Atypical breakpoint in a t(6;17) translocation case of acampomelic campomelic dysplasia

Campomelic dysplasia (CD) is a skeletal dysplasia characterized by Pierre Robin sequence (PRS), shortened and bowed long bones, airway instability, and the potential for sex reversal. A subtype of CD, acampomelic CD (ACD), is seen in approximately 10% of cases and preserves long bone straightness. Both syndromes are caused by alterations in SOX9, with translocations and missense mutations being overrepresented in ACD cases. We report a term infant with PRS, severe cervical spine abnormalities, eleven rib pairs, hypoplastic scapulae, and female genitalia. Chromosome analysis identified a 46,XY,t(6;17)(q25;q24) karyotype. FISH analysis with a series of BAC probes localized the translocation breakpoints to 6q27 and a region at 17q24.3 in the range of 459–379 kb upstream of SOX9. Therefore, this case extends the region classified as the proximal breakpoint cluster. In addition, the comorbidity of acampomelia, complete sex reversal, and severe spinal anomalies in our patient underscores the variability in the level of malformation in the CD/ACD family of disorders.     

Autosomal XX sex reversal caused by duplication of SOX9

SOX9 is one of the genes that play critical roles in male sexual differentiation. Mutations of SOX9 leading to haploinsufficiency can cause campomelic dysplasia and XY sex reversal. We report here evidence supporting that SOX9 duplication can cause XX sex reversal. A newborn infant was referred for genetic evaluation because of abnormal male external genitalia. The infant had severe penile/scrotal hypospadias. Gonads were palpable. Cytogenetic analysis demonstrated a de novo mosaic 46,XX,dup(17)(q23.1q24.3)/46, XX karyotype. Fluorescent in situ hybridization (FISH) with a BAC clone containing the SOX9 gene demonstrated that the SOX9 gene is duplicated on the rearranged chromosome 17. The presence of SRY was ruled out by FISH with a probe containing the SRY gene and polymerase chain reaction with SRY-specific primers. Microsatellite analysis with 13 markers on 17q23-24 determined that the duplication is maternal in origin and defined the boundary of the duplication to be approximately 12 centimorgans (cM) proximal and 4 cM distal to the SOX9 gene. Thus, SOX9 duplication is the most likely cause for the sex reversal in this case because it plays an important role in male sex determination and differentiation. This study suggests that extra dose of SOX9 is sufficient to initiate testis differentiation in the absence of SRY. Other SRY-negative XX sex-reversed individuals deserve thorough investigation of SOX9 gene.     

Isolation of a testis-specific cDNA on chromosome 17q from a region adjacent to the breakpoint of t(12;17) observed in a patient with acampomelic campomelic dysplasia and sex reversal

Campomelic dysplasia (CMPD), a rare congenital disorder, is characterized by a variety of skeletal anomalies, low-set ears and, in nearly half of genotypical-male patients, sex reversal. Observations of chromosomal translocations involving chromosome 17q24-q25 in several CMPD patients have implied that disruption of one or more genes in the breakpoint region is responsible for this disease. Using fluorescence in situ hybridization, we mapped the chromosome-17 breakpoint in a patient with acampomelic CMPD and sex reversal, who carries a de novo constitutional t(12;17) translocation, between two known cosmid markers in the 17q24-q25 region. Through positional cloning, we isolated a 3.5 kb cDNA that is located at a close but distinct position from the SOX9 gene, from the region surrounding this breakpoint. Its mRNA, approximately 3.7 kb long, was expressed specifically in testis among 16 adult tissues examined by Northern blot analysis. As we were unable to find any long open reading frame in the 3.5 kb cDNA sequence or to detect any peptide following an in vitro translation experiment using RNA transcribed from this cDNA, we speculate that this gene may play a critical role in differentiation or sex determination as a functional RNA.      

Adult acampomelic campomelic dysplasia and disorders of sex development due to a reciprocal translocation involving chromosome 17q24.3 upstream of the SOX9 gene

Balanced chromosomal rearrangements with a breakpoint located upstream of the sex determining region Y-box 9 (SOX9) gene on chromosome 17q24.3 are associated with skeletal abnormalities, campomelic dysplasia (CMPD), or acampomelic campomelic dysplasia (ACMPD). We report on a female patient with a reciprocal translocation of t (11; 17) (p15.4; q24.3), who was diagnosed with acampomelic campomelic dysplasia. The 34-year-old Japanese patient presented with distinct skeletal abnormalities, profound intellectual disability, and female phenotype despite the presence of Y chromosome and the sex determining region Y (SRY) gene. Her menarche started at 33 years and 4 months after hormone therapy of estrogen therapy followed by estrogen progesterone therapy. By conducting whole genome sequencing followed by Sanger sequencing validation, we determined the precise breakpoint positions of the reciprocal translocation, one of which was located 203 kb upstream of the SOX9 gene. Considering the phenotypic variations previously reported among the CMPD/ACMPD patients with a chromosomal translocation in the vicinity of SOX9, the identified translocation was concluded to be responsible for all major phenotypes observed in the patient.     

