Variegated silencing through epigenetic modifications of a large Xq region in a case of balanced X;2 translocation with Incontinentia Pigmenti-like phenotype

Molecular mechanisms underlying aberrant phenotypes in balanced X;autosome translocations are scarcely understood. We report the case of a de novo reciprocal balanced translocation X;2(q23;q33) presenting phenotypic alterations highly suggestive of Incontinentia Pigmenti (IP) syndrome, a genodermatosis with abnormal skin pigmentation and neurological failure, segregating as X-linked dominant disorder. Through molecular studies, we demonstrated that the altered phenotype could not be ascribed to chromosome microdeletions or to XIST-mediated inactivation of Xq24-qter. Interestingly, we found that the Xq24-qter region, which translocated downstream of the heterochromatic band 2q34, undergoes epigenetic silencing mediated by DNA methylation and histone alterations. Among the downregulated genes, we found the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase gamma (IKBKG/NEMO), the causative gene of IP. We hypothesize that a mosaic functional nullisomy of the translocated genes, through a Position Effect Variegation-like heterochromatization, might be responsible for the proband's phenotypic anomalies. Partial silencing of IKBKG may be responsible for the skin anomalies observed, thereby mimicking the IP pathological condition. In addition to its clinical relevance, this paper addresses fundamental issues related to the chromatin status and nuclear localization of a human euchromatic region translocated proximally to heterochromatin. In conclusion, the study provides new insight into long-range gene silencing mechanisms and their direct impact in human disease.     

Physical mapping of nine Xq translocation breakpoints and identification of XPNPEP2 as a premature ovarian failure candidate gene

Women with balanced translocations between the long arm of the X chromosome (Xq) and an autosome frequently suffer premature ovarian failure (POF). Two "critical regions" for POF which extend from Xq13-->q22 and from Xq22-->q26 have been identified using cytogenetics. To gain insight into the mechanism(s) responsible for ovarian failure in women with X;autosome translocations, we have molecularly characterized the translocation breakpoints of nine X chromosomes. We mapped the breakpoints using somatic cell hybrids retaining the derivative autosome and densely spaced markers from the X-chromosome physical map. One of the POF-associated breakpoints in a critical region (Xq25) mapped to a sequenced PAC clone. The translocation disrupts XPNPEP2, which encodes an Xaa-Pro aminopeptidase that hydrolyzes N-terminal Xaa-Pro bonds. XPNPEP2 mRNA was detected in fibroblasts that carry the translocation, suggesting that this gene at least partially escapes X inactivation. Although the physiologic substrates for the enzyme are not known, XPNPEP2 is a candidate gene for POF. Our breakpoint mapping data will help to identify additional candidate POF genes and to delineate the Xq POF critical region(s).     

FXY2/MID2, a Gene Related to the X-Linked Opitz Syndrome Gene FXY/MID1, Maps to Xq22 and Encodes a FNIII Domain-Containing Protein That Associates with Microtubules

Opitz G/BBB syndrome (OS) is a genetically heterogeneous disorder with an X-linked locus and an autosomal locus linked to 22q11.2. OS affects multiple organ systems with often variable severity even between siblings. The clinical features, which include hypertelorism, cleft lip and palate, defects of cardiac septation, hypospadias, and anorectal anomalies, indicate an underlying disturbance of the developing ventral midline of the embryo. The gene responsible for X-linked OS, FXY/MID1, is located on the short arm of the human X chromosome within Xp22.3 and encodes a protein with both an RBCC (RING finger, B-box, coiled coil) and a B30.2 domain. The Fxy gene in mice is also located on the X chromosome but spans the pseudoautosomal boundary in this species. Here we describe a gene closely related to FXY/MID1, called FXY2, which also maps to the X chromosome within Xq22. The mouse Fxy2 gene is located on the distal part of the mouse X chromosome within a region syntenic to Xq22. Analysis of genes flanking both FXY/MID1 and FXY2 (as well as their counterparts in mouse) suggests that these regions may have arisen as a result of an intrachromosomal duplication on an ancestral X chromosome. We have also identified in both FXY2 and FXY/MID1 proteins a conserved fibronectin type III domain located between the RBCC and B30.2 domains that has implications for understanding protein function. The FXY/MID1 protein has previously been shown to colocalize with microtubules, and here we show that the FXY2 protein similarly associates with microtubules in a manner that is dependent on the carboxy-terminal B30.2 domain.     

