Atypical deletion of 22q11.2: Detection using the FISH TBX1 probe and molecular characterization with high-density SNP arrays

Despite the heterogeneous clinical presentations, the majority of patients with 22q11.2 deletion syndrome (22q11.2 DS) have either a common recurrent 3 Mb deletion or a less common, 1.5 Mb nested deletion, with breakpoint sites in flanking low-copy repeats (LCR) sequences. Only a small number of atypical deletions have been reported and precisely defined. Haploinsufficiency of the TBX1 gene was determined to be the likely cause of 22q11.2 DS. The diagnostic procedure usually used is FISH using commercially probes (N25 or TUPLE1). However, this test does not contain TBX1, and fails to detect deletions that are either proximal or distal to the FISH probes. Here, we report on two patients with clinical features suggestive of 22q11.2 DS, a male infant with facial dysmorphia, pulmonary atresia, ventricular septal defect, neonatal hypocalcemia, and his affected mother, with facial dysmorphia, learning disabilities, and hypernasal speech. They were tested negative for 22q11.2 DS using N25 or TUPLE1 probes, but were shown deleted for a probe containing TBX1. Delineation of the deletion was performed using high-density SNP arrays (Illumina, 370K). This atypical deletion was spanning 1.89 Mb. The distal breakpoint resided in LCR-D, sharing the same distal breakpoint with the 3 Mb common deletion. The proximal breakpoint was located 105 kb telomeric to TUPLE1, representing a new breakpoint variant that does not correspond to known LCRs of 22q11.2. We conclude that FISH with the TBX1 probe is an accurate diagnostic tool for 22q11.2 DS, with a higher sensitivity than FISH using standard probes, detecting all but the rarest deletions, greatly reducing the false negative rate.     

Array-based comparative genomic hybridization facilitates identification of breakpoints of a novel der(1)t(1;18)(p36.3;q23)dn in a child presenting with mental retardation

Monosomy of distal 1p36 represents the most common terminal deletion in humans and results in one of the most frequently diagnosed mental retardation syndromes. This deletion is considered a contiguous gene deletion syndrome, and has been shown to vary in deletion sizes that contribute to the spectrum of phenotypic anomalies seen in patients with monosomy 1p36. We report on an 8-year-old female with characteristics of the monosomy 1p36 syndrome who demonstrated a novel der(1)t(1;18)(p36.3;q23). Initial G-banded karyotype analysis revealed a deleted chromosome 1, with a breakpoint within 1p36.3. Subsequent FISH and array-based comparative genomic hybridization not only confirmed and partially characterized the deletion of chromosome 1p36.3, but also uncovered distal trisomy for 18q23. In this patient, the duplicated 18q23 is translocated onto the deleted 1p36.3 region, suggesting telomere capture. Molecular characterization of this novel der(1)t(1;18)(p36.3;q23), guided by our clinical array-comparative genomic hybridization, demonstrated a 3.2 Mb terminal deletion of chromosome 1p36.3 and a 200 kb duplication of 18q23 onto the deleted 1p36.3, presumably stabilizing the deleted chromosome 1. DNA sequence analysis around the breakpoints demonstrated no homology, and therefore this telomere capture of distal 18q is apparently the result of a non-homologous recombination. Partial trisomy for 18q23 has not been previously reported. The importance of mapping the breakpoints of all balanced and unbalanced translocations found in the clinical laboratory, when phenotypic abnormalities are found, is discussed.     

Molecular characterization of a novel, de novo, cryptic interstitial deletion on 19p13.3 in a child with a cutis aplasia and multiple congenital anomalies

