Tetrasomy 15q25→qter: Cytogenetic and molecular characterization of an analphoid supernumerary marker chromosome

Tetrasomy for the distal long arm of chromosome 15 is a rare finding. It has been previously described in seven patients, all of whom had a supernumerary marker chromosome (SMC) derived from distal 15q. These SMC contained no apparent centromeres (C‐band/α‐satellite negative), and belong to a novel class of SMC with neocentromeres. We present the oldest surviving patient with tetrasomy for distal 15q. The proposita was a 10‐year‐old girl with moderate to severe mental retardation, absent speech, hypotonia, minor facial anomalies, unusual digits, and pigmentation anomalies. Mosaicism for a symmetrical SMC was identified in metaphases from lymphocytes and fibroblasts. Parental karyotypes were normal, indicating a de novo origin for the SMC. FISH with a whole chromosome paint for chromosome 15 showed that the SMC was derived entirely from chromosome 15. However, C‐banding and FISH with chromosome 15 probes D15Z1, D15S11, SNRPN, and PML were all negative. FISH with the FES probe at 15q26 showed hybridization to both ends of the SMC. The marker was interpreted as an analphoid inverted duplication of 15q25→qter containing a presumed neocentromere. Previous molecular studies suggested either a mitotic or paternal meiotic origin for these distal 15q SMC. However, molecular analysis with chromosome 15 polymorphic markers showed that the analphoid SMC(15) in the proposita originated from a maternal meiotic error. The origins and mechanisms involved in formation of these distal 15q SMC appear to be more diverse than for the proximal pseudodicentic SMC(15). Am. J. Med. Genet. 94:393–398, 2000. © 2000 Wiley‐Liss, Inc.     

Minute supernumerary marker chromosomes identified in two patients with a related,larger pseudodicentric chromosome*

We describe two cases in which a minute supernumerary marker chromosome (SMC) was identified in addition to a larger pseudodicentric chromosome. Case 1, a phenotypically normal male, had mosaicism for a psu dic(15;15)(q11.2;q11.2) chromosome and a minute SMC. Fluorescence in situ hybridization (FISH) showed that the minute SMC was D15Z1 positive, indicating a chromosome 15 origin. Case 2 was a 22‐week fetus with mosaicism for a normal and two abnormal cell lines: one had a psu dic (22;22)(q11.2;q11.2) chromosome containing euchromatin, usually associated with cat eye syndrome; the other a minute SMC. The minute SMC was positive with the D14Z1/D22Z1 α‐satellite probe, indicating a chromosome 14 or chromosome 22 origin. Deletion of centromeric material was proposed as one mechanism of centromere inactivation in dicentric chromosomes. The origin of these two minute SMC suggests that they were derived from one of the centromeres of the larger pseudodicentric chromosome. These stable minute SMC may be the by‐product of a deletion event inactivating one centromere of a dicentric chromosome to generate a pseudodicentric chromosome. Alternatively, the minute SMC may originate from further rearrangement of the larger pseudodicentric chromosome. These cases suggest possible mechanisms for the origin of minute SMC. © 2001 Wiley‐Liss, Inc.     

A woman with an apparent non-mosaic 45,X delivered a 46,X,der(X) liveborn female

A liveborn female with a phenotype suggestive of Down syndrome is reported. Cytogenetic lymphocyte analysis showed a 46,X der(X) karyo-type. Fluorescence in situ hybridization (FISH) with a biotinylated probe specific for chromosome 21 showed no signal on the der(X). This marker was homogeneously painted using a specific probe for X chromosome. In addition, FISH analysis detected telomeres on the rearranged X. Therefore, the proband's karyotype was revaluated as 46,X,del(X) (pter p22.2::p11.3 qter). Cytogenetic analysis of 150 lymphocytes in the mother disclosed a homogeneous 45,X karyotype. FISH analysis of interphase nuclei using the X chromosome painting probe showed two domains of different sizes in 0.8% of cells. This led us to study further metaphases in the mother. In one out of 450 metaphases scored, after FISH with the X chromosome painting probe, the del(X) was observed, confirming that the rearranged X chromosome found in the newborn had segregated from a 45,X/46,X,del(X) mother.     