Disruption of DMD and deletion of ACSL4 causing developmental delay, hypotonia, and multiple congenital anomalies

We have studied a male patient with significant developmental delay, growth failure, hypotonia, girdle weakness, microcephaly, and multiple congenital anomalies including atrial (ASD) and ventricular (VSD) septal defects. Detailed cytogenetic and molecular analyses revealed three de novo X chromosome aberrations and a karyotype 46,Y,der(X)inv(X) (p11.4q11.2)inv(X)(q11.2q21.32 approximately q22.2)del(X)(q22.3q22.3) was determined. The three X chromosome aberrations in the patient include: a pericentric inversion (inv 1) that disrupted the Duchenne muscular dystrophy (DMD) gene, dystrophin, at Xp11.4; an Xq11.2q21.32 approximately q22.2 paracentric inversion (inv 2) putatively affecting no genes; and an interstitial deletion at Xq22.3 that results in functional nullisomy of several known genes, including a gene previously associated with X-linked nonsyndromic mental retardation, acyl-CoA synthetase long chain family member 4 (ACSL4). These findings suggest that the disruption of DMD and the absence of ACSL4 in the patient are responsible for neuromuscular disease and cognitive impairment.     

Mapping of a new SGBS locus to chromosome Xp22 in a family with a severe form of Simpson-Golabi-Behmel syndrome.

Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked overgrowth syndrome with associated visceral and skeletal abnormalities. Alterations in the glypican-3 gene (GPC3), which is located on Xq26, have been implicated in the etiology of relatively milder cases of this disorder. Not all individuals with SGBS have demonstrated disruptions of the GPC3 locus, which raises the possibility that other loci on the X chromosome could be responsible for some cases of this syndrome. We have previously described a large family with a severe form of SGBS that is characterized by multiple anomalies, hydrops fetalis, and death within the first 8 wk of life. Using 25 simple tandem-repeat polymorphism markers spanning the X chromosome, we have localized the gene for this disorder to an approximately 6-Mb region of Xp22, with a maximum LOD score of 3.31 and with LOD scores <-2.0 for all of Xq. These results demonstrate that neither the GPC3 gene nor other genes on Xq26 are responsible for all cases of SGBS and that a second SGBS locus resides on Xp22.     

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.     

Epigenetic control of the critical region for premature ovarian failure on autosomal genes translocated to the X chromosome: a hypothesis

Chromosomal rearrangements in Xq are frequently associated to premature ovarian failure (POF) and have contributed to define a POF “critical region” from Xq13.3 to Xq26. Search for X-linked genes responsible for the phenotype has been elusive as most rearrangements did not interrupt genes and many were mapped to gene deserts. We now report that ovary-expressed genes flanked autosomal breakpoints in four POF cases analyzed whose X chromosome breakpoints interrupted a gene poor region in Xq21, where no ovary-expressed candidate genes could be found. We also show that the global down regulation in the oocyte and up regulation in the ovary of X-linked genes compared to the autosomes is mainly due to genes in the POF “critical region”. We thus propose that POF, in X;autosome balanced translocations, may not only be caused by haploinsufficiency, but also by a oocyte-specific position effect on autosomal genes, dependent on dosage compensation mechanisms operating on the active X chromosome in mammals.     

Regional localization of the genes for human HEXB. PGK, GALA. HPRT, G6PD by somatic cell hybridization

Twenty independent man-mouse (Cl1D,LA/TK-, HPRT-) and man-hamster (CH,HPRT-) hybrids using female human cells with balanced reciprocal translocation XX,t(X;5)(q21;q11) were analyzed for human genes localized on chromosome 5 (HEXB), on chromosome X (PGK, GALA, HPRT, G6PD) and for the different chromosomes in relation with the balanced reciprocal translocation (chr.5, chr.5q-, chr.Xq+, chr.X). The different results obtained indicate that the genes for human markers HEXB, PGK are on Xq+, and that the genes for human markers GALA, G6PD are on 5q-. These data implicate finally the following localizations: HEXB on 5q11 leads to 5qter; PGK on Xq21 leads to Xpter; GALA, HPRT, G6PD on Xq21 leads to Xqter.     

