of human chromosome 19, and contained short fragments homologous to the pericentric region of human chromosome 13. The results may contribute to the current revision of genome evolution concepts in view of numerous segment duplications revealed.     

Molecular cytogenetic evidence for a common breakpoint in the largest inverted duplications of chromosome 15.

Chromosomes from 20 patients were used to delineate the breakpoints of inverted duplications of chromosome 15 (inv dup[15]) that include the Prader-Willi syndrome/Angelman syndrome (PWS/AS) chromosomal region (15q11-q13). YAC and cosmid clones from 15q11-q14 were used for FISH analysis, to detect the presence or absence of material on each inv dup(15). We describe two types of inv dup(15): those that break between D15S12 and D15S24, near the distal boundary of the PWS/AS chromosomal region, and those that share a breakpoint immediately proximal to D15S1010. Among the latter group, no breakpoint heterogeneity could be detected with the available probes, and one YAC (810f11) showed a reduced signal on each inv dup(15), compared with that on normal chromosomes 15. The lack of breakpoint heterogeneity may be the result of a U-type exchange involving particular sequences on either homologous chromosomes or sister chromatids. Parent-of-origin studies revealed that, in all the cases analyzed, the inv dup(15) was maternal in origin.     

Inherited pericentric inversion of chromosome no. 2 with Robertsonian translocation (13q 14q) resulting in trisomy for chromosome 13q

This report includes a patient with an inherited pericentric inversion of chromosome No. 2 in addition to a Robertsonian translocation resulting in trisomy for chromosome 13q. The chromosomal constitution of the proband was 46,XX,inv(2) (pter leads to p11 : : q14 leads to p11 : : q14 leads to qter); t(13,14) (13qter leads to 13p11 : : 14q11 leads to 14qter). Sequential QFQ, RFA and GTG banding techniques were employed on the chromosomes of all family members. The chromosomal constitutions of the father and his first child were normal while the mother had an inversion of chromosome No. 2 [46,XX,inv(2) (pter leads to p11 : : q14 leads to p11 : : q14 leads to qter)]. The proband inherited this abnormal chromosome. In addition, she had a de novo Robertsonian translocation involving chromosomes 13q and 14q resulting in trisomy of chromosome 13q.     

Molecular characterisation of the pericentric inversion that distinguishes human chromosome 5 from the homologous chimpanzee chromosome

Human and chimpanzee karyotypes differ by virtue of nine pericentric inversions that serve to distinguish human chromosomes 1, 4, 5, 9, 12, 15, 16, 17, and 18 from their chimpanzee orthologues. In this study, we have analysed the breakpoints of the pericentric inversion characteristic of chimpanzee chromosome 4, the homologue of human chromosome 5. Breakpoint-spanning BAC clones were identified from both the human and chimpanzee genomes by fluorescence in situ hybridisation, and the precise locations of the breakpoints were determined by sequence comparisons. In stark contrast to some other characterised evolutionary rearrangements in primates, this chimpanzee-specific inversion appears not to have been mediated by either gross segmental duplications or low-copy repeats, although micro-duplications were found adjacent to the breakpoints. However, alternating purine–pyrimidine (RY) tracts were detected at the breakpoints, and such sequences are known to adopt non-B DNA conformations that are capable of triggering DNA breakage and genomic rearrangements. Comparison of the breakpoint region of human chromosome 5q15 with the orthologous regions of the chicken, mouse, and rat genomes, revealed similar but non-identical syntenic disruptions in all three species. The clustering of evolutionary breakpoints within this chromosomal region, together with the presence of multiple pathological breakpoints in the vicinity of both 5p15 and 5q15, is consistent with the non-random model of chromosomal evolution and suggests that these regions may well possess intrinsic features that have served to mediate a variety of genomic rearrangements, including the pericentric inversion in chimpanzee chromosome 4.     