Tetrasomy 15q25-->qter: cytogenetic and molecular characterization of an analphoid supernumerary marker chromosome

Tetrasomy for the distal long arm of chromosome 15 is a rare finding. It has been previously described in seven patients, all of whom had a supernumerary marker chromosome (SMC) derived from distal 15q. These SMC contained no apparent centromeres (C-band/alpha-satellite negative), and belong to a novel class of SMC with neocentromeres. We present the oldest surviving patient with tetrasomy for distal 15q. The proposita was a 10-year-old girl with moderate to severe mental retardation, absent speech, hypotonia, minor facial anomalies, unusual digits, and pigmentation anomalies. Mosaicism for a symmetrical SMC was identified in metaphases from lymphocytes and fibroblasts. Parental karyotypes were normal, indicating a de novo origin for the SMC. FISH with a whole chromosome paint for chromosome 15 showed that the SMC was derived entirely from chromosome 15. However, C-banding and FISH with chromosome 15 probes D15Z1, D15S11, SNRPN, and PML were all negative. FISH with the FES probe at 15q26 showed hybridization to both ends of the SMC. The marker was interpreted as an analphoid inverted duplication of 15q25-->qter containing a presumed neocentromere. Previous molecular studies suggested either a mitotic or paternal meiotic origin for these distal 15q SMC. However, molecular analysis with chromosome 15 polymorphic markers showed that the analphoid SMC(15) in the proposita originated from a maternal meiotic error. The origins and mechanisms involved in formation of these distal 15q SMC appear to be more diverse than for the proximal pseudodicentic SMC(15).     

联合应用多种细胞及分子细胞遗传学技术确诊额外标记染色体

目的应用比较基因组杂交技术（comparative genomic hybridization，CGH）、荧光原位杂交（fluorescence in situ hybridization，FISH）和传统细胞遗传学技术分析1例额外标记染色体（supernumerary marker chromosome，SMC），探讨这些技术的联合使用在识别新发生的标记染色体方面的临床应用。方法1例智力低下患儿，通过常规G显带技术分析其染色体核型。针对发现的SMC，通过CGH分析其起源，通过FISH技术证实。同时应用N带和C带技术分析SMC的随体组成和着丝粒组成。结合已有的文献报道，分析该SMC的表型效应。结果染色体G带分析显示患者为携带SMC的嵌合体，核型描述为mos．47，XX，＋may[31]／48，XX，＋2mar[29]。CGH分析显示患儿基因组中有15q11→q14片段重复，以15q探针与患者中期分裂相进行FISH检测证实SMC来源于15号染色体。应用检测缺失型Prader-wiUi综合征／Angelman综合征的UBE3A探针与患者中期分裂相检测显示SMC有两个UBE3A杂交信号，对照的PML位点没有信号。N带显示SMC携带双随体，c带分析SMC为双着丝粒。综合上述结果，患者核型为：mos．47，xx，＋der（15）（pter＋q14：：q14→pter）[31]／48，XX，＋2der（15）（pter→q14：：q14→pter）[29]．ish der（15）（WCPl5＋，UBE3A＋＋，PML-）。结论CGH对于检测出不平衡染色体结构重排具有提示作用，结合FISH和传统的细胞遗传学技术，为确定SMC的结构组成提供了可信的技术平台，为分析这类异常核型个体的表型、预后和复发风险提供了依据。     

Characterization and meiotic segregation of a supernumerary marker chromosome in sperm of infertile males: Case report and literature review

This couple presented with a 4-year history of primary infertility. The male partner was found to have oligoasthenozoospermia. A supernumerary marker chromosome (SMC) was found. Fluorescent in situ hybridization (FISH) analyses showed that the SMC was a heterochromatic dicentric marker derived from chromosome 22. Further FISH procedures showed the rate of unbalanced spermatozoa containing one chromosome 22 and the SMC to be 15.6%. Due to the low risk of fetal chromosomal imbalance linked to the paternal SMC and the risk of miscarriage linked to the amniocentesis, the couple declined prenatal diagnosis. A healthy newborn baby was obtained after ICSI.     

