PCR protocol to detect parental origin and hidden mosaicism inSex chromosome aneuploidies

In this study, we report an accurate method to determine the parental origin of sex chromosome aneuploidies or polyploidies and to detect low percentage mosaicisms. We have amplified by polymerase chain reaction (PCR) five polymorphic markers along the X chromosome (DXS1283E, DYS II, DMD49, AR and DXS52) and three markers along the Y chromosome (SRY, DYZ3 and DYZ1). False-negative results were discarded by the simultaneous amplification of Y markers and of internal controls. We have applied this protocol to a series of 14 Turner syndrome patients with a 45,X karyotype. We have detected sex chromosome mosaicisms in two patients. The parental origin of the syndrome has been determined in the other 12 patients.     

Cytogenetic and molecular analysis of sex-chromosome monosomy.

X chromosome- and Y chromosome-specific DNA probes were used to study different aspects of the genesis of sex-chromosome monosomy. Using X-linked RFLPs, we studied the parental origin of the single X chromosome in 35 spontaneously aborted and five live-born 45,X conceptions. We determined the origin in 35 cases; 28 had a maternal X (Xm) and seven had a paternal X (Xp). There was a correlation between parental origin and parental age, with the Xp category having a significantly reduced mean maternal age by comparison with the Xm group. Studies aimed at detecting mosaicism demonstrated the presence of a Y chromosome or a second X chromosome in three of 33 spontaneous abortions, a level of mosaicism much lower than that reported for live-born Turner syndrome individuals.     

Study of the parental origin of sexual aneuploidy in ten families using RFLPs

We have studied 10 families with sexual aneuploidy by means of recombinant DNA techniques. The X linked probes were F814 (DXS52), FIX (FVIII gene), pert 87.15 (DXS164), L754 (DXS84) and 47Z (DXYS5). The observed banding patterns allowed us to determine the parental origin of such aneuploidy in nine of our cases. It was paternal in seven cases and maternal in two.     

Familial inheritance of a DXS164 deletion mutation from a heterozygous female.

Restriction-fragment-length-polymorphism analysis was used to examine a female who is segregating for Duchenne muscular dystrophy (DMD) and a deletion of the DXS164 region of the X chromosome. The segregating female has no prior family history of DMD, and she has two copies of the DXS164 region in her peripheral blood lymphocytes. The following two hypotheses are proposed to explain the coincidence of the DMD phenotype and deletion of the DXS164 region in her offspring: (1) she may be a gonadal mosaic for cells with two normal X chromosomes and cells with one normal X chromosome and an X chromosome with a deletion of the DXS164 region; and (2) she may carry a familial X;autosome translocation in which the DXS164 region is deleted from one X chromosome and translocated to an autosome. The segregation of DMD and the DXS164 deletion in this family illustrates the importance of extended pedigree analysis when DXS164 deletions are used to identify female carriers of the DMD gene.     

一个先天性眼球震颤家系致病基因的初步定位

为确定一个X染色体显性遗传先天性眼球震颤家系的致病基因与X染色体的连锁关系, 选用X染色体上的DXS1214、DXS1068、DXS993、DXS8035、DXS1047、DXS8033、DXS1192和DXS1232共8个微卫星DNA标记对该家系进行基因扫描与基因分型,并利用LINKAGE等软件包对基因分型结果进行分析,探讨该家系致病基因与X染色体的连锁关系。 两点连锁分析时X染色体短臂4个基因座最大LOD值均小于-1,不支持与该家系致病基因连锁; X染色体长臂4个基因座中最大LOD值达到2,提示存在较大的连锁可能性。该家系的致病基因可初步定位于X染色体长臂,且提示Xq26-Xq28区间附近可能是先天性眼球震颤一个共同的致病基因座,但区间范围仍较大,仍须进一步选择合适的微卫星标记进行精确的定位以缩小候选基因的筛查范围。Abstract： To investigate the relationship between X chromosome and obligatory gene of a pedigree with congenital nystagmus,we used the following markers: DXS1214、DXS1068、DXS993、DXS8035、DXS1047、DXS8033、DXS1192 and DXS1232.Genome screening and genotyping were conducted in this pedigree of congenital nystagmus, and linkage analysis by LINKAGE package was used to determine the potential location. The linkage was not found on the Xp ( All LOD score <-1) but on Xq (the maximum LOD score=2). The related gene of this pedigree was located on the long arm of X chromosome. We demonstrate that Xq26-Xq28 is a common locus for CMN. It bring us closer to the identification of a gene responsible for X-linked CMN.     