We report on a de novo constitutional deletion within G-band region 19p13.3 in a girl with cutis aplasia of the scalp, facial anomalies, structural heart abnormalities, hypotonia, mild mental retardation and conductive hearing loss which we characterized with chromosomal microarray, fluorescence in situ hybridization (FISH), and SNP analyses. Initial microarray analysis revealed a 6-BAC-clone deletion covering an approximately 1.612?Mb region within 19p13.3. Subsequent BAC FISH studies delineated the proximal deletion breakpoint to within BAC clone RP11-125C3 and the distal deletion breakpoint to within BAC clone RP11-648B14. SNP analysis showed the deletion to be of paternal origin and further refined its distal breakpoint to within a 20?kb region between rs11666694 and novel SNP2 that we identified at g.2,924,845, and its proximal deletion breakpoint to within a 22?kb region between rs35280644 and rs262562. Accordingly, the size of the deletion was revised to 1.89-1.932?Mb in length. We identified many Alu, L1, and L2 repeats, as well as SINE and LINE sequences at both deletion breakpoints. We found the deletion to encompass 71 genes, two of which appear to be good candidates for the patient's observed craniofacial and cardiac anomalies: guanine nucleotide binding protein (G protein), alpha 11 (Gq class)(GNA11), and Transducin-like Enhancer of Split 2 (E(sp1) homolog, Drosophila)(TLE2).     

Submicroscopic deletions at 22q11.2: Variability of the clinical picture and delineation of a commonly deleted region

DiGeorge anomaly (DGA) and velo-cardiofacial syndrome (VCFS) are frequently associated with monosomy of chromosome region 22q11. Most patients have a submicroscopic deletion, recently estimated to be at least 1–2 Mb. It is not clear whether individuals who present with only some of the features of these conditions have the deletion, and if so, whether the size of the deletion varies from those with more classic phenotypes. We have used fluorescence in situ hybridization (FISH) to assess the deletion status of 85 individuals referred to us for molecular analysis, with a wide range of DGA-like or VCFS-like clinical features. The test probe used was the cosmid sc11.1, which detects two loci about 2 Mb apart in 22q11.2. Twenty-four patients carried the deletion. Of the deleted patients, most had classic DGA or VCFS phenotypes, but 6 deleted patients had mild phenotypes, including 2 with minor facial anomalies and velopharyngeal incompetence as the only presenting signs. Despite the great phenotypic variability among the deleted patients, none had a deletion smaller than the 2-Mb region defined by sc11.1 Smaller deletions were not detected in patients with particularly suggestive phenotypes who were not deleted for sc11.1, even when tested with two other probes from the DGA/VCFS region. © 1995 Wiley-Liss, Inc.     

FISH-mapping of telomeric 14q32 deletions: search for the cause of seizures

Ring chromosome 14 is a rare cytogenetic disorder. Individuals with r(14) generally have developmental delay and seizures. Other features include hypotonia, microcephaly, mild facial dysmorphism, and retinal pigmentation. Most of these features are also found in patients with linear terminal deletions of chromosome 14, except for seizures and retinal abnormalities. The objective of the study was to determine if deletion of a specific chromosome region is a possible explanation for the occurrence of seizures in patients with ring chromosome 14. Patients diagnosed either with r(14) (six patients) or a deletion of distal 14q (three patients) were analyzed by FISH (fluorescence in situ hybridization) with BAC probes. We observed differences in the size of deletions in the studied group. In two r(14) patients, we did not detect any deletion; the four other patients had deletions of various sizes, ranging from 0.8 Mb to 5 Mb. Two linear deletions were 3.2 Mb and 5.3 Mb in length, respectively; the third case had an interstitial deletion that did not overlap with the others. The deleted regions in ring chromosomes showed overlap with those in the two linear terminal deletions. We conclude that there is unlikely to be a specific deleted locus in 14q32.3 that predisposes r(14) patients to seizures or retinal pigmentation. The cause is probably related to the formation of the ring itself and the effect this may have on local chromatin structure.     