Preferential involvement of the short arm in chromosome 8‐derived supernumerary markers and ring as identified by chromosome arm painting

We report on the use of fluorescence in situ hybridization (FISH) with specific chromosome 8 arm painting to characterize further small supernumerary chromosome 8‐derived markers/rings (SMC/SRC) identified in three patients. Two patients (patients 1 and 2) who carried the marker (SMC) were evaluated because of mental retardation and minor facial anomalies. The patient (patient 3) who carried the ring (SRC) had ventriculomegaly. Parental blood chromosomes of patients 2 and 3 were normal and unavailable on patient 1. The identification of the SMC/SRC was first characterized by FISH specific alpha‐repeat centromeric probes, second by FISH whole chromosome painting (WCP), and finally by FISH chromosome arm painting (CAP). The latter showed involvement of only the short arm of chromosome 8 in all three SMC/SRC cases, suggesting a U‐type exchange mechanism. Am. J. Med. Genet. 90:276–282, 2000. © 2000 Wiley‐Liss, Inc.     

荧光原位杂交同时检测精子X和Y染色体的方法

用实验手段分离X和Y染色体精子或富集特定性别染色体精子．对于预防性连锁遗传病具有潜在应用价值。建立精子X和Y染色体鉴定方法对于评估分离或富集特定性别染色体效果至关重要。本文介绍采用CY3TM和FlourXTM探针作荧光原位杂交（FISH），同时检测精子Y和X染色体。10份正常精液标本分析结果显示：X染色体精子为50．23％，Y染色体精子为49．77％；有效杂交率达91．99％。FISH方法比传统的精子染色体核型分析和奎纳克林染色检查Y荧光小体更具有简易、特异和快速的优点。     

Minute supernumerary marker chromosomes identified in two patients with a related, larger pseudodicentric chromosome.

We describe two cases in which a minute supernumerary marker chromosome (SMC) was identified in addition to a larger pseudodicentric chromosome. Case 1, a phenotypically normal male, had mosaicism for a psu dic(15;15)(q11.2;q11.2) chromosome and a minute SMC. Fluorescence in situ hybridization (FISH) showed that the minute SMC was D15Z1 positive, indicating a chromosome 15 origin. Case 2 was a 22-week fetus with mosaicism for a normal and two abnormal cell lines: one had a psu dic (22;22)(q11.2;q11.2) chromosome containing euchromatin, usually associated with cat eye syndrome; the other a minute SMC. The minute SMC was positive with the D14Z1/D22Z1 alpha-satellite probe, indicating a chromosome 14 or chromosome 22 origin. Deletion of centromeric material was proposed as one mechanism of centromere inactivation in dicentric chromosomes. The origin of these two minute SMC suggests that they were derived from one of the centromeres of the larger pseudodicentric chromosome. These stable minute SMC may be the by-product of a deletion event inactivating one centromere of a dicentric chromosome to generate a pseudodicentric chromosome. Alternatively, the minute SMC may originate from further rearrangement of the larger pseudodicentric chromosome. These cases suggest possible mechanisms for the origin of minute SMC.     

Analysis of 10 patients with duplications of 15q11q13 region and autism featuresCSCD

Objective: To analyze the clinical and genetic features of 10 unrelated patients with duplications of 15q11q13 region and autism features. Methods: Karyotyping, chromosomal microarray analysis (CMA) and fluorescence in situ hybridiztion (FISH) were carried out for the patients and their parents. Results: Eight patients presented with a supernumerary marker chromosome (SMC) of unknown origin by G-banding analysis and triplication of the 15q11q13 region by high-resolution CMA analysis. Two remaining patients had normal karyotypes but duplications of the 15q11q13 region. All duplications have encompassed the Prader Willi/Angelman syndrome critical region (PWACR). Similar gains in copy number were not detected among the parents of the patients, suggesting a de novo origin for them. Analysis of SNP-array data of the family trios using Chromosome Analysis Suite Software found that the copy number gains have originated from the mothers. The diagnosis of 15q11q13 duplication syndrome was ascertained. For patients with SMC detected by karyotyping analysis, a FISH assay using probes specific for the 15q11q13 region showed that such SMC also derived from chromosome 15q11q13 region and contained two copy numbers, which was consistent with the result of CMA. Conclusion: Ten patients with autism and 15q11q13 duplications were identified with combined karyotyping, CMA and FISH analysis. A phenotype-genotype correlation was established. © 2018 West China University of Medical Sciences. All rights reserved.     