Prenatal molecular cytogenetic diagnosis of partial tetrasomy 10p due to neocentromere formation in an inversion duplication analphoid marker chromosome

Neocentromeres are fully functional centromeres found on rearranged or marker chromosomes that have separated from endogenous centromeres. Neocentromeres often result in partial tri- or tetrasomy because their formation confers mitotic stability to acentric chromosome fragments that would normally be lost. We describe the prenatal identification and characterization of a de novo supernumerary marker chromosome (SMC) containing a neocentromere in a 20-wk fetus by the combined use of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). GTG-banding of fetal metaphases revealed a 47,XY,+mar karyotype in 100% of cultured amniocytes; parental karyotypes were both normal. Although sequential tricolor FISH using chromosome-specific painting probes identified a chromosome 10 origin of the marker, a complete panel of chromosome-specific centromeric satellite DNA probes failed to hybridize to any portion of the marker. The presence of a neocentromere on the marker chromosome was confirmed by the absence of hybridization of an all-human-centromere alpha-satellite DNA probe, which hybridizes to all normal centromeres, and the presence of centromere protein (CENP)-C, which is associated specifically with active kinetochores. Based on CGH analysis and FISH with a chromosome 10p subtelomeric probe, the marker was found to be an inversion duplication of the distal portion of chromosome 10p. Thus, the proband's karyotype was 47,XY,+inv dup(10)(pter-->p14 approximately 15::p14 approximately 15-->neo-->pter), which is the first report of partial tetrasomy 10p resulting from an analphoid marker chromosome with a neocentromere. This study illustrates the use of several molecular strategies in distinguishing centric alphoid markers from neocentric analphoid markers.     

Molecular characterization of the pericentric inversion that causes differences between chimpanzee chromosome 19 and human chromosome 17

A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development. The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres. Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level. During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17. Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs). By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements. Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation. No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences. Next to the break, at a distance of 10.2-39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13). The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes.     

Sperm chromosome analysis in a man heterozygous for a paracentric inversion of chromosome 7 (q11q22)

Summary Human sperm chromosomes were studied in a man heterozygous for a paracentric inversion of chromosome 7 (q11q22). The pronuclear chromosomes were analysed after in vitro penetration of golden hamster (Mesocricetus auratus) eggs. Ninety-four sperm chromosome spreads were examined, of which 34 contained the normal number 7 chromosome and 59 the inverted 6. This segregation was significantly different from the expected 1:1 ratio. The number of X- to Y-bearing sperm was 48 and 46 respectively. No sperm contained a recombinant chromosome caused by a crossover within the inversion. The frequency of chromosomal abnormalities in other chromosomes was 9.6%, which is not significantly different from the frequency observed in normal donors (8.9%) in our laboratory. These result suggest that the risk of chromosomally unbalanced sperm is not high for this paracentric inversion.     

Molecular cytogenetic identification and characterization of a de novo supernumerary neocentromeric derivative chromosome 13

We report a young girl with microphthalmia, conductive deafness, aortic isthmus stenosis, laryngomalacia, and laryngeal stenosis carrying a de novo supernumerary neocentromeric derivative chromosome 13. For the precise identification and characterization of the eu- and heterochromatic content of the marker chromosome, straightforward molecular cytogenetic analyses were performed, such as chromosome microdissection, FISH with different probes (e.g. wcp, alphoid centromeric probes, BAC), centromere-specific multicolor FISH (cenM-FISH), and multicolor banding (MCB). The analyses demonstrated that the marker consisted of an inverted duplication (partial tetrasomy) of the distal portion of chromosome 13 that was separated from the endogenous chromosome 13 centromere. Using an all-centromere probe and multicolor cenM-FISH, no alpha-satellite DNA hybridization signal was detectable on any portion of the derivative chromosome. The presence of a functional and active neocentromere on the derivative chromosome 13 was confirmed by positive immunofluorescence signals with CENP-C antibodies. BAC-FISH confirmed the cytogenetic localization of the neocentromere in band 13q31.3. Thus the patient had a mosaic conventional karyotype mos 47,XX,+inv dup(13)(qter-->q21.3::q21.3-->q31.3-->neo-->q31.3-->qter)[6]/46,XX [49].     

Some clinical and cytogenetic observations on a ring chromosome 13 (p11 q34).