Two novel translocation breakpoints upstream of SOX9 define borders of the proximal and distal breakpoint cluster region in campomelic dysplasia

The semilethal skeletal malformation syndrome campomelic dysplasia (CD) with or without XY sex reversal is caused by mutations within the SOX9 gene on 17q24.3 or by chromosomal aberrations (translocations, inversions or deletions) with breakpoints outside the SOX9 coding region. The previously published CD translocation breakpoints upstream of SOX9 fall into two clusters: a proximal cluster with breakpoints between 50-300 kb and a distal cluster with breakpoints between 899-932 kb. Here, we present clinical, cytogenetic and molecular data from two novel CD translocation cases. Case 1 with karyotype 46,XY,t(1;17)(q42.1;q24.3) has characteristic symptoms of CD, including mild tibial bowing, cryptorchidism and hypospadias. By standard fluorescence in situ hybridization (FISH) and by high-resolution fiber FISH, the 17q breakpoint was mapped 375 kb from SOX9, defining the centromeric border of the proximal breakpoint cluster region. Case 2 with karyotype 46,X,t(Y;17)(q11.2;q24.3) has the acampomelic form of CD and complete XY sex reversal. By FISH and somatic cell hybrid analysis, the 17q breakpoint was mapped 789 kb from SOX9, defining the telomeric border of the distal breakpoint cluster region. We discuss the structure of the 1 Mb cis-control region upstream of SOX9 and the correlation between the position of the 14 mapped translocation breakpoints with respect to disease severity and XY sex reversal.     

A 1-Mb critical region in six patients with 9q34.3 terminal deletion syndrome

Patients with 9q34.3 terminal deletion usually show a clinically recognizable phenotype characterized by specific facial features (microcephaly, flat face, arched eyebrows, hypertelorism, short nose, anteverted nostrils, carp mouth and protruding tongue) in combination with severe mental retardation, hypotonia, and other anomalies. We analyzed six unrelated patients with a various 9q34.3 terminal deletion. While having different-sized 9q34.3 deletions, all of these patients shared several distinctive anomalies. These anomalies are likely to arise from a commonly deleted region at distal 9q34.3. Fluorescence in situ hybridization (FISH) analysis using a dozen BAC clones mapped at the 9q34.13-q34.3 region defined the shortest region of deletion overlap (SRO) as a 1-Mb segment proximal to 9qter containing eight known genes. Possible candidate genes delineating specific phenotypes of the 9q34.3 terminal deletion syndrome are discussed.     

Subtelomeric 6.7 Mb trisomy 10p and 5.6 Mb monosomy 21q detected by FISH and array-CGH in three related patients

Cryptic subtelomeric chromosomal aberrations are responsible for 5-10% of moderate/severe and 1% of mild intellectual disability. Unbalanced subtelomeric chromosomal rearrangements result in variable phenotypes which seem to be highly influenced by both the size of the duplication/deletion and the chromosomes involved in the translocation. We report on three related patients with moderate intellectual disability, language delay, hypotonia, facial dysmorphism, cardiac anomalies, scoliosis, and kyphosis in whom a familial (maternal) unbalanced submicroscopic translocation was found by subtelomeric fluorescence in situ hybridization (FISH). This rearrangement resulted in a partial trisomy 10pter and partial monosomy 21qter. The karyotype was 46,XY.ish der(21)t(10;21)(p14;q22.2). Confirmation of a 6.7?Mb size distal duplication of the p15.3-14 region of chromosome 10 and a 5.6?Mb distal deletion of the q22.2-22.3 region of chromosome 21 was obtained by array-CGH. To our best knowledge, such a composition of subtelomeric unbalanced translocations has not yet been published. Detection of this aberration in successive pregnancies of carrier members of the family by prenatal FISH could prevent the recurrence of the disease. Furthermore, detection of the rearrangements and identification of genes located in the chromosomal regions involved might be of interest.     

Array-CGH analysis and clinical description of 2q37.3 de novo subtelomeric deletion