Apparently unrelated cytogenetic abnormalities among 462 probands referred for the detection of del(22q) by FISH

Laboratory-based reports of the cytogenetic abnormalities detected during the course of testing for deletion del(22q) are scant. We report our findings from the testing with FISH of 462 patients suspected to have del(22q) between 1994 and 2000. Of these, 447 had a normal karyotype. An apparently unrelated cytogenetic abnormality was detected in 15 (3.2%). Two of these abnormalities involved reciprocal translocation with chromosome 22q and one of these showed del(22q) with FISH. The other abnormalities included sex chromosome aneuploidies and unbalanced rearrangements of various chromosomal segments. There was no commonality among these abnormalities and no correlation with other reported cases. Among those with a normal karyotype, an unexpected deletion of the control arylsulphatase A (ARSA) probe was found, providing a definite frequency of 1/262 for ARSA deletions among patients suspected to have del(22q). Of the 462 referrals, 48 (10%) had one or more additional diagnoses, and in this group, 4 (8%) had del(22q) and 2 (4%) had an apparently unrelated cytogenetic abnormality. The data highlight the importance of initial cytogenetic analysis in patients suspected of del(22q) and negates the use of interphase FISH screening by itself for del(22q). The finding of 3.2% unrelated cytogenetic abnormalities is noteworthy. FISH should be used in any structural rearrangement to ascertain if the relevant locus is deleted or not. The continued reporting of patients diagnosed with del(22q) found to have an unrelated cytogenetic abnormality will expand the phenotypic spectrum and possible gene mapping may refine phenotypic specificity.     

Supernumerary marker chromosomes (SMCs) in Turner syndrome are mostly derived from the Y chromosome

DNA and FISH (fluorescence in situ hybridization) analysis were carried out in 12 patients with stigmata of Turner syndrome to determine whether the Supernumerary M arker C hromosome (SMC) found cytogenetically in each of these patients was derived from the Y chromosome. The presence of a Y chromosome in these patients may predispose them to develop gonadoblastoma. PCR-Southern blot analysis, followed by FISH, was used to detect the presence of Y chromosome material. The S ex determining R egion Y (SRY), T estis S pecific P rotein Y -encoded (TSPY) and Y -chromosome R NA R ecognition M otif (YRRM) genes, which map at Yp11.31, Yp11.1–11.2 and Yp11.2/Yq11.21–11.23, respectively, were selected as markers, because they span the whole Y chromosome, and more importantly, they are considered to be involved in the development of gonadoblastoma. It was shown that in 12 patients, all of whom had an SMC, the SMC of 11 was derived from the Y chromosome. Furthermore, the presence of the SRY, TSPY and YRRM gene sequences was determined and FISH analysis confirmed the Y origin of the SMCs. The methodology described in this report is a rapid, reliable and sensitive approach which may be easily applied to determine the Y origin of an SMC carried in Turner syndrome. The identification of an SMC is important for the clinical management and prognostic counseling of these patients with Turner syndrome.     

A patient with Prader-Willi syndrome and a supernumerary marker chromosome r(15)(q11.1-13p11.1)pat and maternal heterodisomy

We report on a Prader-Willi patient with a de novo supernumerary marker chromosome (SMC) in 16% of the cells. The SMC was a ring chromosome and it included the PWS/AS critical region as was demonstrated by FISH. Segregation analysis indicated that the SMC originated from a paternal chromosome 15 and the two normal chromosomes 15 of the patients were of the maternal homologues. Namely, the patient had maternal heterodisomy in 85% of the cells and triplication of the PWS/AS region in 15% of the cells. The Prader-Willi features were the result of the low mosaicism of the SMC. The evolution of the maternal heterodisomy and the SMC were two unrelated events, the occurrence of both events in the same embryo rescued it from lethality.     