Testis development in the absence of SRY: chromosomal rearrangements at SOX9 and SOX3

Duplications in the ~2 Mb desert region upstream of SOX9 at 17q24.3 may result in familial 46,XX disorders of sex development (DSD) without any effects on the XY background. A balanced translocation with its breakpoint falling within the same region has also been described in one XX DSD subject. We analyzed, by conventional and molecular cytogenetics, 19 novel SRY-negative unrelated 46,XX subjects both familial and sporadic, with isolated DSD. One of them had a de novo reciprocal t(11;17) translocation. Two cases carried partially overlapping 17q24.3 duplications ~500 kb upstream of SOX9, both inherited from their normal fathers. Breakpoints cloning showed that both duplications were in tandem, whereas the 17q in the reciprocal translocation was broken at ~800 kb upstream of SOX9, which is not only close to a previously described 46,XX DSD translocation, but also to translocations without any effects on the gonadal development. A further XX male, ascertained because of intellectual disability, carried a de novo cryptic duplication at Xq27.1, involving SOX3. CNVs involving SOX3 or its flanking regions have been reported in four XX DSD subjects. Collectively in our cohort of 19 novel cases of SRY-negative 46,XX DSD, the duplications upstream of SOX9 account for ~10.5% of the cases, and are responsible for the disease phenotype, even when inherited from a normal father. Translocations interrupting this region may also affect the gonadal development, possibly depending on the chromatin context of the recipient chromosome. SOX3 duplications may substitute SRY in some XX subjects.     

Recurrent rearrangements in the proximal 15q11-q14 region: a new breakpoint cluster specific to unbalanced translocations

Unbalanced translocations, that involve the proximal chromosome 15 long arm and the telomeric region of a partner chromosome, result in a karyotype of 45 chromosomes with monosomy of the proximal 15q imprinted region. Here, we present our analysis of eight such unbalanced translocations that, depending on the parental origin of the rearranged chromosome, were associated with either Prader-Willi or Angelman syndrome. First, using FISH with specific BAC clones, we characterized the chromosome 15 breakpoint of each translocation and demonstrate that four of them are clustered in a small 460 kb interval located in the proximal 15q14 band. Second, analyzing the sequence of this region, we demonstrate the proximity of a low-copy repeat 15 (LCR15)-duplicon element that is known to facilitate recombination events at meiosis and to promote rearrangements. The presence, in this region, of both a cluster of translocation breakpoints and a LCR15-duplicon element defines a new breakpoint cluster (BP6), which, to our knowledge, is the most distal breakpoint cluster described in proximal 15q. Third, we demonstrate that the breakpoints for other rearrangements including large inv dup (15) chromosomes do not map to BP6, suggesting that it is specific to translocations. Finally, the translocation breakpoints located within BP6 result in very large proximal 15q deletions providing new informative genotype-phenotype correlations.     

Localization of the oncogene c-erbA1 immediately proximal to the acute promyelocytic leukaemia breakpoint on chromosome 17

Using in situ hybridization, c- erb A1 has been mapped immediately distal to the translocation breakpoint on chromosome 17 in fibroblasts with a karyotype 46, XX, t(15;17)(q22;q11). Previous work has shown that c- erb A1 is proximal to the translocation breakpoint on chromosome 17 in the t(15;17)(q22;q12–21) in acute promyelocytic leukaemia. The oncogene can therefore be localized to the region of chromosome 17 between the breakpoints in 17q11 and 17q12–21.     

Distinct breakpoints in band 11q23 of the t(4;11) and t(11;14) associated with leukocyte malignancy.

