Application of a comprehensive subtelomere array in clinical diagnosis of mental retardation

In 2-8% of patients with mental retardation, small copy number changes in the subtelomeric region are thought to be the underlying cause. As detection of these genomic rearrangements is labour intensive using FISH, we constructed and validated a high-density BAC/PAC array covering the first 5 Mb of all subtelomeric regions and applied it in our routine screening of patients with idiopathic mental retardation for submicroscopic telomeric rearrangements. The present study shows the efficiency of this comprehensive subtelomere array in detecting terminal deletions and duplications but also small interstitial subtelomeric rearrangements, starting from small amounts of DNA. With our array, the size of the affected segments, at least those smaller than 5 Mb, can be determined simultaneously in the same experiment. In the first 100 patient samples analysed in our diagnostic practice by the use of this comprehensive telomere array, we found three patients with deletions in 3p, 10q and 15q, respectively, four patients with duplications in 9p, 12p, 21q and Xp, respectively, and one patient with a del 6q/dup 16q. The patients with del 3p and 10q and dup 12p had interstitial rearrangements that would have been missed with techniques using one probe per subtelomeric region chosen close to the telomere.     

Cryptic telomere imbalance: a 15-year update

It has been 15 years since we proposed that assays of telomere integrity might reveal cryptic translocations and deletions as a significant cause of mental retardation (MR) in patients with normal G-banded karyotypes. Development of unique genomic probes adjacent to the subtelomeric repeats of each chromosome arm allowed multiplex FISH analyses that confirmed such cryptic telomeric imbalances in 3-6% of all unexplained MR. Although such "telomere FISH" analysis quickly became standard of care, limitations of this technology platform included a lack of information on the size and gene content of the deleted/duplicated segments and the failure to detect interstitial deletions not involving the most distal unique clone. The development of "molecular ruler" clone sets for every human telomere provided the foundation for accurate determination of size and gene content of each imbalance, as well as the detection of interstitial deletions within these regions. Array comparative genomic hybridization (aCGH) has emerged as a powerful technology to assess single copy changes (monosomy or trisomy) at targeted loci such as telomeres or across the whole genome. This technology now replaces multiplex FISH for the assessment of telomere integrity in unexplained MR and has the advantage of efficiently determining the size and gene content of the imbalance, as well as detecting interstitial deletions near telomeres or anywhere else in the genome covered by the array design. The application of aCGH in several studies of unexplained MR has confirmed that telomere imbalances are overrepresented compared to "average" chromosomal regions, although this is likely due to random chromosome breakage rather than specific molecular mechanisms associated with the genomic architecture of human telomeres. Telomere imbalances are significantly larger than initially envisioned ( approximately 40% are >5 Mb in size), and indicate the analytic sensitivity of the G-banded karyotype is much lower than previously thought. Finally, experience with smaller benign variants compared to larger pathogenic imbalances at telomeres serves as a model for approaching whole-genome aCGH in a clinical setting.     

17p11.2p12 triplication and del(17)q11.2q12 in a severely affected child with dup(17)p11.2p12 syndrome

Multiple congenital anomalies/mental retardation syndromes due to genomic rearrangements involving chromosome 17p11.2 include deletion resulting in Smith-Magenis syndrome and a reciprocal duplication of the same region resulting in the 17p11.2 duplication syndrome. We present the clinical and molecular analysis of an 8-year-old male with a dup(17p11.2p12) who was evaluated for unusual severity of the phenotype. Fluorescent in situ hybridization (FISH) analysis not only confirmed the 17p duplication but also identified an approximately 25% mosaicism for tetrasomy 17p11.2p12. Whole-genome array comparative genomic hybridization (aCGH) was performed to identify other genomic rearrangements possibly contributing to the severe phenotype and the unusual features in the patient. The 17p duplication was determined by FISH and aCGH to encompass approximately 7.5 Mb, from COX10 to KCNJ12. An approximately 830 Kb deletion of 17q11.2q12, including exon 1 of an amiloride-sensitive cation channel neuronal gene, ACCN1, was also identified by aCGH; breakpoints of the deletion were confirmed by FISH. Sequencing the non-deleted allele of ACCN1 did not show any mutations. Western analysis of human tissue-specific proteins revealed that ACCN1 is expressed not only in the brain as previously reported but also in all tissues examined, including heart, liver, kidneys, and spleen. The large-sized 17p11.2p12 duplication, partial triplication of the same region, and the 17q11.2q12 deletion create a complex chromosome 17 rearrangement that has not been previously identified. This is the first case of triplication reported for this chromosome. Our study emphasizes the utility of whole-genome analysis for known cases with deletion/duplication syndromes with unusual or severe phenotypes.     