We report on a 13-year-old girl with normal karyotype and a de novo cryptic terminal deletion of chromosome 2q, detected by subtelomeric FISH analysis. Further investigation with array-CGH analysis using the 1Mb resolution Spectral Chip 2600 (Spectral Genomics) confirmed the deletion and also showed a deletion of four additional clones. No other abnormalities were detected by array-CGH. FISH studies using 8 BAC-probes were performed for fine mapping of the deletion and confirmed the array results. FISH analysis showed that the deletion breakpoint lies between clones RP11-84G18 and RP11-83N2 (physical distance between clones 0.36Mb) and extends to the telomere. The size of the deletion was estimated to be about 6.4-6.7Mb. Clinical findings include: developmental delay, severe behavioural disturbances, growth-pubertal retardation, congenital conductive mild hearing loss, growth hormone deficiency, compensate hypothyroidism, dysmorphic facial features, excessive joint hypermobility, brachymetaphalangy, abnormal dermatoglyphics and a history of neonatal laryngomalacia, hypotonia and umbilical hernia. The phenotype of our patient is in keeping with those of the literature, with the exception of cardiovascular, urogenital, neurological anomalies and eczema, which were not observed. The report of the clinical and molecular presentation of similar cases will allow accurate phenotype-genotype correlation and proper genetic counseling of the family.     

Deletion size characterization of der(9) deletions in Philadelphia-positive chronic myeloid leukemia

About 95% of the CML patients with chronic myeloid leukemia (CML) have a Philadelphia chromosome resulting from a reciprocal translocation between bands 9q34 and 22q11.2 that juxtaposes the 3′ region of the ABL gene to the 5′ region of BCR. Over the past few years, submicroscopic deletions due to the loss of sequences proximal to chromosome 9 breakpoint or distal to chromosome 22 breakpoint have been found using fluorescence in situ hybridization (FISH). Among 150 CML bone marrow samples analyzed by molecular cytogenetics in our laboratory, 11 had a der(9) deletion detectable by FISH (deletion of the 5′ABL region and 3′BCR region in 10 samples and deletion of the 5′ABL region solely in 1 sample). To delineate the size of the deletions, FISH mapping was performed using 22 bacterial artificial chromosomes (BACs), 11 on either side of the breakpoints, the mean distance between BACs being 0.5 Mb. The deletion size of the 5′ABL region on the der(9) extended from 2 to 5 Mb, the minimal deletion size being localized between BACs RP11-101E3 and RP11-83J21. In two patients, the deletion size of the 3′BCR region was about 500 kb (between RP11-80O7 and RP11-681C06). The poor prognosis associated with these deletions was postulated by several workers to be explained by haploinsufficiency of a tumor suppressor gene. However, in our cases, the hypothetical deletion of one or more tumor suppressor genes is not sufficient to explain the poor response to interferon therapy, but the good response to imatinib treatment. We think that there could be one or more genes coding for interferon receptors or for proteins acting directly or indirectly with these receptors in the deleted regions.     

Two cases of partial trisomy 21 (pter-q22.1) without the major features of Down syndrome

We report two cases of partial trisomy 21 with clinical features distinct from Down syndrome (DS). These patients presented with moderate mental retardation and short stature, but the typical facial appearance of DS was not observed. Each patient had a similarly sized extra chromosome 21. We performed FISH analysis to examine whether deletions of reported approximately 5 Mb DS critical region (DSCR) might be associated with unusual clinical features in these cases. The results showed that each of their extra chromosomes 21 contained a distal part of chromosome 3p or 14q at the telomeric region of chromosome 21q. The translocation breakpoint of 21q for each patient was located on the centromeric side of DSCR (DSCR was deleted) and the sizes of partial trisomy 21 in respective patients are approximately 34.5 (21pter-q22.12) and approximately 33.0 Mb (21pter-q22.11). In one patient, the additional region of the short arm of chromosome 3 was 3pter-p26.1 from maternal origin, measuring approximately 9 Mb in size. The second patient had an extra 14q32.1-qter of maternal origin, measuring approximately 14 Mb in size. These are one of the shortest partial distal trisomy among reported cases. Taken together, two patients with partial trisomy 21 lack all of DSCR on 21q22, and their distinct clinical features are likely caused by the genes located at 21pter-q22.1 and the distal part of chromosome 3p or 14q.     