Hexasomy of the Prader-Willi/Angelman critical region, including the OCA2 gene, in a patient with pigmentary dysplasia: case report

Derivatives of chromosome 15, often referred to as inv dup(15), represent the most common supernumerary marker chromosome (SMC). SMC(15)s can be classified into two major groups according to their length: small SMC(15) and large ones. Depending on the amount of euchromatin, the carriers may either present with a normal phenotype or with a recognizable syndrome. Here we describe a patient with severe mental retardation, epilepsy, dysmorphic features and pigmentary dysplasia. His karyotype was 47,XY,+mar[41]/46,XY[9]. Chromosomal fluorescence in situ hybridization (FISH) showed the SMC to be originating from chromosome 15, dicentric and containing four copies of the Prader-Willi/Angelman Syndrome Critical Region (PWACR), including the OCA2 gene. Molecular studies indicated that it is maternally derived. This report supports the previous observations assuming that severity of phenotype in patients with SMC(15) depends on the dosage of the PWACR and that skin pigmentation is correlated to OCA2 gene copy number.     

Application of fluorescent in situ hybridization with X and Y chromosome specific probes to buccal smear analysis

Conventional X- and Y-chromatin and fluorescent in situ hybridization (FISH) analysis based on X- and Y-chromosome specific probes were conducted from buccal smear, on 15 normal males, 15 normal females, and 9 cases suspected of sex chromosome anomalies. The proportion of X- and Y-chromatin in normal females and males was 12% ± 3% and 51.5% ± 4.9%, respectively, by the conventional X- and Y-chromatin procedure. The CEP-X/Y analysis by FISH for the same specimens provided a proportion of 98.8% ± 0.7% cells with XX signals in the normal females and 99.8% ± 0.4% cells with XY signals in the normal males. The FISH method was superior to the conventional procedure in nine cases suspected of sex chromosome anomalies, including one case of mosaicism. The results of CEP-X/Y will sometimes be false; it will not detect structural anomalies of sex chromosomes, and it is not intended to detect low level mosaicism. However, the test is useful for rapid screening of sex chromosome aneuploidy at a fraction of the cost for chromosome analysis. The FISH test is also appropriate to detect tissue specific sex chromosome mosaicism, especially if it is relatively high. This FISH test is best used as an adjunct to chromosome analysis whenever possible. © 1996 Wiley-Liss, Inc.     

两例Y染色体部分缺失胎儿的产前诊断

目的对两例Y染色体部分缺失胎儿进行产前诊断。方法采用常规G显带及C显带技术分析胎儿及父亲的核型,采用荧光原位杂交(fluorescence in situ hybridization,FISH)、染色体拷贝数变异检测技术(copy number varaition sequencing,CNV-seq)性别决定基因(sex region of Y chromosome,SRY)检测技术及无精子因子(azoospermia factor,AZF)检测技术检测胎儿DNAO结果2例胎儿羊水染色体在320〜400条带水平均提示46,XN,del(Y)(qll.2),Y染色体着丝粒探针FISH检测结果均提示Y染色体数目未见异常。2例胎儿父亲外周血染色体核型均未见明显异常。胎儿羊水DNA拷贝数检测提示一例胎儿Y染色体q 11.221-ql2处缺失12.88 Mb,涉及全部AZFb+AZFc区域;另一例胎儿Y染色体qll.21-ql2处缺失14.84 Mb,涉及全部AZF区域。2例胎儿羊水SRY基因检测提示SRY基因阳性,SKY基因编码区未检测到已报道的致病点突变。2例胎儿基因检测提示存在AZF部分或全部缺失。结论联合多种技术有助于明确诊断Y染色体结构异常。CNV-seq检测有利于快速筛查胎儿Y染色体微缺失,可做为对染色体核型分析的补充和验证的方法。     