Screening for subtelomeric rearrangements using automated fluorescent genotyping of microsatellite markers: a Lebanese study

A screening for submicroscopic rearrangements using specific polymorphic microsatellite markers from the subtelomeric regions of all chromosome arms was performed in 34 independent Lebanese families, including 45 patients with idiopathic mental retardation plus additional features. Five cryptic rearrangements were found in five different families, but subsequent FISH studies confirmed only three of those, showing a proportion of nearly 9% of subtelomeric rearrangements in our population. Two patients presented a de novo deletion from paternal origin, one involving telomere 3p, and another telomere 7p. An unbalanced paternally inherited translocation was detected in two patients from the same family resulting in both trisomy for telomere 5q and monosomy for telomere 6p.     

Report of interstitial 22q13.1q13.2 microduplication in two siblings with distinctive dysmorphic features,heart defect and mental retardation

We present two siblings (a boy and a girl) with a submicroscopic 4 Mb duplication at 22q13.1q13.2. Both children manifested infantile hypotonia and delayed motor milestones, congenital heart defect, growth deficiency, and strikingly similar and distinctive craniofacial dysmorphism including brachycephaly, blepharophimosis, short broad-based nose and wide mouth with thin upper lip. The boy had also a submucous cleft palate. Both had fair skin and hair compared with their parents. Both had moderate mental retardation associated with a short attention span. A 4-Mb interstitial duplication at 22q13.1q13.2 was detected by whole genome microarray comparative genomic hybridisation (array CGH) in both children. The duplication was confirmed by fluorescence in situ hybridisation (FISH) analysis. Their parents had normal array CGH results. FISH analysis revealed that the father was a carrier of a balanced interchromosomal submicroscopic insertion of 22q13 into chromosome 11q23, explaining the unbalanced aberration detected in both children. This report narrows down the critical region at 22q13.1q13.2, which is associated with mental retardation, pre- and post-natal growth retardation, hippocampal malformation, psychiatric symptoms such as short attention span and facial dysmorphism including hypertelorism, epicanthal folds and low set/abnormal ears.     

Detection of a cryptic translocation in a family with mental retardation using FISH and telomere region-specific probes

Cryptic rearrangements involving the telomeres are thought to account for a substantial number of patients with unexplained mental retardation and multiple congenital anomalies, although the exact incidence of these rearrangements is still unclear. With the advent of chromosome-specific telomeric probes and the use of FISH (fluorescence in situ hybridization), it is now possible to identify submicroscopic rearrangements of the distal ends of chromosomes that may otherwise go undetected using conventional cytogenetic studies. We report on a 4 1/2 year-old girl with severe mental retardation and minor anomalies who inherited the unbalanced product of a cryptic translocation involving chromosomes 2 and 17 from her father. The family history was significant for early pregnancy losses, stillbirths, and mental retardation in many other family members, suggesting segregation of a familial translocation. This translocation was detected using chromosome-specific telomere FISH probes, and not visible using conventional cytogenetic methods. Collectively, this case and those previously reported clearly demonstrate the value of a systematic search for cryptic chromosome rearrangements in patients with unexplained mental retardation with previously reported normal chromosome studies; and in particular those with a family history of mental retardation, birth defects, or early pregnancy losses.     

Submicroscopic deletion 9(q34.3) and duplication 19(p13.3): identified by subtelomere specific FISH probes

Submicroscopic rearrangements involving chromosome ends are responsible for the unexplained mental retardation and multiple congenital anomalies observed in a number of patients. We have studied a patient with mental retardation, significant microcephaly, alopecia universalis, and other anomalies who carries an unbalanced segregant from a cryptic reciprocal translocation involving chromosomes 9 and 19. FISH studies using subtelomere specific probes revealed a derivative chromosome 9 in which the 9q subtelomeric sequence has been replaced by 19p subtelomeric sequence. As a result, the patient has partial monosomy 9q and partial trisomy 19p. The patient inherited the derivative 9 from his father, who carries a cryptic apparently balanced reciprocal translocation involving the terminal regions of 9q and 19p. This case is exceptional in that reports of rearrangements involving distal chromosome 9q and 19p are rare. This study demonstrates the utility of subtelomere specific FISH probes for detecting cryptic subtelomeric rearrangements in patients with idiopathic mental retardation and normal appearing karyotypes.     

