FISH analysis for apparently simple terminal deletions of the X chromosome: Identification of hidden structural abnormalities

We report on fluorescence in situ hybridization (FISH) analysis in 30 mosaic or nonmosaic females diagnosed as having apparently simple terminal X deletions by standard G‐banding analysis. FISH studies for DXZ1, the Xp and Xq telomere regions, and the whole X chromosome painting were carried out for the 30 females, indicating rearranged X chromosomes with signal patterns discordant with terminal deletions in 6 cases: one dic(X)(DXZ1++) chromosome, two der(X)(qtel++) chromosomes, one Xq? (qtel+) chromosome, and two der(X)(ptel++) chromosomes. Additional FISH studies were performed for the 6 cases using probes defining 12 loci on the X chromosome, showing large Xp deletion and small Xp duplication in the dic(X)(DXZ1++) chromosome, partial Xp deletions and partial Xq duplications in the two der(X)(qtel++) chromosomes, an interstitial Xq deletion in the Xq? (qtel+) chromosome, and partial Xq deletions and partial Xp duplications in the two der(X)(ptel++) chromosomes. Clinical assessment of the 6 cases revealed tall and normal stature in the two mosaic cases with the der(X)(ptel++) chromosomes that were shown to be associated with SHOX duplication. The results suggest that unusual X chromosome rearrangements are often misinterpreted as simple terminal X deletions, and that FISH analysis is useful for precise structural determination and better genotype‐phenotype correlation of the X chromosome aberrations. © 2001 Wiley‐Liss, Inc.     

Cytogenetic and clinical characteristics of a case involving complete duplication of Xpter-->Xq13.

True isochromosomes for Xp probably do not exist in a liveborn. We describe a rare case of complete Xp duplication and retention of the inactivation centre at Xq13. Cytogenetically, it is described as a nonmosaic 46,X,psu idic(X)(q13). Complete duplication of Xpter-->Xq13 was confirmed by banded analysis and FISH probes for X centromere, Xp21, XIST locus, and whole chromosome paints for X and Y. The abnormal X was always late replicating. Clinically, the patient was short statured, had primary amenorrhoea, and incomplete development of secondary sexual characteristics, but otherwise was phenotypically normal. There are no non-mosaic reported cases with complete duplication of i(Xp) confirmed by FISH or molecular techniques. Those cases with partial duplication of Xp and presence of the inactivation centre share the traits of amenorrhoea and poor secondary sexual development. To develop a clinical profile of duplication of Xp (in presence of Xq13) there is a need to study more cases.     

A duplication of distal Xp associated with hypogonadotrophic hypogonadism, hypoplastic external genitalia, mental retardation, and multiple congenital abnormalities.

An unusual familial case of three sibs with a partial duplication of distal Xp sequences is described. The proband, an 18 year old boy, showed mental retardation, severe dysmorphic features, hypogonadotrophic hypogonadism (HHG), and hypoplastic external genitalia. His karyotype was 46,Y,inv dup(X) (p22.11-->p 22.32). The proband has two sisters each with the same inv dup(Xp) chromosome. Both sisters presented with short stature but were otherwise phenotypically normal. The abnormal X chromosome was inactive in the majority of cells examined. Southern blot dosage analysis indicated a duplication of distal Xp sequences. The proximal breakpoint is located between DXS28 and DXS41, and is therefore at least 2 Mb distal to the DSS locus. The relationship between the phenotype and the Xp duplication is discussed.     

Array-CGH characterization of a prenatally detected de novo 46,X,der(Y)t(X;Y)(p22.3;q11.2) in a male fetus

We report on an apparently normal 5-month-old boy with a X;Y complex rearrangement identified first on prenatal diagnosis and found on array-CGH to have a 7.6?Mb duplication of Xp22.3 chromosome and a deletion of Yq chromosome, distal to the AZFa locus. Karyotype analysis on amniotic fluid cell cultures revealed a de novo homogenous chromosome marker that we interpreted as an isochromosome Yp. FISH analysis using SRY probe revealed only one signal on the derivative Y chromosome. The final karyotype was interpreted as 46,X,der(Y)t(X;Y)(p22.31;q11.22). Translocation Xp22;Yq11 in male are very rare event and only 4 cases have been published, all showing mental retardation and malformations. Herein we discussed some possible explanation for this apparent phenotypic variability.     

