Genomic and genetic definition of a functional human centromere.

The definition of centromeres of human chromosomes requires a complete genomic understanding of these regions. Toward this end, we report integration of physical mapping, genetic, and functional approaches, together with sequencing of selected regions, to define the centromere of the human X chromosome and to explore the evolution of sequences responsible for chromosome segregation. The transitional region between expressed sequences on the short arm of the X and the chromosome-specific alpha satellite array DXZ1 spans about 450 kilobases and is satellite-rich. At the junction between this satellite region and canonical DXZ1 repeats, diverged repeat units provide direct evidence of unequal crossover as the homogenizing force of these arrays. Results from deletion analysis of mitotically stable chromosome rearrangements and from a human artificial chromosome assay demonstrate that DXZ1 DNA is sufficient for centromere function. Evolutionary studies indicate that, while alpha satellite DNA present throughout the pericentromeric region of the X chromosome appears to be a descendant of an ancestral primate centromere, the current functional centromere based on DXZ1 sequences is the product of the much more recent concerted evolution of this satellite DNA.

Large-insert clone/STS contigs in Xq11-q12, spanning deletions in patients with androgen insensitivity and mental retardation.

An integrated large-insert clone map of the region Xq11-q12 is presented. A physical map containing markers within a few hundred kilobases of the centromeric locus DXZ1 to DXS1125 spans nearly 5 Mb in two contigs separated by a gap estimated to be approximately 100-250 kb. The contigs combine 75 yeast artificial chromosome clones, 12 bacterial artificial chromosome clones, and 17 P1-derived artificial chromosome clones with 81 STS or EST markers. Overall marker density across this region is approximately 1 STS/60 kb. Mapped within the contigs are 12 ESTs as well as 5 known genes, moesin (MSN), hephaestin (HEPH), androgen receptor (AR), oligophrenin-1 (OPHN1), and Eph ligand-2 (EPLG2). Orientation of the contigs on the X chromosome, as well as marker order within the contigs, was unambiguously determined by reference to a number of X chromosome breakpoints. In addition, the distal contig spans deletions from chromosomes of three patients exhibiting either complete androgen insensitivity (CAI) or a contiguous gene syndrome that includes CAI, impaired vision, and mental retardation.

Deletions in Xq26.3-q27.3 including FMR1 result in a severe phenotype in a male and variable phenotypes in females depending upon the X inactivation pattern.

High resolution cytogenetics, microsatellite marker analyses, and fluorescence in situ hybridization were used to define Xq deletions encompassing the fragile X gene, FMR1, detected in individuals from two unrelated families. In Family 1, a 19-year-old male had facial features consistent with fragile X syndrome; however, his profound mental and growth retardation, small testes, and lover limb skeletal defects and contractures demonstrated a more severe phenotype, suggestive of a contiguous gene syndrome. A cytogenetic deletion including Xq26.3-q27.3 was observed in the proband, his phenotypically normal mother, and his learning-disabled non-dysmorphic sister. Methylation analyses at the FMR1 and androgen receptor loci indicated that the deleted X was inactive in > 95% of his mother's white blood cells and 80-85% of the sister's leukocytes. The proximal breakpoint for the deletion was approximately 10 Mb centromeric to FMR1, and the distal breakpoint mapped 1 Mb distal to FMR1. This deletion, encompassing approximately 13 Mb of DNA, is the largest deletion including FMR1 reported to date. In the second family, a slightly smaller deletion was detected. A female with moderate to severe mental retardation, seizures, and hypothyroidism, had a de novo cytogenetic deletion extending from Xq26.3 to q27.3, which removed approximately 12 Mb of DNA around the FMR1 gene. Cytogenetic, and molecular data revealed that approximately 50% of her white blood cells contained an active deleted X. These findings indicate that males with deletions including Xq26.3-q27.3 may exhibit a more severe phenotype than typical fragile X males, and females with similar deletions may have an abnormal phenotype if the deleted X remains active in a significant proportion of the cells. Thus, important genes for intellectual and neurological development, in addition to FMR1, may reside in Xq26.3-q27.3. One candidate gene in this region, SOX3, is thought to be involved in neuronal development and its loss may partly explain the more severe phenotypes of our patients.