Mapping of locus for X-linked congenital stationary night blindness (CSNB1) proximal to DXS7.

A recombinant chromosome in a male affected with X-linked congenital stationary night blindness (CSNB1) provides new information on the location of the CSNB1 locus. A four-generation family with five males affected with X-linked CSNB was analyzed with five polymorphic markers for four X-chromosome loci spanning the region OTC (Xp21.1) to DXS255 (Xp11.22). Four of the males inherited the same X chromosome; one male inherited a chromosome that from OTC to DXS7, inclusive, was derived from the normal X chromosome of his unaffected grandfather and that from a location between DXS7 and DXS426 proximally was derived from the chromosome carrying the CSNB1 locus. This recombinant maps the CSNB1 locus in this family to a region on the short arm of the X chromosome proximal to the DXS7 locus.     

Genetic polymorphisms of 10 X-STR among four ethnic populations in northwest of China

In the present study, 10 short tandem repeats (STR) (HPRTB, DXS6789, DXS6799, DXS6804, DXS7130, DXS7132, DXS7133, DXS7423, DXS7424, and DXS8378) on X chromosome were investigated among four ethnic populations (431 individuals) in northwest of China, in order to learn about the genetic diversity, forensic suitability, and possible genetic affinities of the populations. The polymorphism information content (PIC) values for the ten loci range from 0.340 to 0.810, and the observed heterozygosity values for the ten loci range from 0.397 to 0.860, the results indicated that the ten loci have a moderate degree of variability in the four populations, and each population has its own characteristics of genetic structure. A Neighbor-joining (NJ) tree constructed on the basis of the generated data shows very low genetic distance between Han, Mongolian and Tibetan, Dongxiang and Tu as well as between Salar and Bonan populations. Our results based on genetic distance analysis are consistent with the results of earlier studies based on linguistics and the immigration history and origin of these populations. These STR loci on the X chromosome studied here are not only useful in showing significant genetic variation between the populations, but also are suitable for human identity testing among the four populations.     

Survival of XO mouse fetuses: effect of parental origin of the X chromosome or uterine environment?

Using a recombinant product from the structurally abnormal Y chromosome, Y*, female mice with a single X of either maternal or paternal origin were generated. The two types of females were produced on the same genetic background and differ only in the origin of the X chromosome. Hence it has been possible to assess the effect of parental origin of the X on survival of females with a single X chromosome. A highly significant prenatal loss of females with a single X of paternal origin, but no comparable loss of females with a single X of maternal origin was observed. The reduced viability of females with a paternally derived X could be mediated by the parental origin of the X (i.e. X chromosome imprinting) or alternatively, since the mothers of females with a single paternally derived X have only a single X chromosome, the effect could be mediated by the genotype of the mother (i.e. maternal uterine effect).     

New distal marker closely linked to the fragile X locus

Summary We have isolated II-10, a new X-chromosomal probe that identifies a highly informative two-allele TaqI restriction fragment length polymorphism at locus DXS466. Using somatic cell hybrids containing distinct portions of the long arm of the X chromosome, we could localize DXS466 between DXS296 and DXS304, both of which are closely linked distal markers for fragile X. This regional localization was supported by the analysis, in fragile X families, of recombination events between these three loci, the fragile X locus and locus DXS52, the latter being located at a more distal position. DXS466 is closely linked to the fragile X locus with a peak lod score of 7.79 at a recombination fraction of 0.02. Heterozygosity of DXS466 is approximately 50%. Its close proximity and relatively high informativity make DXS466 a valuable new diagnostic DNA marker for fragile X.     