Several non-random translocation breakpoints associated with leukemia or lymphoma have been shown to occur in chromosome band 11q23 between the genes CD3G and PBGD, a distance of approximately 750 kb. A combination of yeast artificial chromosome (YAC) cloning, in situ hybridization, and pulsed field gel electrophoresis (PFGE) experiments has further refined the interval containing one of these breakpoints, t(4;11)(q21;q23), to within 200 kb of CD3G. We have extended the PFGE analysis to show that the t(4;11) breakpoint lies in a region of approximately 100 kb, situated 100 kb distal to CD3G. Furthermore, we show that a second 11q23 breakpoint, t(11;14)(q23;q32), which was also previously mapped between CD3G and PBGD, is distinct from that of the t(4;11) chromosome. The 11q23 sequences that are involved at the t(11;14) breakpoint are not present in a YAC containing the t(4;11) breakpoint. The t(11;14) breakpoint has been localized on the PFGE map of the CD3G-PBGD interval and is at least 110 kb distal to the t(4;11) breakpoint, thus demonstrating heterogeneity among 11q23 breakpoints.     

Mandibulofacial dysostosis in a patient with a de novo 2;17 translocation that disrupts the HOXD gene cluster

Treacher Collins syndrome (TCS) is the prototypical mandibulofacial dysostosis syndrome, but other mandibulofacial dysostosis syndromes have been described. We report an infant with mandibulofacial dysostosis and an apparently balanced de novo 2;17 translocation. She presented with severe lower eyelid colobomas requiring skin grafting, malar and mandibular hypoplasia, bilateral microtia with external auditory canal atreasia, dysplastic ossicles, hearing loss, bilateral choanal stenosis, cleft palate without cleft lip, several oral frenula of the upper lip/gum, and micrognathia requiring tracheostomy. Her limbs were normal. Chromosome analysis at the 600-band level showed a 46,XX,t(2;17)(q24.3;q23) karyotype. Sequencing of the entire TCOF1 coding region did not show evidence of a sequence variation. High-resolution genomic microarray analysis did not identify a cryptic imbalance. FISH mapping refined the breakpoints to 2q31.1 and 17q24.3-25.1 and showed the 2q31.1 breakpoint likely affects the HOXD gene cluster. Several atypical findings and lack of an identifiable TCOF1 mutation suggest that this child has a provisionally unique mandibulofacial dysostosis syndrome. The apparently balanced de novo translocation provides candidate loci for atypical and TCOF1 mutation negative cases of TCS. Based on the agreement of our findings with one previous case of mandibulofacial dysostosis with a 2q31.1 transocation, we hypothesize that misexpression of genes in the HOXD gene cluster produced the described phenotype in this patient.     

Further evidence for the involvement of human chromosome 6p24 in the aetiology of orofacial clefting.

Chromosomal translocations affecting the 6p24 region have been associated with orofacial clefting. Here we present a female patient with cleft palate, severe growth retardation, developmental delay, frontal bossing, hypertelorism, antimongoloid slant, bilateral ptosis, flat nasal bridge, hypoplastic nasal alae, protruding upper lip, microretrognathia, bilateral, low set, and posteriorly rotated ears, bilateral microtia, narrow ear canals, short neck, and a karyotype of 46,XX,t(6;9)(p24;p23). The translocation chromosomes were analysed in detail by FISH and the 6p24 breakpoint was mapped within 50-500 kb of other breakpoints associated with orofacial clefting, in agreement with the assignment of such a locus in 6p24. The chromosome 9 translocation breakpoint was identified to be between D9S156 and D9S157 in 9p23-p22, a region implicated in the 9p deletion syndrome.     

Characterization of complex chromosomal rearrangements by targeted capture and next-generation sequencing

Translocations are a common class of chromosomal aberrations and can cause disease by physically disrupting genes or altering their regulatory environment. Some translocations, apparently balanced at the microscopic level, include deletions, duplications, insertions, or inversions at the molecular level. Traditionally, chromosomal rearrangements have been investigated with a conventional banded karyotype followed by arduous positional cloning projects. More recently, molecular cytogenetic approaches using fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH), or whole-genome SNP genotyping together with molecular methods such as inverse PCR and quantitative PCR have allowed more precise evaluation of the breakpoints. These methods suffer, however, from being experimentally intensive and time-consuming and of less than single base pair resolution. Here we describe targeted breakpoint capture followed by next-generation sequencing (TBCS) as a new approach to the general problem of determining the precise structural characterization of translocation breakpoints and related chromosomal aberrations. We tested this approach in three patients with complex chromosomal translocations: The first had craniofacial abnormalities and an apparently balanced t(2;3)(p15;q12) translocation; the second has cleidocranial dysplasia (OMIM 119600) associated with a t(2;6)(q22;p12.3) translocation and a breakpoint in RUNX2 on chromosome 6p; and the third has acampomelic campomelic dysplasia (OMIM 114290) associated with a t(5;17)(q23.2;q24) translocation, with a breakpoint upstream of SOX9 on chromosome 17q. Preliminary studies indicated complex rearrangements in patients 1 and 3 with a total of 10 predicted breakpoints in the three patients. By using TBCS, we quickly and precisely defined eight of the 10 breakpoints.     