Exome sequencing identified a de novo mutation of PURA gene in a patient with familial Xp22.31 microduplication

The clinical significance of Xp22.31 microduplication is controversial as it is reported in subjects with developmental delay (DD), their unaffected relatives and unrelated controls. We performed multifaceted studies in a family of a boy with hypotonia, dysmorphic features and DD who carried a 600?Kb Xp22.31 microduplication (7515787-8123310bp, hg19) containing two genes, VCX and PNPLA4. The duplication was transmitted from his cognitively normal maternal grandfather.We found no evidence of the duplication causing the proband's DD and congenital anomalies based on unaltered expression of PNPLA4 in the proband and his mother in comparison to controls and preferential activation of the paternal chromosome X with Xp22.31 duplication in proband's mother. However, a de novo, previously reported deleterious, missense mutation in Pur-alpha gene (PURA) (5q31.2), with a role in neuronal differentiation was detected in the proband by exome sequencing.We propose that the variability in the phenotype in carriers of Xp22.31 microduplication can be due to a second and more deleterious genetic mutation in more severely affected carriers. Widespread use of whole genome next generation sequencing in families with Xp22.31 CNV could help identify such cases.     

Parental origin and mechanism of formation of X chromosome structural abnormalities: four cases determined with RFLPs

Parental origin and mechanism of formation of X chromosome structural abnormalities were studied in one each case of dup(X)(pter----p11.4::p22.1----qter), del(X)(qter----p11:), i(X)(qter----cen----qter), and inv dup(X) (pter----q22::q22----pter) using various X-linked RFLPs as genetic markers. Segregation and densitometric analyses on polymorphic DNAs revealed that the dup(Xp) and the del(Xp) are both of paternal origin and the i(Xq) and i dic(X) are of maternal origin. The dup(Xp) had arisen by an unequal sister chromatid exchange and the del(Xp) had occurred through an intrachromosomal breakage-reunion mechanism, both in the paternal X chromosome. The i(Xq) had arisen either through centromere fission of a maternal X chromosome, followed by duplication of its long-arm, or through a translocation between two maternal X chromosomes after meiotic crossing-over. The inv dup(X) arose through sister chromatid breakage and reunion in a maternal X chromosome. These results, together with those of previous studies, suggest that the de novo abnormalities due to events involving centromere disruption arise predominantly during oogenesis, while those due to simple breakage-reunion events occur preferentially during spermatogenesis.     

First reported case of intrachromosomal cryptic inv dup del Xp in a boy with developmental retardation

We report here on a 6-year-old boy referred to the laboratory for karyotyping and SHOX microdeletion testing. The most significant clinical findings in this boy were small stature, Madelung deformity, facial dysmorphism, mild mental retardation and behavioral problems. R-, G- and RTBG-banding chromosome analysis showed a normal male karyotype. Fine molecular characterization, by FISH, of terminal Xp microdeletion revealed an associated partial duplication. Further refinement of the molecular analysis indicated an inverted duplication of the Xp22.31-Xp22.32 (13.7 Mb) region including the STS, VCX-A and KAL1 genes, associated with a terminal Xp deletion Xp22.33-Xpter (3.6 Mb) encompassing the SHOX and ARSE genes. Such rearrangements have been characterized for other chromosomal pairs, but this is the first reported male patient involving the short arm of the X chromosome. Molecular analysis of the maternal and patient's microsatellite markers showed interchromatid mispairing leading to non-allelic homologous recombination to be the most likely mechanism underlying this rearrangement. This case highlights the importance of clinically driven FISH investigations in order to uncover cryptic micro-rearrangements.     

Pure de-novo 5 Mb duplication at Xp11.22–p11.23 in a male: phenotypic and molecular characterization

Males with duplications within the short arm of the X chromosome are rare and most cases are inherited from a maternal heterozygote. Here we describe the first detailed characterization of a de-novo Xp duplication delineated to Xp11.22→Xp11.23 in a 15-year-old male with moderate mental impairment, autistic-like behaviour, short stature, and mild dysmorphic features. Chromosome analysis (550 band resolution) was normal and comparative genomic hybridization (CGH) analysis on metaphase spreads detected duplication on Xp11. Further characterization of the duplication by array CGH, FISH experiments with specific BAC probes, and genotyping with microsatellite markers helped to determine proximal and distal breakpoints giving a size of the duplication of approximately 5 Mb. As far as we are aware this is the first described male with isolated microduplication on Xp11.22–Xp11.23. Among the genes included within the duplicated region, and particularly those which are outside copy number polymorphisms, we discuss the relationship of FTSJ1, PQBP1 and HDAC6 with the clinical symptoms of our patient.     