A girl of 9-10/12 years of age with 46,XX,r(13)(p11q34) karyotypes was studied. She presented some clinical and radiological features, such as pectus excavatum, scalp alopecic area, 12th rib agenesis, hypoplastic pelvis, small gluteal pits and hypoplasia of the external genitalia in a female, which have never been previously described in other cases with ring 13 chromosomes. Cytogenetically, in vivo and in vitro viability of complete monosomic and partially trisomic and tetrasomic cells was found. The presence of nucleolus organizer regions and association of the ring 13 with other acrocentrics question the exclusivity of these attributes to acrocentrics p12.     

Segregation analysis in a man heterozygous for a pericentric inversion of chromosome 7 (p13;q36) by sperm chromosome studies.

We have analyzed 140 sperm chromosome complements from a subfertile man heterozygous for an inv(7)(p13;q36). Seventy-five percent of the chromosome complements were not recombinant: 37.9% contained the normal chromosome 7, and 37.1% contained the inverted chromosome 7. Twenty-five percent of the 140 were recombinant: 7.1% carried a recombinant chromosome 7 with a duplication p and deletion q, 17.1% carried a recombinant chromosome 7 with a duplication q and deletion p, and 0.7% carried both recombinant chromosomes. The frequency of structural chromosomal aberrations unrelated to the inversion was 11.4%, and the frequency of aneuploidy was 2.9%. Both frequencies were not significantly different from those in control donors. Two sperm complements with a second independent, contiguous inversion involving one of the original breakpoints (q36) were observed (1.4%). The risk of producing chromosomally abnormal offspring or spontaneous abortions would be 34.3%. The proportion of X-bearing and Y-bearing sperm was 46.8% and 53.2%, respectively, not significantly different from the expected 1:1 ratio.     

Candidate genes required for embryonic development: a comparative analysis of distal mouse chromosome 14 and human chromosome 13q22

Mice homozygous for the Ednrb(s-1Acrg) deletion arrest at embryonic day 8.5 from defects associated with mesoderm development. To determine the molecular basis of this phenotype, we initiated a positional cloning of the Acrg minimal region. This region was predicted to be gene-poor by several criteria. From comparative analysis with the syntenic human locus at 13q22 and gene prediction program analysis, we found a single cluster of four genes within the 1.4-to 2-Mb contig over the Acrg minimal region that is flanked by a gene desert. We also found 130 highly conserved nonexonic sequences that were distributed over the gene cluster and desert. The four genes encode the TBC (Tre-2, BUB2, CDC16) domain-containing protein KIAA0603, the ubiquitin carboxy-terminal hydrolase L3 (UCHL3), the F-box/PDZ/LIM domain protein LMO7,and a novel gene. On the basis of their expression profile during development, all four genes are candidates for the Ednrb(s-1Acrg) embryonic lethality. Because we determined that a mutant of Uchl3 was viable, three candidate genes remain within the region.     

Molecular genetic delineation of a deletion of chromosome 13q12-->q13 in a patient with autism and auditory processing deficits

In a sporadic case of autism and language deficit due to auditory processing defects, molecular genetic studies revealed that a chromosomal deletion occurred in the 13q12-->q13 region. No chromosome abnormalities were detected in the parents. We determined that the deletion occurred on the paternally derived chromosome 13. There are two previous reports of chromosome 13 abnormalities in patients with autism. The deletion in the subject described in this paper maps between the two chromosome 13 linkage peaks described by Bradford et al. (2001) in studies of subjects with autism and language deficits. The 9-Mb region deleted in the patient described here contains at least four genes that are expressed in brain and that play a role in brain development. They are NBEA, MAB21L1, DCAMKL1 and MADH9. These genes therefore represent candidate genes for autism and specific language deficits.     

Molecular cytogenetic characterization of a case of primary amenorrhea with intrachromosomal triplication of the X chromosome q arm

This is a unique case of intrachromosomal triplication of the X chromosome q arm detected with cytogenetic and spectral karyotyping in a 21-year-old woman with primary amenorrhea, who had been referred because of primary hypergonadotropic hypogonadism and Mullerian hypoplasia. Intrachromosomal triplications are rare rearrangements resulting in partial tetrasomy. Since 1993, at least 34 cases of intrachromosomal triplications involving 9 different chromosomes have been reported. The vast majority of the reported triplications are on the 15th chromosome, arised de novo and had middle inverted repetitions. In this report the genotype-fenotype correlation in a case of primary amenorrhea associated with triplication of the X chromosome q arm and the possible mechanisms of this rearrangement are discussed. Further the clinical usability of SKY analysis as a molecular cytogenetic tool in searching for genomic instability arising from cytogenetic rearrangements is highlighted.     