Highly Conserved Non-Coding Sequences and the 18q Critical Region for Short Stature: A Common Mechanism of Disease?

Background

Isolated growth hormone deficiency (IGHD) and multiple pituitary hormone deficiency (MPHD) are heterogeneous disorders with several different etiologies and they are responsible for most cases of short stature. Mutations in different genes have been identified but still many patients did not present mutations in any of the known genes. Chromosomal rearrangements may also be involved in short stature and, among others, deletions of 18q23 defined a critical region for the disorder. No gene was yet identified.

Methodology/Principal Findings

We now report a balanced translocation X;18 in a patient presenting a breakpoint in 18q23 that was surprisingly mapped about 500 Kb distal from the short stature critical region. It separated from the flanking SALL3 gene a region enriched in highly conserved non-coding elements (HCNE) that appeared to be regulatory sequences, active as enhancers or silencers during embryonic development.

Conclusion

We propose that, during pituitary development, the 18q rearrangement may alter expression of 18q genes or of X chromosome genes that are translocated next to the HCNEs. Alteration of expression of developmentally regulated genes by translocation of HCNEs may represent a common mechanism for disorders associated to isolated chromosomal rearrangements.     

X-autosome translocation with a breakpoint in Xq22 in a fertile woman and her 47,XXX infertile daughter

Summary An unusual case is presented of a fertile woman heterozygous for a balanced X-autosome translocation t(X;12) (q22;p12) with a break-point (Xq22) in the critical region of the X chromosome. The karyotypes of her daughter, who is infertile, and one of her two sons are 47,XXX,t(X;12)(q22;p12) and 46,XY,t(X;12)(q22;p12) respectively. The literature on balanced X-autosome translocations in males and females involving both arms of the X chromosome is reviewed. All 23 of the 36 cases of females with balanced Xq-autosome translocation, that exhibited gonadal failure have a break-point between bands Xq13 and Xq26.     

Identification of Novel Genomic Aberrations in AML-M5 in a Level of Array CGH

To assess the possible existence of unbalanced chromosomal abnormalities and delineate the characterization of copy number alterations (CNAs) of acute myeloid leukemia-M5 (AML-M5), R-banding karyotype, oligonucelotide array CGH and FISH were performed in 24 patients with AML-M5. A total of 117 CNAs with size ranging from 0.004 to 146.263 Mb was recognized in 12 of 24 cases, involving all chromosomes other than chromosome 1, 4, X and Y. Cryptic CNAs with size less than 5 Mb accounted for 59.8% of all the CNAs. 12 recurrent chromosomal alterations were mapped. Seven out of them were described in the previous AML studies and five were new candidate AML-M5 associated CNAs, including gains of 3q26.2-qter and 13q31.3 as well as losses of 2q24.2, 8p12 and 14q32. Amplication of 3q26.2-qter was the sole large recurrent chromosomal anomaly and the pathogenic mechanism in AML-M5 was possibly different from the classical recurrent 3q21q26 abnormality in AML. As a tumor suppressor gene, FOXN3, was singled out from the small recurrent CNA of 14q32, however, it is proved that deletion of FOXN3 is a common marker of myeloid leukemia rather than a specific marker for AML-M5 subtype. Moreover, the concurrent amplication of MLL and deletion of CDKN2A were noted and it might be associated with AML-M5. The number of CNA did not show a significant association with clinico-biological parameters and CR number of the 22 patients received chemotherapy. This study provided the evidence that array CGH served as a complementary platform for routine cytogenetic analysis to identify those cryptic alterations in the patients with AML-M5. As a subtype of AML, AML-M5 carries both common recurrent CNAs and unique CNAs, which may harbor novel oncogenes or tumor suppressor genes. Clarifying the role of these genes will contribute to the understanding of leukemogenic network of AML-M5.     