Random X-inactivation in a girl with duplication Xp11.21-p21.3: report of a patient and review of the literature.

We describe a 10-month-old girl with abnormal clinical findings and Xp duplication. She showed poor weight gain and developmental retardation, and had several minor anomalies including pigmentary dysplasia (hypomelanosis of Ito). She had a partial short arm duplication in the paternally derived X chromosome, 46,X,dup(X)(p11. 21p21.3), with the normal and duplicated X chromosomes randomly inactivated. These findings indicate that gross functional imbalance in the cells with an active dup(X) chromosome has caused global developmental defects in the patient, and that functional chromosomal mosaicism with respect to the duplicated Xp region has resulted in pigmentary dysplasia. Literature review of 52 patients with partial X duplications revealed (1) random or skewed but not completely selective X-inactivation in 9 of 45 patients examined for the X-inactivation pattern, independently of the size or location of duplicated segments, (2) apparently normal phenotype in 6 of 9 patients with random or skewed X-inactivation, and (3) an abnormal phenotype in 13 of 35 patients with completely selective inactivation of dup(X) chromosomes.     

荧光原位杂交联合微阵列比较基因组杂交分析一例原发性闭经患者的染色体畸变

目的分析1例原发性闭经患者的染色体畸变,探讨该患者原发性闭经的可能原因。方法采集临床已确诊的原发性闭经患者外周血,并抽提基因组DNA,进行荧光原位杂交和微阵列比较基因组杂交,分析染色体异常。结果微阵列比较基因组杂交显示患者染色体Xp22.31区域存在长1.637Mb片段的三倍体,Xp21.2-q21.1区域存在长52.156 Mb片段的重复片段。结论微阵列比较基因组杂交技术可以检测染色体微小畸变,值得临床推广应用。     

Random X-inactivation in a girl with duplication Xp11.21–p21.3: Report of a patient and review of the literature

We describe a 10-month-old girl with abnormal clinical findings and Xp duplication. She showed poor weight gain and developmental retardation, and had several minor anomalies including pigmentary dysplasia (hypomelanosis of Ito). She had a partial short arm duplication in the paternally derived X chromosome, 46,X,dup(X)(p11.21p21.3), with the normal and duplicated X chromosomes randomly inactivated. These findings indicate that gross functional imbalance in the cells with an active dup(X) chromosome has caused global developmental defects in the patient, and that functional chromosomal mosaicism with respect to the duplicated Xp region has resulted in pigmentary dysplasia. Literature review of 52 patients with partial X duplications revealed (1) random or skewed but not completely selective X-inactivation in 9 of 45 patients examined for the X-inactivation pattern, independently of the size or location of duplicated segments, (2) apparently normal phenotype in 6 of 9 patients with random or skewed X-inactivation, and (3) an abnormal phenotype in 13 of 35 patients with completely selective inactivation of dup(X) chromosomes. Am. J. Med. Genet. 86:44–50, 1999. © 1999 Wiley-Liss, Inc.     

Partial Xp duplication in a girl with dysmorphic features: the change in replication pattern of late-replicating dupX chromosome

In this paper we present the case of a girl at the age of 32 months with dysmorphic features, including general muscular hypotonia, developmental delay and mental retardation. The cytogenetic analysis revealed de novo partial duplication of Xp: 46,X,dup(X)(p11.23-->p22.33: :p11.23-->p22.33). To characterize the duplication, X painting, Kallman (KAL), yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) covering Xp11.23-->Xp22.33 region were used. Selective inactivation of the abnormal X chromosome using HpaII digestion of the AR gene was evident. After BrdU incorporation the abnormal X was late-replicating in all lymphocytes examined. There was one peculiar exception observed: the break-point region was consistently early replicating. The replicating pattern of this region corresponded to the active X chromosome. Methylation pattern of late replicating X chromosome was studied also using antibodies against 5-methylcytosine. The pattern corresponded to the normally inactive X chromosome, with the exception of the previously observed break-point region which revealed an early replicating pattern with strong fluorescent signal, similar to the pattern of the active X chromosome. The observed phenomenon could lead to the abnormal phenotype of the patient, with some normally inactive genes of the break-point region escaping the inactivation process. The abnormal clinical findings could also be due to tissue-dependent differences in the inactivation pattern.     