Chromosome 18 breakpoint in t(11;18)(q21;q21) translocation associated with MALT lymphoma is proximal to BCL2 and distal to DCC

The t(11;18)(q21;q21) translocation has recently been identified as a recurring chromosomal abnormality in a subset of extranodal marginal zone B‐cell lymphoma, a low‐grade lymphoma of mucosa‐associated lymphoid tissue (MALT). Neither the 11q21 nor the 18q21 breakpoints have been characterized by molecular genetic analysis. As a prelude to isolation of the gene(s) involved in this translocation, we have mapped the 18q21 breakpoint region by fluorescence in situ hybridization (FISH) of YAC and PAC clones. We mapped 37 YACs assigned to a 29‐cM region within the chromosomal band 18q21. Using nine of these YACs in single‐ and/or dual‐color FISH to analyze three cases of MALT lymphomas with the t(11;18)(q21;q21) translocation, we localized the breakpoints within a 1.6‐Mb nonchimeric YAC (938E1). This YAC is useful for the detection of the translocation in metaphase and in interphase cells. A nonchimeric YAC contig of an 8‐cM region around the breakpoint comprising nine YACs and a PAC contig of YAC 938E1 were constructed, which enabled the refinement of the breakpoint region in the proximal region of the YAC within a <820‐kb segment. This breakpoint is proximal to the BCL2 locus and distal to DCC and DPC4 loci in chromosomal band 18q21. Genes Chromosomes Cancer 24:156–159, 1999. © 1999 Wiley‐Liss, Inc.     

t(1;18)(q32.1;q22.1) associated with genitourinary malformations

We report a male infant who has impaired penile development, hypospadias, and mild developmental delay with a 46,XY,t(1;18)(q32.1;q22.1) karyotype. Fluorescent in situ hybridization (FISH) was performed to more precisely map the translocation breakpoint. The translocation breakpoint maps to a region that has been implicated in genitourinary malformations in the 18q- syndrome. This case report suggests that a gene involved in genitourinary development maps at or near the chromosome 18 translocation breakpoint.     

Acampomelic campomelic syndrome and sex reversal associated with de novo t(12;17) translocation

The association of rare chromosomal rearrangements involving a specific 17q breakpoint with campomelic syndrome (CMPS) and or sex reversal (SR) has led to an assignment of the CMPS1 SRA1 locus to 17q24.3→q25.1. We describe a patient with multiple anomalies and SR, who had a de novo t(12;17) translocation. The phenotype was consistent with that of CMPS except for the lack of lower limb bowing and talipes equinovarus. Chromosome painting indicated that the breakpoints appeared to have occurred at 12q21.32 and 17q24.3 or q25.1. This study suggests that acampomelic CMPD with SR represents a variant of the CMPS1/SRA1 locus disorder. We emphasize the likelihood that CMPS may be a contiguous gene syndrome. © 1995 Wiley-Liss, Inc.     

Interphase fluorescence in situ hybridization for detection of 8q24/MYC breakpoints on routine histologic sections: validation in Burkitt lymphomas from three geographic regions

A chromosomal translocation involving the MYC gene is characteristic of Burkitt lymphoma (BL) and represents a molecular disease marker with diagnostic and clinical implications. The detection of MYC breakpoints is hampered by technical problems, including the distribution of the breakpoints over a very large genomic region of approximately 1,000 kb. In this article, we report on the testing and validation of a segregation fluorescence in situ hybridization (FISH) assay for MYC breakpoints on a large series of BLs. A contig of overlapping genomic clones was generated, and two probe sets flanking the MYC gene were selected. Both probe sets were tested in an interphase FISH segregation assay on 8 B-cell lymphoma cell lines and 32 lymphoma samples with proved 8q24/MYC abnormalities and validated in 47 BLs from The Netherlands, Brazil, and Uganda. MYC translocation breakpoints were identified in 98% of the tumors of the test series and in 89% of the cases of the validation series. In 89% of all positive samples, the breakpoints were located between 190 kb 5' and 50 kb 3' of MYC. Nine cases had more distant breakpoints, and in one patient an insertion of MYC into the IGH region was detected. In two of the three BLs lacking CD10 expression, no breakpoint could be detected, suggesting that CD10 is a discriminative marker of BL. We did not find consistent differences between BL and atypical BL in incidence of an MYC breakpoint.     