Deletion (X)(q26.1-->q28) in a proband and her mother: molecular characterization and phenotypic-karyotypic deductions.

During a routine prenatal diagnosis we detected a female fetus with an apparent terminal deletion of an X chromosome with a karyotype 46,X,del(X)(q25); the mother, who later underwent premature ovarian failure, had the same Xq deletion. To further delineate this familial X deletion and to determine whether the deletion was truly terminal or, rather, interstitial (retaining a portion of the terminal Xq28), we used a combination of fluorescence in situ hybridization (FISH) and Southern analyses. RFLP analyses and dosage estimation by densitometry were performed with a panel of nine probes (DXS3, DXS17, DXS11, DXS42, DXS86, DXS144E, DXS105, DXS304, and DXS52) that span the region Xq21 to subtelomeric Xq28. We detected a deletion involving the five probes spanning Xq26-Xq28. FISH with a cosmid probe (CLH 128) that defined Xq28 provided further evidence of a deletion in that region. Analysis with the X chromosome-specific cocktail probes spanning Xpter-qter showed hybridization signal all along the abnormal X, excluding the possibility of a cryptic translocation. However, sequential FISH with the X alpha-satellite probe DXZ1 and a probe for total human telomeres showed the presence of telomeres on both the normal and deleted X chromosomes. From the molecular and FISH analyses we interpret the deletion in this family as 46,X,del(X) (pter-->q26::qter). In light of previous phenotypic-karyotypic correlations, it can be deduced that this region contains a locus responsible for ovarian maintenance.     

Determination of the parental origin of sex-chromosome monosomy using restriction fragment length polymorphisms.

The parental origin of the single X chromosome in sex-chromosome monosomy was evaluated by comparing restriction fragment length polymorphisms (RFLPs) of 10 spontaneous aborted 45,X conceptions with those of their parents. Seven X-linked marker loci were used, and we were able to specify the origin of the X in nine cases, with six being maternally and three paternally derived. These results demonstrate the efficiency of the technique and show that the single X chromosome in 45,X spontaneous abortions can be derived from either parent.     

RFLPs研究三例X染色体结构异常的起源和形成机理

本文对三例X染色体结构异常46,X,dup(X)(p21);46,X,del(X)(p11);46,X,i(Xq)患者及其父母,用X染色体短臂或长臂上的限制性片段长度多态性(RFLPs)作为遗传标记,研究了异常X染色体的起源和形成机理。结果表明,dup(X)(p21)和del(X)(p11)起源于父方,而i(Xq)起源于母方。dup(X)(p21)是由X染色体姊妹染色单体不均等的互换所引起的,del(X)(p11)是由于X染色体断裂后丢失所致,i(Xq)的发生是由于卵母细胞X染色体着丝粒错分裂。     

Mapping of the MYCL2 processed gene to Xq22-23 and identification of an additional L MYC-related sequence in Xq27.2

We report here the identification of a human genomic sequence from the q27.2 region of the X chromosome which shows a high homology to the L-MYC proto-oncogene. This sequence is not the MYCL2 homology, previously mapped to the long arm of the X chromosome at q22-qter by Morton et al., as we located the MYCL2-processed gene in Xq22-23, using a panel containing a combination of hybrid DNA carrying different portions of the human X chromosome. Based on computer analysis, the MYC-like sequence (MYCL3) is 98.2% identical to a portion of exon 3 of the MYCL1 gene and maps to the Xq27.2 region, between the DXS312 and DXS292 loci.     