Molecular cytogenetic characterization of an insertional translocation, ins(6;7)(p25;q33q34): deletion/duplication of 7q33-34 and clinical correlations

A balanced insertional translocation between chromosomes 6 and 7, ins(6;7)(p25;q33q34) has been extensively investigated. The insertional translocation was found in several members of a three-generation family, where some were healthy balanced carriers while others had clinical symptoms due to deletion or duplication of 7q33-34. The deleted/duplicated segment could only be detected using high resolution banding and fluorescent in situ hybridization. A number of BAC/PAC clones located on chromosome 6 and 7 were used to characterize the breakpoint regions in detail and to determine the size of the deletion, which was 7.6 Mb, containing up to 68 genes. However, the insert on chromosome 6 was only 7.4 Mb, due to a deletion of 227 kb at the distal breakpoint on 7q. This small deletion was also found in the "balanced" carriers, and although the chromosome segment contains at least eight genes, none of the carriers seem to be affected by haploinsufficiency, since the phenotype is apparently normal. This is the first detailed characterization and phenotype correlation of such a deletion/duplication of distal 7q.     

Clinical and molecular-cytogenetic evaluation of a family with partial Jacobsen syndrome without thrombocytopenia caused by an approximately 5 Mb deletion del(11)(q24.3)

Clinical manifestations of Jacobsen syndrome (JBS) depend on the size of the 11qter deletion, which usually varies between approximately 7 and 20 Mb. Typical JBS features include developmental delay/mental retardation, short stature, congenital heart defects, thrombocytopenia, and characteristic dysmorphic facial features. We report on a family in which a 4-year-old girl as well as her mother and maternal uncle present with subtle features of JBS. Notably, neither thrombocytopenia nor congenital anomalies were detected in this family. Cytogenetic analyses revealed normal karyotypes. Using fluorescence in situ hybridization (FISH) and whole-genome oligonucleotide array CGH analyses, we identified an approximately 5 Mb deletion of the terminal part of chromosome 11q in all the three affected family members. The deletion breakpoint was mapped between 129,511,419 and 129,519,794 bp. This is the smallest deletion reported in a JBS patient. Interestingly, the FLI1 (friend leukemia virus integration 1) hematopoiesis factor gene located approximately 6.5 Mb from 11qter and usually deleted in patients with JBS, is intact. Our data support previous hypotheses that FLI1 haploinsufficiency is responsible for thrombocytopenia in patients with JBS.     

A familial inverted duplication/deletion of 2p25.1-25.3 provides new clues on the genesis of inverted duplications

We studied a family in which the same 10 Mb inverted duplication of 2p25.3-p25.1 segregates in two children and their father, all showing a trisomy phenotype. As FISH analysis demonstrated that the duplication was inverted, we suspected that a contiguous terminal deletion was also present, according to the classical inv dup del type of rearrangements. Although FISH with 2p and 2q subtelomeric probes gave normal results, 100 kb resolution array-C/GH (aCGH) showed that, beside the duplication, a 273 kb deletion was also present. The presence of a single-copy region between the deleted and duplicated regions was further suspected through high-resolution aCGH analysis (approximately 20 kb), although only one informative spot having a normal log ratio was detected. The precise structure of the rearrangement was re-defined by real-time PCR and breakpoint cloning, demonstrating the presence of a 2680 bp single-copy sequence between deleted and duplicated regions and the involvement of a simple repeat with the potential for forming a non-B DNA structure. The rearrangement was not mediated by segmental duplications or short inverted repeats, and the double-strand break might have been repaired by non-homologous end joining or microhomology-mediated intrastrand repair. These data highlight the fact that concomitant deletions associated with inverted duplications are very likely to be more frequent than classical cytogenetic methods alone have been able to demonstrate. The phenotypic effects of the trisomy and of the terminal 2p deletion are discussed.     

De novo subtelomeric deletion of 15q associated with satellite translocation in a child with developmental delay and severe growth retardation

We report on a case of satellited 15q with subtelomeric deletion in a girl with delayed development and severe growth retardation. The patient also has a triangular face, downturned angles of the mouth, micrognathia, and minor limb malformations including mild talipes equinovarus, genu recurvatum, and increased dorsiflexion of both limbs. Cytogenetic analysis using standard GTG banding showed a female karyotype with a satellited-like structure at the distal long arm of one chromosome 15. Silver staining of the nucleolar organizing region (AgNOR) confirmed the presence of a satellite DNA translocation at the lesion. Analysis using fluorescent in situ hybridization (FISH) detected a subtelomeric deletion of the terminal 15q. Additional molecular analysis using microsatellite markers along the long arm of chromosome 15 defined a maximally deleted region at approximately 4.7 Mb. Haploinsufficiency of the IGF1R gene expression is thought to be the cause of growth delay in all 15q terminal deletion including our patient.     