Detection of a cryptic translocation in a family with mental retardation using FISH and telomere region‐specific probes

Cryptic rearrangements involving the telomeres are thought to account for a substantial number of patients with unexplained mental retardation and multiple congenital anomalies, although the exact incidence of these rearrangements is still unclear. With the advent of chromosome‐specific telomeric probes and the use of FISH (fluorescence in situ hybridization), it is now possible to identify submicroscopic rearrangements of the distal ends of chromosomes that may otherwise go undetected using conventional cytogenetic studies. We report on a 4½ year‐old girl with severe mental retardation and minor anomalies who inherited the unbalanced product of a cryptic translocation involving chromosomes 2 and 17 from her father. The family history was significant for early pregnancy losses, stillbirths, and mental retardation in many other family members, suggesting segregation of a familial translocation. This translocation was detected using chromosome‐specific telomere FISH probes, and not visible using conventional cytogenetic methods. Collectively, this case and those previously reported clearly demonstrate the value of a systematic search for cryptic chromosome rearrangements in patients with unexplained mental retardation with previously reported normal chromosome studies; and in particular those with a family history of mental retardation, birth defects, or early pregnancy losses. Am. J. Med. Genet. 92:250–255, 2000. © 2000 Wiley‐Liss, Inc.     

Identification of an unbalanced X-autosome translocation by array CGH in a boy with a syndromic form of chondrodysplasia punctata brachytelephalangic type

Screening of a large series of patients with unexplained mental retardation with a 1 Mb BAC array resulted in the detection of several cryptic chromosomal imbalances. In this paper we present the findings of array CGH screening in a 14-year-old boy with the brachytelephalangic type of chondrodysplasia punctata, mental retardation and obesity. On several occasions, cytogenetic analysis of this boy revealed a normal karyotype. Subsequent screening with array CGH resulted in the detection of a distal 9p trisomy and distal Xp nullisomy caused by an unbalanced X;9 translocation: 46,Y,der(X)t(X;9)(p22.32;p23). The identification of this de novo chromosomal rearrangement not only made accurate genetic counselling possible but also explained most of the phenotypic abnormalities observed in this patient. This study confirms the power of array CGH in the detection of subtle or submicroscopic chromosomal changes.     

采用多重连接探针扩增技术检测智力低下儿童的基因缺陷

目的 探讨原因不明智力低下儿童的发病与染色体亚端粒基因重组间的关系.方法 采用多重连接探针扩增(multiplex ligation-dependent probe amplification,MLPA)技术检测30名原因不明的综合征性智力低下患儿的染色体亚端粒区域.结果 检测到5例患儿存在染色体亚端粒的基因缺失或重复突变,分别为4p缺失,21q重复,10p重复、4p缺失,15p重复,3p重复、9p缺失.结论 不明原因智力低下儿童的发病与染色体亚端粒基因重组密切相关.MLPA技术可以作为一种高效、特异的方法对智能障碍儿童进行基因缺陷筛查.     

Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation (array-CGH)

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include growth and developmental delay and dysmorphism. Novel high resolution, whole genome technologies, such as array based comparative genomic hybridisation (array-CGH), improve the detection rate of submicroscopic chromosomal abnormalities allowing re-investigation of cases where conventional cytogenetic techniques, Spectral karyotyping (SKY), and FISH failed to detect abnormalities. We performed a high resolution genome-wide screening for submicroscopic chromosomal rearrangements using array-CGH on 41 children with idiopathic mental retardation (MR) and dysmorphic features. The commercially available microarray from Spectral Genomics contained 2600 BAC clones spaced at approximately 1 Mb intervals across the genome. Standard chromosome analysis (>450 bands per haploid genome) revealed no chromosomal rearrangements. In addition, multi-subtelomeric FISH screening in 30 cases and SKY in 11 patients did not detect any abnormality. Using array-CGH we detected chromosomal imbalances in four patients (9.8%) ranging in size from 2 to 14 Mb. Large scale copy number variations were frequently observed. Array-CGH has become an important tool for the detection of chromosome aberrations and has the potential to identify genes involved in developmental delay and dysmorphism. Moreover, the detection of genomic imbalances of clinical significance will increase knowledge of the human genome by performing genotype-phenotype correlation.     