Evidence to exclude SOX9 as a candidate gene for XY sex reversal without skeletal malformation.

The skeletal malformation syndrome campomelic dysplasia (CMD1) is caused by mutations within the SOX9 gene or chromosomal rearrangement breakpoints outside SOX9. Approximately three quarters of cases of CMD1 in XY subjects show complete or partial sex reversal. As some mutations cause CMD1 alone and others cause CMD1 and sex reversal, it is conceivable that some mutations might cause sex reversal in the absence of CMD1. In this study, we have investigated this possibility by screening the entire coding region of SOX9 in 30 patients with a spectrum of XY sex reversal phenotypes. No mutations were identified, suggesting that SOX9 should not be considered a candidate gene for XY sex reversal without skeletal malformation.     

Acampomelic campomelic syndrome

Campomelic syndrome (or campomelic dysostosis, CD; MIM *114290) is an autosomal dominant skeletal malformation syndrome characterized by shortness and bowing of long bones, especially of the lower limbs. Additional radiological and clinical findings are 11 pairs of ribs and a bell‐shaped thorax, hypoplastic scapulae, narrow iliac wings, non‐mineralized thoracic pedicles, clubbed feet, Robin sequence, typical facial anomalies and tracheomalacia. The disorder is frequently lethal due to respiratory distress. Sex reversal occurs in most patients with an XY karyotype. CD is caused by heterozygous mutations in the SOX9 gene, an SRY‐related gene at 17q24.3–q25.1 with pleiotropic effects on the skeletal and genital systems. In addition, cases with chromosomal rearrangements involving 17q have been described that are most likely caused by disturbing one or more cis‐regulatory elements from an extended control region. Campomelia (bowed limbs) is seen in most but not all patients, defining a so‐called acampomelic campomelic dysostosis (ACD). Half of the CD cases with 17q rearrangements have no or mild campomelia. Furthermore, campomelia is absent or only mildly present in a small subgroup of cases with a normal karyotype. We present a chromosomally normal boy with ACD and his clinical follow‐up up to the age of 2 years, in whom a heterozygous SOX9 missense mutation (H165Y) was identified. A SOX9 missense mutation was published in two other patients with ACD. Although up to now a general genotype‐phenotype correlation could not be established for CD, a correlation emerges for the ACD variant that needs further confirmation. © 2001 Wiley‐Liss, Inc.     

Molecular characterization of the breakpoint region associated with a constitutional t(2;15)(q34;q26) in a patient with multiple myeloma

The molecular cloning of the translocation breakpoints from constitutional chromosome rearrangements in patients with a variety of human diseases has consistently led to the isolation of genes important in the development of the phenotype. We used fluorescence in situ hybridization (FISH) to analyze the breakpoint region of a constitutional chromosome translocation involving regions 2q34 and 15q26 observed in a patient with multiple myeloma (MM), a malignant disorder of plasma cells secreting monoclonal immunoglobulin. FISH analysis of this rearrangement showed that the chromosome 2-specific yeast artificial chromosome (YAC) 914E7 and the chromosome 15-specific YAC 757H6 span the translocation breakpoints, respectively. In order to characterize the location of the breakpoints further, somatic cell hybrids were constructed between mouse NIH3T3 cells and t(2;15)-bearing lymphoblastoid cells. Using these somatic cell hybrids, we have shown that the breakpoint on chromosome 2 lies between D2S3007 and D2S3004 and the chromosome 15 breakpoint lies between D15S107 and WI5967 (D15S836). YAC fragmentation has been used to define a 350 kb region containing the 15q26 breakpoint.     

Localisation of a 10q breakpoint within the PAX2 gene in a patient with a de novo t(10;13) translocation and optic nerve coloboma-renal disease.

We describe a 5 year old boy with a de novo t(10;13) translocation and optic nerve coloboma-renal disease (ONCR). On the basis of GTG banding analysis of prometaphase chromosomes, the patient's karyotype was interpreted as either 46,XY,t(10;13)(q24.3;q12.3) or t(10;13) (q25.2;q14.1). Fluorescence in situ hybridisation (FISH) studies using a YAC clone containing the PAX2 gene and YAC clones adjoining FRA10B at 10q25.2 showed that the 10q breakpoint had occurred just within the PAX2 gene and was proximal to FRA10B. These FISH results suggest that the translocation causes a disruption of the PAX2 gene and leads to ONCR, in agreement with the recent reports of PAX2 mutations in two unrelated families with ONCR. Furthermore, we refined the regional mapping of the human PAX2 gene to the junction of bands 10q24.3 and 10q25.1.