An interstitial,apparently-balanced chromosomal insertion in the etiology of Langer–Giedion syndrome in an Asian family

Langer–Giedion syndrome (LGS; MIM 150230), also called trichorhinophalangeal syndrome type II (TRPS2), is a contiguous gene syndrome caused by a one-copy deletion in the chromosome 8q23-q24 region, spanning the genes TRPS1 and EXT1. We identified an LGS family with two affected and two unaffected siblings from unaffected parents. To investigate the etiology of recurrence of LGS in this family, array CGH was performed on all family members. We identified a 7.29 Mb interstitial deletion at chromosome region 8q23-q24 in the two affected siblings, but no such deletion in the unaffected family members. However, the mother and one of the two unaffected siblings carried a 1.29 Mb deletion at chromosome region 8q24.1, sharing the distal breakpoint with the larger deleted segment found in the affected siblings. Another unaffected sibling had a 6.0 Mb duplication, sharing the proximal breakpoint of the deletion in the affected siblings. Karyotypic and FISH analyses in the unaffected mother revealed an insertional translocation of 8q23-q24 genomic material into chromosome 13: 46,XX,ins(13;8)(q33;q23q24). This insertional translocation in the mother results in the recurrence of LGS in this family, highlighting the importance of submicroscopic rearrangements in the genetic counseling for LGS.     

Chromosome aberrations involving 10q22: report of three overlapping interstitial deletions and a balanced translocation disrupting C10orf11

Interstitial deletions of chromosome band 10q22 are rare. We report on the characterization of three overlapping de novo 10q22 deletions by high-resolution array comparative genomic hybridization in three unrelated patients. Patient 1 had a 7.9 Mb deletion in 10q21.3–q22.2 and suffered from severe feeding problems, facial dysmorphisms and profound mental retardation. Patients 2 and 3 had nearly identical deletions of 3.2 and 3.6 Mb, the proximal breakpoints of which were located at an identical low-copy repeat. Both patients were mentally retarded; patient 3 also suffered from growth retardation and hypotonia. We also report on the results of breakpoint analysis by array painting in a mentally retarded patient with a balanced chromosome translocation 46,XY,t(10;13)(q22;p13)dn. The breakpoint in 10q22 was found to disrupt C10orf11, a brain-expressed gene in the common deleted interval of patients 1–3. This finding suggests that haploinsufficiency of C10orf11 contributes to the cognitive defects in 10q22 deletion patients.     

Familial 14-Mb deletion at 21q11.2-q21.3 and variable phenotypic expression

We report a familial case with a proximal interstitial deletion of chromosome 21q [del(21q)]. Although the mother in the family was phenotypically normal, her first child was affected with both sensorineural hearing loss and moderate mental retardation, and the second affected child had mild mental retardation but not sensorineural hearing loss. We determined breakpoints of the del(21q) in the mother and her two affected children by fluorescence in situ hybridization analysis using 45 DNA clones and the molecular analysis using 21 DNA markers. The proximal breakpoint of the del(21q) was located at a region between 0.33 Mb and 0.46 Mb distal to the centromere, and the distal breakpoint was at a region between 14.6 Mb and 14.9 Mb. The finding indicates that the three individuals had an approximate 14-Mb deletion within 21q11.2-q21.3. Molecular analysis showed that both affected children shared the same maternal haplotype of their del(21q), but a crossover was detected in the paternally inherited normal chromosome 21. These data suggest that unmasking of deleterious genes on the paternally derived chromosome 21 of the two children as a result of the deletion may affect the extent of their mental retardation and/or sensorineural hearing loss. Usher syndrome 1E may be a candidate disease locus related to the sensorineural hearing loss of the first child.