The influence of parental origin of X chromosome genes on the stature of patients with 45 X Turner syndrome

Thirty-seven 45 X Turner syndrome patients with confirmed peripheral blood lymphocyte karyotype were initially selected to determine the origin of the retained X chromosome and to correlate it with their parents' stature. Blood samples were available in 25 families. The parental origin of the X chromosome was determined in 24 informative families through the analysis of the exon 1-CAG repeat variation of the androgen receptor gene. In 70.8% of the cases, the retained X chromosome was maternal in origin and 29.2% was paternal. When we classified the patients according to maternal (Xm) or paternal (Xp) X chromosome, there was a positive correlation between patients' and maternal heights only in the Xm group. There was no correlation with paternal height in either group, and a significant correlation with target height was only observed in the Xm group. In conclusion, maternal height is the best variable correlating with the height of 45 X Turner syndrome patients who retain the maternal X chromosome, suggesting a strong influence of genes located on the maternal X chromosome on stature.     

Linkage studies do not confirm the cytogenetic location of incontinentia pigmenti on Xp11

Summary Linkage studies have been performed in 5 incontinentia pigmenti (IP) families totaling 29 potentially informative meioses. Ten probes of the Xp arm were used, six of them were precisely localized on the X chromosome, using hamster x human somatic cell hybrids containing a broken X chromosome derived from an incontinentia pigmenti patient carrying an X;9 translocation [46,XX,t(X;9)(p11.21;q34)]. The following order for probes is proposed: pter-(DXS7, DXS146, DXS255)-IP1-(DXS14, DXS90)-DXS106-qter. The negative lod scores obtained exclude the possibility that in the families studied, the gene for IP is located in Xp11 or in the major part of the Xp arm.     

Lowe oculocerebrorenal syndrome in a female with a balanced X;20 translocation: mapping of the X chromosome breakpoint.

A Hispanic girl with Lowe oculocerebrorenal syndrome (OCRL), an X-linked recessive condition characterized by cataracts, glaucoma, mental retardation, and proteinuria, is reported. A balanced X;20 chromosomal translocation with the X chromosome breakpoint at q26.1 was found with high-resolution trypsin-Giemsa banding. Somatic cell hybridization was used to separate the X chromosome derivative and the chromosome 20 derivative in order to position, with respect to the translocation breakpoint, several DNA loci that are linked to the Lowe syndrome locus (Xq24-q26). DXS10 and DXS53 were found to be distal to the breakpoint, whereas DXS37 and DXS42 were located proximal to it. These studies suggest that the OCRL locus lies in the region between these probes. The translocation chromosome originated from an unaffected male without a visible translocation, indicating that the most likely cause of OCRL in this patient is the de novo translocation that disrupted the OCRL locus.     

Fine structure mapping of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene region of the human X chromosome (Xq26).

The Xq26-q27 region of the X chromosome is interesting, as an unusually large number of genes and anonymous RFLP probes have been mapped in this area. A number of studies have used classical linkage analysis in families to map this region. Here, we use mutant human T-lymphocyte clones known to be deleted for all or part of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene, to order anonymous probes known to map to Xq26. Fifty-seven T-cell clones were studied, including 44 derived from in vivo mutation and 13 from in vitro irradiated T-lymphocyte cultures. Twenty anonymous probes (DXS10, DXS11, DXS19, DXS37, DXS42, DXS51, DXS53, DXS59, DXS79, DXS86, DXS92, DXS99, DXS100d, DXS102, DXS107, DXS144, DXS172, DXS174, DXS177, and DNF1) were tested for codeletion with the hprt gene by Southern blotting methods. Five of these probes (DXS10, DXS53, DXS79, DXS86 and DXS177) showed codeletion with hprt in some mutants. The mutants established the following unambiguous ordering of the probes relative to the hprt gene: DXS53-DXS79-5'hprt3'-DXS86-DXS10-DXS177 . The centromere appears to map proximal to DXS53. These mappings order several closely linked but previously unordered probes. In addition, these studies indicate that rather large deletions of the functionally haploid X chromosome can occur while still retaining T-cell viability.     