A 7 Mb duplication at 22q13 in a girl with bipolar disorder and hippocampal malformation

We identified a duplication of 22q13.1-q13.2 in a 10-year-old girl and demonstrated that this duplication was the recombinant product of a maternal intrachromosomal insertion. Phenotypic characteristics included prominent forehead, small low-set ears, hypertelorism, epicanthal folds, small palpebral fissures, short philtrum, and syndactyly. MRI of the brain revealed high signal abnormalities in the periventricular white matter, a hypoplastic corpus callosum, under-rotated hippocampus on the left and atrophic hippocampus on the right. Since age 5, the child's behavior has shown cyclic maniacal episodes with severely disorganized mood and behavior. Psychiatric and cognitive assessment led to a diagnosis of bipolar disorder not otherwise specified, manic episodes, attention deficit hyperactivity disorder and moderate mental retardation. Array-CGH revealed an interstitial duplication of 6.9 Mb at chromosome 22q: dup(22)(q13.1q13.2). FISH using BAC clones confirmed the array-CGH results and demonstrated that the duplication was inverted. G-banding analysis in the proposita's mother revealed a banding pattern suggestive of an intrachromosomal insertion, as demonstrated by dual-color FISH with BACs that were duplicated in the proposita and multicolor-banding (MCB) based on microdissection derived region-specific libraries for chromosome 22. Our findings suggest that in both seemingly de novo deletions and duplications, the parent transmitting the imbalance should be investigated for possible balanced rearrangements. This report reinforces previous evidence that chromosome imbalances, and thus gene dosage effects, may be at the basis of some psychiatric disorders. Stringent correlations between submicroscopic imbalances, specific behavioral phenotypes and brain imaging will possibly help in dissecting complex behavioral traits.     

Familial 4.3 Mb duplication of 21q22 sheds new light on the Down syndrome critical region

A 4.3 Mb duplication of chromosome 21 bands q22.13-q22.2 was diagnosed by interphase fluorescent in-situ hybridisation (FISH) in a 31-week gestational age baby with cystic hygroma and hydrops; the duplication was later found in the mother and in her 8-year-old daughter by the same method and confirmed by array comparative genomic hybridisation (aCGH). All had the facial gestalt of Down syndrome (DS). This is the smallest accurately defined duplication of chromosome 21 reported with a DS phenotype. The duplication encompasses the gene DYRK1 but not DSCR1 or DSCAM, all of which have previously been implicated in the causation of DS. Previous karyotype analysis and telomere screening of the mother, and karyotype analysis and metaphase FISH of a chorionic villus sample, had all failed to reveal the duplication. The findings in this family add to the identification and delineation of a "critical region" for the DS phenotype on chromosome 21. Cryptic chromosomal abnormalities can be missed on a routine karyotype for investigation of abnormal prenatal ultrasound findings, lending support to the use of aCGH analysis in this setting.     

"Molecular rulers" for calibrating phenotypic effects of telomere imbalance

As a result of the increasing use of genome wide telomere screening, it has become evident that a significant proportion of people with idiopathic mental retardation have subtle abnormalities involving the telomeres of human chromosomes. However, during the course of these studies, there have also been telomeric imbalances identified in normal people that are not associated with any apparent phenotype. We have begun to scrutinize cases from both of these groups by determining the extent of the duplication or deletion associated with the imbalance. Five cases were examined where the telomere rearrangement resulted in trisomy for the 16p telomere. The size of the trisomic segment ranged from approximately 4-7 Mb and the phenotype included mental and growth retardation, brain malformations, heart defects, cleft palate, pancreatic insufficiency, genitourinary abnormalities, and dysmorphic features. Three cases with telomeric deletions without apparent phenotypic effects were also examined, one from 10q and two from 17p. All three deletions were inherited from a phenotypically normal parent carrying the same deletion, thus without apparent phenotypic effect. The largest deletion among these cases was approximately 600 kb on 17p. Similar studies are necessary for all telomeric regions to differentiate between those telomeric rearrangements that are pathogenic and those that are benign variants. Towards this goal, we are developing "molecular rulers" that incorporate multiple clones at each telomere that span the most distal 5 Mb region. While telomere screening has enabled the identification of telomere rearrangements, the use of molecular rulers will allow better phenotype prediction and prognosis related to these findings.     