Formation of de novo centromeres and construction of first-generation human artificial microchromosomes.

We have combined long synthetic arrays of alpha satellite DNA with telomeric DNA and genomic DNA to generate artificial chromosomes in human HT1080 cells. The resulting linear microchromosomes contain exogenous alpha satellite DNA, are mitotically and cytogenetically stable in the absence of selection for up to six months in culture, bind centromere proteins specific for active centromeres, and are estimated to be 6-10 megabases in size, approximately one-fifth to one-tenth the size of endogenous human chromosomes. We conclude that this strategy results in the formation of de novo centromere activity and that the microchromosomes so generated contain all of the sequence elements required for stable mitotic chromosome segregation and maintenance. This first-generation system for the construction of human artificial chromosomes should be suitable for dissecting the sequence requirements of human centromeres, as well as developing constructs useful for therapeutic applications.

Molecular characterization of isochromosomes of Xq.

We have undertaken a study of 35 patients with i(Xq) to determine whether those that are maternally derived originate by similar mechanisms to those that are paternally derived. Isochromosome formation is not associated with increased parental age and > 90% of i(Xq) contain proximal Xp sequences suggesting that centromere misdivision is not a common mechanism of formation. Our results indicate that the majority of i(Xq) originate from a single X chromosome and the usual mechanisms of formation do not appear to differ according to the parent of origin.

Three genes that escape X chromosome inactivation are clustered within a 6 Mb YAC contig and STS map in Xp11.21-p11.22.

In order to study the distribution of genes that escape X chromosome inactivation, a high density yeast artificial chromosome (YAC) contig and STS map spanning approximately 6 Mb has been constructed in Xp11.21-p11.22. The contig contains 113 YACs mapped with 53 markers, including 10 genes. Four genes have been assayed for their expression status on both the active and inactive human X chromosomes, and these data have been combined with previous results on two other genes in the contig. Three of these genes escape X inactivation and have been localized to a single YAC clone of approximately 1075 kb. The other three genes are subject to inactivation, with two of them lying among the genes that escape inactivation. These results suggest that there are both regional control signals as well as gene-specific elements that determine the X inactivation status of genes on the proximal short arm of the human X chromosome.

Partial X chromosome trisomy with functional disomy of Xp due to failure of X inactivation.

A 5-month-old girl with mild phenotypic abnormalities, developmental delay, and seizures was found to have the de novo karyotype 46,XX,-13,+der(13)t(X;13)(p21.2;p11.1). The partial trisomy of Xp21.2-->pter was confirmed with fluorescence in situ hybridization, using an X chromosome painting probe and several cosmid and YAC probes for Xp sequences. Replication banding showed that one of the structurally normal X chromosomes was late-replicating, but that the Xp segment of the der(13) was early-replicating in all cells examined. Since segments of the X chromosome separated from the X inactivation center in Xq13.2 cannot undergo X inactivation, the result is functional disomy of distal Xp. As the loss of short arm material from chromosome 13 is not considered to be clinically significant, the genomic imbalance of Xp expressed in this patient most likely accounts for her abnormal phenotype.

Mapping of the distal boundary of the X-inactivation center in a rearranged X chromosome from a female expressing XIST.