Translocation (X;13)(p11.21;q12.3) in a girl with incontinentia pigmenti and bilateral retinoblastoma

A de novo t(X;13)(p11.21;q12.3) translocation is described in an 19-month-old girl with incontinentia pigmenti (IP) and bilateral retinoblastoma. Based on previously reported two girls and this patient, each with a structural X chromosome abnormality and IP, it was assumed that the locus for IP is at Xp11.21. Q-banding analysis revealed that the translocated chromosomes were of paternal origin. The derivative X chromosome was late-replicating in 9% of cultured peripheral blood lymphocytes and in 1% of skin fibroblasts. The erythrocyte esterase D activity in the patient was normal. Several possibilities were considered for possible causative relationship between the X/13 translocation and the development of retinoblastoma. One possibility involved functional monosomy of 13q14 in a minority of retinoblasts due to the spreading of inactivation of the translocated X chromosome segment.     

Infantile hypertrophic pyloric stenosis: evaluation of three positional candidate genes, <Emphasis Type="Italic">TRPC1</Emphasis>, <Emphasis Type="Italic">TRPC5</Emphasis> and <Emphasis Type="Italic">TRPC6</Emphasis>, by association analysis and re-sequencing

Infantile hypertrophic pyloric stenosis (IHPS) is the most common inherited form of gastrointestinal obstruction in infancy with a striking male preponderance. Infants present with vomiting due to gastric outlet obstruction caused by hypertrophy of the smooth muscle of the pylorus. Two loci specific to extended pedigrees displaying autosomal dominant inheritance have been identified. A genome scan identified loci on chromosomes 11q14–q22 and Xq23–q24 which are predicted to be responsible for a subset of smaller families with IHPS demonstrating non-Mendelian inheritance. The two linked chromosomal regions both harbour functional candidate genes which are members of the canonical transient receptor potential (TRPC) family of ion channels. Both TRPC5 (Xq23–q24) and TRPC6 (11q14–q22) have a potential role in smooth muscle control and hypertrophy. Here, we report suggestive evidence for a third locus on chromosome 3q12–q25 (Z _max = 2.7, p < 0.004), a region which harbours a third TRPC gene, TRPC1. Fine mapping of all three genes using a tagSNP approach and re-sequencing identified a SNP in the promoter region of TRPC6 and a missense variant in exon 4 of TRPC6 which may be putative causal variants.     

A dynamic study in two new cases of X chromosome translocations

Summary The authors discuss the clinical and cytogenetic problems raised in two new cases of X-chromosome translocations.The first case involves a child who presented marked growth retardation, behavioral anomalies, and discrete facial malformations at age 3 months. Chromosome analysis revealed the presence of a translocation between a 22 and X chromosome resulting in partial X monosomy and partial trisomy 22: 46,X,der(X),t(X;22)(q112;q13)mat. The balanced translocation form was detected in the mother. Dynamic study after 5-Brdu treatment revealed inactivation of the translocated X chromosome in the proband, while in the mother the normal X chromosome was inactivated.In addition to magnesium dependent hypocalcemia resulting from a specific absorption anomaly, Case 2 presented discrete malformations and psychomotor retardation. Chromosome analysis revealed an apparently balanced translocation between a 9 and X chromosome: 46,X,r(9;X)(q12; p22). Treatment with 5-Brdu demonstrated that the translocated X chromosome was inactivated but that inactivation did not extend to the translocated part of chromosome 9. Finally, a pericentric inversion of a 9 chromosome was detected in the father, grandfather, and brother of the proband.     

Cytogenetic and molecular analysis of an unbalanced translocation (X;7) (q28;p15) in a dysmorphic girl

Summary A severely retarded and dysmorphic girl, carrying an unbalanced X/7 translocation with breakpoints at Xq28 and 7p14, was analyzed by cytogenetic, biochemical and molecular techniques. The X/7 translocated chromosome was found to replicate consistently late in the 105 metaphases analyzed. In 83 of these cells, late replication was limited to the X portion of the abnormal chromosome, whereas in 22 cells incomplete spreading into the autosomal fragment was observed. Southern blot and in situ hybridization experiments with probe G80 (locus D7S373) (previously localized to 7p13–15) and G98 (localized to 7p14–15) assigns the former to 7p15 and the latter to 7p14, thus suggesting the order 7ter-G80-G98-cen. The activity of the enzyme phosphoserine phosphatase localized to 7pter p14 was increased. Southern blotting experiments with 19 probes spanning the entire X chromosome demonstrated that the translocated chromosome had lost a portion of Xq28 (locus DXS51) but still retained part of Xq27 (F9 locus). The results confirm that the proband is trisomic for the region 7p15-pter and monosomic for the region Xq28-qter. Comparing her phenotype with those of other cases of partial trisomy or monosomy 7p, we confirm that band 7p21 is probably involved in skull development.     