Cryptic chromosomal rearrangement screening in 30 patients with mental retardation and dysmorphic features

Mental retardation affects 1-3% of the general population, and the genetic causes in many cases are unknown. Cytogenetically undetected chromosomal imbalances have been indicated as an explanation. Nowadays, due to the development of molecular cytogenetic techniques, it is possible to identify cryptic rearrangements involving the ends of chromosomes. We report a screening using chromosome-specific telomere fluorescence in-situ hybridization (FISH) probes, in a group of 30 patients with a well-characterized phenotype including mental retardation, dysmorphic features, and a normal karyotype. Among them, two subtelomeric rearrangements have been detected and characterized. One of them is a de novo deletion of 1p36, which has been previously described as a new contiguous gene syndrome. The second is an unbalanced product of a cryptic translocation involving chromosomes 1 and 13, which results in a partial 1q trisomy and partial 13q monosomy. These findings highlight, the importance of searching for cryptic subtelomeric rearrangements in non-syndromic mentally retarded patients.     

Identification of chromosome abnormalities in subtelomeric regions by microarray analysis: a study of 5,380 cases

Subtelomeric imbalances are a significant cause of congenital disorders. Screening for these abnormalities has traditionally utilized GTG-banding analysis, fluorescence in situ hybridization (FISH) assays, and multiplex ligation-dependent probe amplification. Microarray-based comparative genomic hybridization (array-CGH) is a relatively new technology that can identify microscopic and submicroscopic chromosomal imbalances. It has been proposed that an array with extended coverage at subtelomeric regions could characterize subtelomeric aberrations more efficiently in a single experiment. The targeted arrays for chromosome microarray analysis (CMA), developed by Baylor College of Medicine, have on average 12 BAC/PAC clones covering 10 Mb of each of the 41 subtelomeric regions. We screened 5,380 consecutive clinical patients using CMA. The most common reasons for referral included developmental delay (DD), and/or mental retardation (MR), dysmorphic features (DF), multiple congenital anomalies (MCA), seizure disorders (SD), and autistic, or other behavioral abnormalities. We found pathogenic rearrangements at subtelomeric regions in 236 patients (4.4%). Among these patients, 103 had a deletion, 58 had a duplication, 44 had an unbalanced translocation, and 31 had a complex rearrangement. The detection rates varied among patients with a normal karyotype analysis (2.98%), with an abnormal karyotype analysis (43.4%), and with an unavailable or no karyotype analysis (3.16%). Six patients out of 278 with a prior normal subtelomere-FISH analysis showed an abnormality including an interstitial deletion, two terminal deletions, two interstitial duplications, and a terminal duplication. In conclusion, genomic imbalances at subtelomeric regions contribute significantly to congenital disorders. Targeted array-CGH with extended coverage (up to 10 Mb) of subtelomeric regions will enhance the detection of subtelomeric imbalances, especially for submicroscopic imbalances.     

Unusual 8p inverted duplication deletion with telomere capture from 8q

Inverted 8p duplication deletions are recurrent chromosomal rearrangements that are mediated through non-allelic homologous recombination (NAHR) between olfactory receptor (OR) gene clusters at 8p23.1. These rearrangements result in a proximal inverted duplication of various extent, a single copy region between the OR gene clusters and a terminal 8p deletion. The terminal deletions are stabilized by direct addition of telomeric repeats, so called telomere healing. Here, we report a patient with an unusual inverted duplication deletion of 8p. Stabilization of the broken chromosome end was achieved by telomere capture instead of telomere healing, resulting in an additional duplication of 8q24.13→qter on the short arm of chromosome 8. Moreover, the inverted duplication was only 3.4 Mb in size (restricted to band 8p22) and thus cytogenetically undetectable. To the best of our knowledge this is the smallest inverted duplication reported hitherto. We describe the molecular characterization by FISH and array CGH of this unusual inv dup del (8p) and a previously reported patient with a similar 8q duplication and review the literature on cases associated with telomere capture.     