Cytogenetic heterogeneity of translocations associated with Duchenne muscular dystrophy

Lymphoblastoid cell lines have been established from nine female patients with Duchenne muscular dystrophy who had previously been reported to have chromosome translocations with breakpoints in the Xp21 region. A detailed cytogenetic comparison of prometaphase chromosomes in these cell lines revealed that six of the translocations had X chromosome breakpoints in the sub-band Xp212 and that one further breakpoint could be assigned to either Xp212 or Xp213. These findings confirm and extend previous observations and provide strong evidence for Xp212 as the site of the Duchenne and Becker loci. For the remaining two translocations the simplest explanation for the observed banding pattern is that the X chromosome breakpoint lies a few thousand kilobases away, in the sub-band Xp211. Other explanations which assume breaks in Xp212 combined with complex local chromosome rearrangements are also presented. It is also possible that the altered banding pattern in these two cases is due to the influence of local sequences on the staining or uncoiling properties of the chromatin.     

Sex reversal in a child with a 46,X,Yp+ karyotype: support for the existence of a gene(s), located in distal Xp, involved in testis formation.

We report on a sex reversed Japanese child with a 46,X,Yp+ karyotype, minor dysmorphic features, and no testicular development. The Yp+ chromosome was derived by translocation of an Xp fragment (Xp21-Xp22.3) to Yp11.3. This has resulted in deletion of distal part of the Y chromosome pseudoautosomal region (DXYS15-telomere) and duplication of the X specific region (DXS84-PABX) and proximal part of the pseudoautosomal region (MIC2-DXYS17). No deletion of the Y specific region was detected nor was any mutation found in SRY. Cytogenetic analysis suggests that the proximal part of the Xp fragment is the most distal part of the short arm of the Yp+ chromosome (Xp21----Xp 22.3::Yp11.3----Yqter). No chromosomal mosaicism was detected. These results are similar to previous reports of sex reversal in four subjects with a 46,Y,Xp+ karyotype. We conclude that the sex reversal is a direct, or indirect, consequence of having two active copies of the distal part of Xp and may indicate the presence of a gene(s) which acts in the testis determination or differentiation pathway.     

Characterization of interstitial Xp duplications in two families by tiling path array CGH

Duplications of the short arm of the X chromosome in male patients are rare. We report on the clinical features of mentally retarded patients in two families with different interstitial duplications of Xp and their characterization by tiling path array comparative genomic hybridization (array CGH). In Family A, we detected a duplication of 9.3 Mb in Xp11p21 in a male with severe mental retardation [karyotype 46,XY,dup(X)(p11.3p21.1)] and his healthy mother. The clinical features of this patient--severe mental retardation, obesity, macrocephaly--are in accordance with those of a previously reported patient with a similar duplication. In Family B, a duplication of 8.5 Mb was diagnosed in Xp22 in three male patients with mental retardation [karyotype 46,XY,dup(X)(p22.11p22.2)] and two healthy females. Characterization of the duplications by array CGH enabled the identification of the genes within these intervals. These comprise known mental retardation genes such as MAOA, NDP, TM4SF2, NDP, RSK2, and CDKL5. Duplication of MAOA will be discussed as a possible cause of obesity.     