An Xp22 microdeletion associated with ocular albinism and ichthyosis: approximation of breakpoints and estimation of deletion size by using cloned DNA probes and flow cytometry.

Ocular albinism of the Nettleship-Falls type (OA1) and X-linked ichthyosis (XI) due to steroid sulfatase (STS) deficiency are cosegregating in three cytogenetically normal half-brothers. The mother has patchy fundal hypopigmentation consistent with random X inactivation in an OA1 carrier. Additional phenotypic abnormalities that have been observed in other STS "deletion syndromes" are not present in this family. STS is entirely deleted on Southern blot in the affected males, but the loci MIC2X, DXS31, DXS143, DXS85, DXS43, DXS9, and DXS41 are not deleted. At least part of DXS278 is retained. Flow cytometric analysis of cultured lymphoblasts from one of the XI/OA1 males and his mother detected a deletion of about 3.5 million bp or about 2% of the X chromosome. Southern blot and RFLP analysis in the XI/OA1 family support the order tel-[STS-OA1-DXS278]-DXS9-DXS41-cen. An unrelated patient with the karyotype 46,X,t(X;Y) (p22;q11) retains the DXS143 locus on the derivative X chromosome but loses DXS278, suggesting that DXS278 is the more distal locus and is close to an XI/OA1 deletion boundary. If a contiguous gene deletion is responsible for the observed XI/OA1 phenotype, it localizes OA1 to the Xp22.3 region.     

An interstitial duplication of the X chromosome in a male allows physical fine mapping of probes from the Xq13-q22 region

Summary An insertional translocation into the proximal long arm of the X chromosome in a boy showing muscular hypotony, growth retardation, psychomotor retardation, cryptorchidism, and Pelizaeus-Merzbacher disease (PMD) was identified as a duplication of the Xq21–q22 segment by employing DNA probes. With densitometric scanning for quantitation of hybridization signals, 15 Xq probes were assigned to the duplicated region. Analysis of the duplication allowed us to dissect the X-Y homologous region physically at Xq21 and to refine the assignments of the loci for DXYS5, DXYS12, DXYS13, DXS94, DXS95, DXS96, DXS111, and DXS211. Furthermore, we demonstrated the presence of two different DXYS13, and DXS17 alleles in genomic DNA of our patient, suggesting that the duplication resulted from a meiotic recombination event involving the two maternal X chromosomes.     

注意缺损多动障碍的X染色体基因组扫描分析

摘 要：注意缺损多动障碍（ADHD）是儿童期多见行为障碍。男孩发病多于女孩。家系、双生儿和寄养子研究显示该障碍发生具有遗传基础。但是病因尚不清楚。分子遗传学和药理学研究表明ADHD涉及到多巴胺和去甲肾上腺素等神经递质系统,一系列报告发现ADHD与多巴胺D4受体（DRD4）、多巴胺转运体（DAT1）和儿茶酚-O-甲基转移酶（COMT）等基因相关联。我们以往研究表明ADHD与X染色体上DXS7位点和MAOA基因相关联,而DXS7是紧密连锁于MAO基因。依此假设,我们应用基因组扫描技术探讨ADHD在X染色体上易感位点。采用TDT方法分析X染色体上48个DNA标志的多态性与中国人群中84个ADHD核心家系间的连锁关系,ADHD诊断依据DSM-III-R标准。TDT分析结果观察到如下位点与ADHD相连锁,DXS1214(TDT:χ2=18.1,df=7, P<0.01), DXS8102(TDT: χ2=7.9, df=3, P<0.05),DXS1068(TDT: χ2=21.9, df=9, P<0.01), DXS8015(TDT:χ2=14.6, df=7, P<0.05),DXS1059(TDT: χ2=27.8, df=10, P<0.01) 和DXS8088(TDT:χ2=20.4, df=3, P<0.01).研究资料提示X染色体上Xp11.4-Xp21和Xq23区域可能存在ADHD的易感基因。