Co‐existence of 9p deletion and Silver‐Russell syndromes in a patient with maternally inherited cryptic complex chromosome rearrangement involving chromosomes 4, 9, and 11

We report a patient with a maternally inherited unbalanced complex chromosomal rearrangement (CCR) involving chromosomes 4, 9, and 11 detected by microarray comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). This patient presents with clinical features of 9p deletion syndrome and Silver‐Russell syndrome (SRS). Chromosome analysis performed in 2000 showed what appeared to be a simple terminal deletion of chromosome 9p22.1. aCGH performed in 2010 revealed a 1.63 Mb duplication at 4q28.3, a 15.48 Mb deletion at 9p24.3p22.3, and a 1.95 Mb duplication at 11p15.5. FISH analysis revealed a derivative chromosome 9 resulting from an unbalanced translocation between chromosomes 9 and 11, a chromosome 4 fragment inserted near the breakpoint of the translocation. The 4q28.3 duplication does not contain any currently known genes. The 9p24.3p22.3 deletion region contains 36 OMIM genes including a 3.5 Mb critical region for the 9p‐phenotype. The 11p15.5 duplication contains 49 OMIM genes including H19 and IGF2. Maternal aCGH was normal. However, maternal chromosomal and FISH analyses revealed an apparently balanced CCR involving chromosomes 4, 9, and 11. To the best of our knowledge, this is the first report of a patient with maternally inherited trans‐duplication of the entire imprinting control region 1 (ICR1) among the 11p15.5 duplications reported in SRS patients. This report supports the hypothesis that the trans‐duplication of the maternal copy of ICR1 alone is sufficient for the clinical manifestation of SRS and demonstrates the usefulness of combining aCGH with karyotyping and FISH for detecting cryptic genomic imbalances. © 2012 Wiley Periodicals, Inc.     

Subtelomeric deletions detected in patients with idiopathic mental retardation using multiplex ligation-dependent probe amplification (MLPA)

Subtelomeric rearrangements are responsible for 5% to 10% of cases of unexplained mental retardation. Despite their clinical relevance, methods to screen for these cytogenetically invisible abnormalities on a routine base are scarce. We screened patients with idiopathic mental retardation for subtelomeric aberrations using multiplex ligation-dependent probe amplification (MLPA). This recently developed technique is based on PCR amplification of ligated probes hybridized to chromosome ends. Currently, 41 telomeres can be screened in just two multiplex reactions. Four subtelomeric rearrangements (5.3%) were detected in a group of 75 patients with mild to severe mental retardation in combination with dysmorphic features and/or a familial history of mental retardation: two terminal 1p deletions, a terminal 1q deletion, and a terminal 3p deletion. Deletions could be verified by FISH and marker analysis. In one case the MLPA indicated a terminal 21q deletion due to a 3-bp deletion at the site of the probe, giving a false-positive rate of 1.3%. This study demonstrates that MLPA is a fast and reliable screening method, potentially suitable for use in routine diagnostics.     

dup(8p)/del(8q) recombinant chromosome in a girl with hepatic focal nodular hyperplasia

A 15-year-old girl had exertion dyspnea, focal nodular hyperplasia of the liver, portal vein hypoplasia, portopulmonary hypertension, mental retardation, and minor facial abnormalities. Cytogenetic analysis demonstrated an abnormal chromosome 8 with 8p22-pter duplication and 8q24.3-qter deletion, with the duplicated 8p segment attached to band 8q24.3. Her mother had a pericentric inversion of chromosome 8, inv(8)(p22q24.3). Therefore, the girl's abnormal chromosome 8 was a recombinant of maternal inversion chromosome: 46,XX,rec(8)dup(8p)inv(8)(p22q24.3)mat. Further characterization of the recombinant chromosome, using array CGH and regional FISH analyses, defined 15 Mb distal 8p duplication and 0.5 Mb 8q deletion. Possible correlation of the recombinant chromosome and hepatic focal nodular hyperplasia in the patient is discussed.