Cloning of ZNF237, a novel member of the MYM gene family that maps to human chromosome 13q11-->q12

We have cloned a novel, widely expressed human gene, ZNF237, that shows extensive similarity to the N-terminal region of ZNF198. Two alternatively spliced regions were identified by RT-PCR; the major splice variant is predicted to encode a 383 amino acid protein that contains a single diverged MYM domain. ZNF237 maps to 13q11-->q12, immediately proximal to ZNF198.     

Neurofibromatosis pseudogene amplification underlies euchromatic cytogenetic duplications and triplications of proximal 15q

Cytogenetically visible interstitial duplications of proximal 15q, which lack the Prader-Willi Angelman critical region (PWACR) frequently segregate in families without phenotypic effect, but the nature of the extra euchromatin has remained unclear. We used comparative genome hybridisation to confirm that the extra material in a cytogenetic triplication originated from proximal 15q. A PAC clone containing sequences specific for the type-1 neurofibromatosis (NF-1) pseudogenes, which map to 15q11.2, hybridised along the length of the enlarged region between the PWACR and the centromere. Computerised measurement of the fluorescent signal from the enlarged and normal chromosomes gave an average ratio of 9.85:1, consistent with amplification. In a second family, an amplified P1–4 signal co-segregated with a cytogenetic duplication and the average ratio between amplified and normal signals in the proband was 8.22:1. Ratios in non-carrier family members and control individuals were close to unity in most cases, but significantly greater than one in at least one instance. Our results provide a novel explanation for cytogenetic variation in 15q11.2. They also suggest that NF-1 pseudogene copy number may be polymorphic in the normal population, and that high copy numbers can produce G bands which do not reflect those of the normal constitutional karyotype. Received: 7 May 1998 / Accepted: 22 July 1998     

Prenatal detection of a paracentric inversion 16(q11.2q13)

We report the prenatal detection of an inherited paracentric inversion 16(q11.2q13).     

Molecular cytogenetic characterization of breakpoints involving pericentric inversions of human chromosome 9

Pericentric inversions involving the secondary constriction (qh) region of chromosome 9 are considered to be normal variants. The evolutionary mechanisms and conservation of these inversions via Mendelian fashion have been investigated since the advent of banding techniques. Routine cytogenetic techniques cannot provide the fine characterization necessary to determine the type of genetic material involved in these rearrangements. Therefore, the fluorescence in situ hybridization technique with the human centromere-specific alpha satellite and the beta satellite (D9Z5) and classical satellite (D9Z1) human DNA probes were used to identify the breakpoints of chromosome 9 pericentric inversions. Four unique types of pericentric inversions involving the 9qh region were observed, and the mechanism may be due to breakage and reunion at the proposed breakpoints. They are: type A inversions consist of breakpoints within the alpha and beta satellite DNA regions; type B consist of breakpoints within the beta satellite DNA region and band 9q13; type C involve breakage within the beta and classical satellite DNA regions, and type D have breakpoints within the alpha and classical satellite DNA regions. Obviously, reshuffling of satellite DNA sequences has occurred, which has given rise to a variety of heteromorphisms whose clinical significance remains obscure. Received: 21 December 1995 / Revised: 30 May 1996     

A neocentromere in the DAZ region of the human Y chromosome

We describe a novel rearranged human Y chromosome consisting of an inverted duplication of the long arm heterochromatin and a small amount of euchromatin: rea(Y)(qter-q11.2::q11.2-qter). The normal centromere has been deleted and a neocentromere containing CENP-A, -C, -E and Mad2 but not CENP-B has formed close to the breakpoint. A 2.7 Mb yeast artificial chromosome contig spanning the breakpoint was constructed and the breakpoint was localised to a region of <120 kb close to the DAZ gene cluster. Combined immunofluorescence and fluorescence in situ hybridisation showed that the centromeric protein-binding domain of the neocentromere was located near the breakpoint and within the DAZ cluster.