High-resolution mapping of mouse Chromosome 8 identifies an evolutionary chromosomal breakpoint

The central region of mouse Chromosome (Chr) 8, containing the myodystrophy (myd) locus, is syntenic with human Chr 4q28-qter. The human neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD) maps to Chr 4q35, and myd has been proposed as a mouse homolog of FSHD. We have employed a comparative mapping approach to investigate this relationship further by extending the mouse genetic map of this region. We have ordered 12 genes in a single cross, 8 of which have human homologs on 4q28-qter. The results confirm a general relationship between the most distal genes on human 4q and the most proximal genes in the mouse 8 syntenic region. Despite chromosomal rearrangements of syntenic groups in this region, conservation of gene order is maintained between the group of genes in the human telomeric region of 4q35 and MMU8. Furthermore, this conserved telomeric HSA4q35 syntenic group maps proximal to the myd mutation and is flanked by genes with homologs on HSA8p22. At the proximal boundary of the MMU8 linkage group we have identified a single 300-kb YAC containing the genes Frgl and Pcml, which have human homologs on 4q35 and 8p22, respectively. Thus, this YAC spans an evolutionary chromosomal breakpoint. As well as providing clues about chromosomal evolution, this map of the FSHD syntenic mouse region should prove invaluable in the isolation of candidate genes for this disease. Received: 20 January 1998 / Accepted: 10 April 1998     

The similarity of phenotypic effects caused by Xp and Xq deletions in the human female: a hypothesis

Summary We have collected from the literature adult nonmosaic women with the following aberrant X chromosomes: Xp- (52), Xq- (67), idic(Xp-)(10), idic(Xq-)(9), and interstitial deletions (12). Lack of Xp, and especially Xcen-Xp11 (b region), may cause full-blown Turner syndrome. However, individual Turner symptoms, including gonadal dysgenesis, otherwise seem to be randomly distributed with respect to the different Xp and Xq deletions, although breakpoints distal to Xq25 do not give rise to any phenotypic anomalies except in a few cases of secondary amenorrhea or premature menopause. Of the carriers of an Xp- or Xq- chromosome, 65% and 93%, respectively, suffer from ovarian dysgenesis, whereas all idic(Xp-) and idic(Xq-) chromosomes cause primary or secondary amenorrhea. Xq deletions do not induce specific symptoms different from those caused by Xp deletions. Lack of the tip of Xp has led in 46/52 cases to short stature, but 43% of the Xq- carriers are also short. To explain these observations, we propose the following hypothesis. Since deletions of truly inactivated regions do not seem to cause any symptoms, we assume that the b region (Xcen-p11) always stays active in a normal inactive X, but is inactivated in deleted X chromosomes, especially in Xq- chromosomes. In some cases, inactivation may spread to the tip of Xp; this would explain the apparently variable behavior of the Xg and STS genes, and the short stature of some Xq- carriers. Full chromosome pairing seems to be a prerequisite for the viability of oocytes and thus for gonadal development. Deleted X chromosomes necessarily leave a portion of the normal X unpaired and isodicentrics probably interfere with pairing, resulting in atresia of oocytes. The role played by the critical region (Xq13–q24) in ovarian development is still unclear.     

Regional assignment of arylsulfatase A,mitochondrial aconitase and NADH-cytochrome b5 reductase by somatic cell hybridization

Summary By using somatic cell hybrids between HPRT deficient hamster cells and fibroblasts derived from a patient with a X/22 translocation t(X;22)(q13;q112), we have assigned the genes for human ARSA, DIA 1, and ACO 2 to region q112qter of human chromosome 22 and the gene for human PGK close to the breakpoint in band Xq13.