A female patient with primary amenorrhea, immature secondary sexual characteristics, and tall stature was found to have a normal X chromosome and a rearranged X [rea(X)] chromosome that resembled an 'isochromosome' Xp, but retained the proximal portion of Xq. The rea(X) was interpreted as rec(X)dup p,inv(X)(p11.4q13). Replication studies demonstrated that the rea(X) was always the late-replicating and, therefore, presumably inactive X chromosome, which must contain the X-inactivation center. Consistent with this interpretation, fluorescence in situ hybridization demonstrated that the rea(X) retained the XIST gene, and reverse transcription polymerase chain reaction analysis showed that XIST was expressed in the patient's cells. By fluorescence in situ hybridization with previously mapped probes, the breakpoint of the rea(X) was located within an approximately 500-kb region located approximately 200 to 700 kb distal to the XIST locus. This is the closest breakpoint distal to XIST in an inactivated X chromosome and, therefore, defines a new distal boundary for the X-inactivation center in humans.

The presence of interstitial telomeric sequences in constitutional chromosome abnormalities.

We describe a novel chromosome structure in which telomeric sequences are present interstitially, at the apparent breakpoint junctions of structurally abnormal chromosomes. In the linear chromosomes with interstitial telomeric sequences, there were three sites of hybridization of the telomere consensus sequence within each derived chromosome: one at each terminus and one at the breakpoint junction. Telomeric sequences also were observed within a ring chromosome. The rearrangements examined were constitutional chromosome abnormalities with a breakpoint assigned to a terminal band. In each case (with the exception of the ring chromosome), an acentric segment of one chromosome was joined to the terminus of an apparently intact recipient chromosome. One case exhibited apparent instability of the chromosome rearrangement, resulting in somatic mosaicism. The rearrangements described here differ from the telomeric associations observed in certain tumors, which appear to represent end-to-end fusion of two or more intact chromosomes. The observed interstitial telomeric sequences appear to represent nonfunctional chromosomal elements, analogous to the inactivated centromeres observed in dicentric chromosomes.

Diagnosis of tetrasomy 18p using in situ hybridization of a DNA probe to metaphase chromosomes.

We identified an isochromosome of 18p [47,XY, +i(18p)] conclusively by in situ hybridization of an 18p-specific probe (B74; D18S3) to metaphase chromosomes of an affected patient. Clinical findings included mental retardation, microcephaly, and an atrial septal defect. Although there is similarity to patients previously described with tetrasomy 18p, it is impossible to rule out a low frequency of misdiagnoses in karyotypes determined solely by standard cytogenetic analyses. We expect the ability to conclusively identify an i(18p) to lead to the delineation of tetrasomy 18p as a distinct clinical syndrome.

Prenatal diagnosis of Turner syndrome using cells cultured from cystic hygromas in two pregnancies with normal maternal serum alpha-fetoprotein.

In two cases of prenatally detected cystic hygroma with oligohydramnios, successful cytogenetic diagnosis of Turner syndrome was achieved using cells obtained from direct aspiration of the cystic hygroma. Exceptionally high levels of alpha-fetoprotein were found in the cystic hygroma fluid, as might be expected. However, the maternal serum alpha-fetoprotein levels were within normal limits. Elevated alpha-fetoprotein levels in 'amniotic fluid' noted previously in the literature may have resulted because of inadvertent tapping of the cystic hygroma. It is clear from our cases that maternal serum levels of alpha-fetoprotein will not necessarily be elevated and will not serve as a screening mechanism for cystic hygromas.

Cytogenetic and flow cytometric studies of cells from patients with Fanconi's anemia.

Cells from patients wtih Fanconi's anemia are unusually sensitive to agents which are capable of crosslinking DNA. This increased sensitivity can be detected both by cytogenetic and flow cytometric methods. An elevated frequency of chromosome aberrations, which is further exaggerated by exposure of cells to DNA crosslinking agents, is a general feature of Fanconi's anemia. Information about the formation of sister chromatid exchanges in this disease is less consistent. Cytogenetic analysis of cells from patients with Fanconi's anemia can be compromised by a low mitotic index. This is reflected in an accumulation of cells In the G2 phase of the cycle, after exposure to the bifunctional alkylating agent, mitomycin C. New methods for differentiating individuals with Fanconi's anemia from unaffected individuals should be of empirical use and might also facilitate mechanistic studies of this disease.