用双色荧光原位杂交检测人精子染色体非整倍体率

目的检测人精子染色体非整倍体率。方法采用双色荧光原位杂交（ＦＩＳＨ）方法,取少量精标本经洗后制片,用二硫苏糖醇（ＤＴＴ）和二碘水杨酸锂（ＬＩＳ）处理,使精子头部染色质去凝集。然后,与生物素标记的α卫星Ｘ染色体特异ＤＮＡ探针（ＤＸＺ１）和地高辛标记的α卫星Ｙ染色体特异ＤＮＡ探针（ＤＹＺ３）进行原位杂交。用ＣＹ３－链亲和素、山羊抗链亲和素检测Ｘ染色体探针杂交信号;用鼠抗地高辛抗体、与荧光素结合的兔抗鼠抗体检测Ｙ染色体探针杂交信号。结果在Ｎｉｋｏｎ荧光显微镜下可以清楚看到精子头部的杂交信号,头部有１个红色荧光杂交信号的精子为Ｘ染色体精子（Ｘ精子）,有１个绿色荧光杂交信号的精子为Ｙ染色体精子（Ｙ精子）。精子头部有２个荧光杂交信号的精子为染色体数目异常精子。若用１条常染色体探针和１条性染色体探针进行ＦＩＳＨ,可以区别头部有２个相同颜色荧光杂交信号的精子属非整倍体精子或二倍体精子。结论双色荧光原位杂交（ＦＩＳＨ）方法,可以用于测定接触致突变剂和非整倍体诱导剂后,人精子染色体非整倍体率的变化。     

A molecular and FISH approach to determining karyotype and phenotype correlations in six patients with supernumerary marker(22) chromosomes

In a cytogenetic, molecular, and clinical study of patients with autosomal supernumerary marker chromosomes (SMC), 6 out of 72 (8.3%) were shown by fluorescence in situ hybridisation (FISH) to be derived from chromosome 22. PCR microsatellite analysis and FISH using primers and cosmids from proximal 22q showed 3 of the 6 to contain euchromatin. The first, a de novo nonmosaic bisatellited, dicentric SMC, was acsertained in a patient with cat eye syndrome and Duane anomaly. Microsatellite analysis showed the SMC was maternal in origin with euchromatin extending to D22S427, i.e., proximal to the DiGeorge syndrome critical region (DGSCR). The second, a nonmosaic bisatellited, dicentric marker, was found in a child with severe hypotonia and developmental delay and had been inherited from the patient's phenotypically normal father. FISH showed the SMC to contain euchromatin extending into the DGSCR. The third, a de novo SMC, was ascertained antenatally and was shown to contain 22q euchromatin extending distal to the DGSCR. The 19-week terminated fetus was phenotypically normal at autopsy. Two of the three SMC(22)s not containing detectable proximal 22q euchromatin were ascertained coincidentally in phenotypically normal individuals, whereas the third, the only mosaic with a minority euploid cell line, was found in a patient with mild developmental delay. These results suggest that SMC(22)s devoid of proximal 22q euchromatin are not associated with adverse phenotypic effects whereas SMC(22)s containing euchromatin may be found in individuals with phenotypes ranging from cat eye syndrome to normal. Am. J. Med. Genet. 72:440–447, 1997. © 1997 Wiley-Liss, Inc.     

FISH and molecular studies of autosomal supernumerary marker chromosomes excluding those derived from chromosome 15: II. Review of the literature

Using fluorescence in situ hybridization (FISH), supernumerary marker chromosomes (SMC) from all the human autosomes except chromosome 5, have now been described, most being derived from the acrocentric autosomes. This review summarizes the results of 168 cases of autosomal SMC excluding those from chromosome 15 where FISH has been used to define the chromosomal origin of the SMC and from which phenotypic information is available. Although the number of reported cases from some of the chromosomal SMC groups remains small, the pooled data suggest that the risk of an abnormal phenotype associated with a randomly ascertained de novo SMC derived from the acrocentric autosomes (excluding 15s) is ∼7% compared with ∼28% for SMCs derived from the nonacrocentric autosomes. Am. J. Med. Genet. 75:367-381, 1998. © 1998 Wiley-Liss, Inc.     

A chromosome painting method for human sperm chromosomes using fluorescentin situ hybridization

Summary A method of chromosome painting on human sperm chromosomes using fluorescentin situ hybridization (FISH) is introduced. Sperm chromosome slides were prepared afterin vitro fertilization of hamster eggs with human spermatozoa. The slides were treated by RNase A before FISH. Chromosome 4 was clearly and specifically painted in a majority of sperm-derived metaphase plates after an application of whole chromosome painting DNA probes of this chromosome. This is the first report of successful painting on human sperm chromosomes.