47, XY, +der(Y),t(X;Y)(p21.1;p11.2): a unique case of XY sex reversal

Translocations involving the short arms of the X and Y chromosomes are rare and can result in a functional disomy of the short arm of the X chromosome, including the dosage-sensitive sex reversal (DSS) locus. A result of such imbalance may be sex reversal with multiple congenital anomalies. We present the clinical and cytogenetic evaluation of a newborn infant with DSS and additional clinical findings of minor facial anomalies, left abdominal mass, 5th finger clinodactyly, and mild hypotonia. The external genitalia appeared to be normal female. The infant had bilateral corneal opacities and findings suggestive of anterior segment dysgenesis. Ultrasonography showed a small uterus with undetectable ovaries, and a left multicystic dysplastic kidney. High-resolution chromosome analysis identified the presence of a derivative Y chromosome, 47,XY, +der(Y)t(X;Y)(p21.1;p11.2), which was confirmed by fluorescence in situ hybridization studies. Array CGH showed a 35.1 Mb copy number gain of chromosome region Xp22.33-p21.1 and a 52.2 Mb copy number gain of Yp11.2-qter, in addition to the intact X and Y chromosomes. Previously reported patients with XY sex reversal have not had DSS with corneal opacities, dysgenesis of the anterior segment of the eye, and unilateral multicystic dysplastic kidney. These findings represent a new form of XY sex reversal due to an Xp duplication.     

Schizophrenia susceptibility gene locus at Xp22.3

Multiple genetic loci have been implicated in the search for schizophrenia susceptibility genes, none having been proven as causal. Genetic heterogeneity is probable in the polygenic etiology of schizophrenia. We report on two unrelated Caucasian women with paranoid schizophrenia (meeting Diagnostic and Statistical Manual of Mental Disorders (DSM IV) criteria) who have an Xp22.3 overlapping deletion characterized by fluorescence in situ hybridization (FISH). Patient 1 was previously reported by us (Wyandt HE, Bugeau-Michaud L, Skare JC, Milunsky A. Partial duplication of Xp: a case report and review of previously reported cases. Amer J Med Genet 1991: 40: 280-283) to have a de novo partial duplication of Xp. At that time, she was a 24-year-old woman with short stature, irregular menses, other abnormalities suggestive of Turner syndrome, and paranoid schizophrenia. Recently, FISH analysis demonstrated that she has an inverted duplication (X)(p22.1p11.2) and a microscopic deletion (X)(p22.2p22.3) between DXS1233 and DXS7108 spanning approximately 16-18 cM. Patient 2 is a 14-year-old girl with short stature, learning disabilities, and paranoid schizophrenia. High-resolution chromosome analysis revealed a de novo deletion involving Xp22. FISH analysis showed that the deletion (X)(p22.2p22.3) spanned 10-12 cM between AFMB290XG5 and DXS1060. Given that deletions of Xp22 are not common events, the occurrence of two unrelated schizophrenia patients with an overlapping deletion of this region would be extraordinarily rare. Hence, the deletion within Xp22.3 almost certainly contains a gene involved in the pathogenesis of paranoid schizophrenia.     

Familial inv(X)(p22q22): ovarian dysgenesis in two sisters with del Xq and fertility in one male carrier

A recombinant chromosome with Xp duplication and Xq deletion was found in two sisters with normal height and gonadal dysgenesis. Their mother and other four relatives, including a fertile male, carried an inv(X)(p22q22); the inverted X was randomly inactivated in one female carrier. The abnormal X chromosome showed inactivation in all the examined cells. This is the tenth report of a recombinant X chromosome. A review of the literature shows that: i) most female carriers of inv(X) are phenotypically normal and fertile; ii) recombinants having short-arm duplication and long-arm deletion are associated with ovarian failure and normal or tall stature, whereas the reciprocal recombinants are compatible with fertility but cause short stature; and (ü) except for one index case, all male carriers have a normal phenotype and 11 of them (from eight families) are of proven fertility. Moreover, no instance of male infertility has been documented.     

Duplication Xp22.2 and pseudoisodicentric Yq detected by FISH and PCR in a sterile male

A chromosome mosaicism with two cell lines was diagnosed in a sterile man. One cell line had a 45,—Y, dup (X) (p22.2) karyotype and accounted for 83% of lymphocytes analyzed. Fluorescence in situ hybridization (FISH) analysis with specific X and Y probes excluded a translocation between the short arms of the X and Y chromosomes and showed that Xp duplication involved a region containing the DXS85 locus, distal to the ZFX and DSS sites. The other cell line consisted of a diploid karyotype with a rearranged Y chromosome, which was shown to be a pseudoisodicentric Yq by FISH. Moreover, FISH with a specific probe for the AZF locus and polymerase chain reaction using Yq SY108 and SY121 primers showed no signals for this region, possibly accounting for the azoospermia in this patient.