Subtelomeric imbalances in phenotypically normal individuals

Subtelomeric imbalances are identified in approximately 5% of patients with idiopathic mental retardation (MR) and multiple congenital anomalies (MCA). Because of this high incidence, screening for subtelomeric anomalies became part of the routine genetic evaluation of MCA/MR patients. In contrast to the general view that subtelomeric imbalances cause MCA/MR, we report here 15 subtelomeric copy-number changes in 12 families in which the imbalance is inherited from a phenotypically normal parent. We detected inherited deletions at subtelomeres 2q, 3p, 4p, 4q, 6q, 10q, 17p, 17q, Xp, and Yq and duplications at 1q, 4q, 10q, and 11q. Interestingly, in addition to small deletions (<1 Mb) also unexpected large deletions and duplications up to 7.8 Mb were detected. Taken together with previous reports, a total of 16 subtelomeric duplications and 18 deletions inherited from a phenotypically normal parent have now been reported. Clearly, more extensive genotype-phenotype correlations are needed to better understand the phenotypic consequences of these subtelomeric copy number variations and to resolve the current uncertainty for genetic counseling in postnatal and prenatal diagnosis.     

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.     

Development of a comparative genomic hybridization microarray and demonstration of its utility with 25 well-characterized 1p36 deletions

Chromosomal abnormalities, such as deletions and duplications, are characterized by specific and often complex phenotypes resulting from an imbalance in normal gene dosage. However, routine chromosome banding is not sensitive enough to detect subtle chromosome aberrations (<5-10 Mb). Array-based comparative genomic hybridization (array CGH) is a powerful new technology capable of identifying chromosomal imbalance at a high resolution by co-hybridizing differentially labeled test and control DNAs to a microarray of genomic clones. We used a previously assembled contig of large-insert clones that span 10.5 Mb of the most distal region of 1p36 to design a microarray. The array includes 97 clones from 1p36, 41 clones from the subtelomeric regions of all human chromosomes, and three clones from each of the X and Y chromosomes. We used this microarray to study 25 subjects with well-characterized deletions of 1p36. All array CGH results agree with the deletion sizes and locations of the breakpoints in these subjects as determined previously by FISH and microsatellite analyses. Terminal deletions, interstitial deletions, derivative chromosomes and complex rearrangements were also identified. We anticipate that array CGH will change the diagnostic approach to many congenital and acquired genetic diseases such as mental retardation, birth defects and cancer.     

Detection and calibration of microdeletions and microduplications by array-based comparative genomic hybridization and its applicability to clinical genetic testing.

PURPOSE: Genome-wide telomere screening by fluorescence in situ hybridization (FISH) has revealed that approximately 6% of unexplained mental retardation is due to submicroscopic telomere imbalances. However, the use of FISH for telomere screening is labor intensive and time consuming, given that 41 telomeres are interrogated. We have evaluated the use of array-based Comparative Genomic Hybridization (aCGH) as a more efficient tool for identifying telomere rearrangements. METHODS: In this study, 102 individuals with unexplained mental retardation, with either normal or abnormal FISH results, were selected for a blinded retrospective study using aCGH. Results between the two methodologies were compared to ascertain the ability of aCGH to be used in a clinical diagnostics setting. RESULTS: We detected 100% of all imbalances previously identified by FISH (n = 17) and identified two additional abnormalities, a 10q telomere duplication and an interstitial duplication of 22q11. Interphase FISH analysis verified all abnormal array results. We also demonstrated that aCGH can accurately calibrate the size of telomere imbalances by using an array with "molecular rulers" for the telomeric regions of 1p, 16p, 17p, and 22q. CONCLUSION: This study demonstrates that aCGH is an equivalent methodology to telomere FISH for detecting submicroscopic deletions. In addition, small duplications that are not easily visible by FISH can be accurately detected using aCGH. Because aCGH allows simultaneous interrogation of hundreds to thousands of DNA probes and is more amenable to automation, it offers an efficient and high-throughput alternative for detecting and calibrating unbalanced rearrangements, both of the telomere region, as well as other genomic locations.     

Inverted duplications deletions: underdiagnosed rearrangements??

Molecular techniques led to the discovery that several chromosome rearrangements interpreted as terminal duplications were in fact inverted duplications contiguous to terminal deletions. Inv dup del rearrangements originate through a symmetric dicentric chromosome that, after asymmetric breakage, generates an inv dup del and a deleted chromosome. In recurrent inverted duplications the dicentric chromosome is formed at meiosis through non‐allelic homologous recombination. In non‐recurrent inv dup del cases, dicentric intermediates are formed by non‐homologous end joining or intrastrand annealing. Some authors hypothesized that in these cases the dicentric may have been formed directly in the zygote. Healing of the broken dicentric chromosomes can occur not only in a telomerase‐dependent way but also through telomere capture and circularization thus creating translocated or ring inv dup del chromosomes. In all the cases reported up to now, the duplicated region was always longer than the deleted one, but we can safely assume that there is another group of rearrangements where the deleted region is longer than the duplicated portion. In general, in these cases, the cytogeneticist will suspect the presence of a deletion and confirm it by FISH with a subtelomeric probe, but he/she will almost certainly miss the duplication. It is likely that the conventional analysis techniques used until now have led to a substantial underestimate of the frequency of inv dup del rearrangements and that the widespread use of array‐CGH in routine analysis will allow a more realistic estimate. Obviously, the concomitant presence of deletion and duplication has important consequences in genotype/phenotype correlations.     

Screening for subtelomeric rearrangements in 210 patients with unexplained mental retardation using multiplex ligation dependent probe amplification (MLPA)

Background: Subtelomeric rearrangements contribute to idiopathic mental retardation and human malformations, sometimes as distinct mental retardation syndromes. However, for most subtelomeric defects a characteristic clinical phenotype remains to be elucidated.

Objective: To screen for submicroscopic subtelomeric aberrations using multiplex ligation dependent probe amplification (MLPA).

Methods: 210 individuals with unexplained mental retardation were studied. A new set of subtelomeric probes, the SALSA P036 human telomere test kit, was used.

Results: A subtelomeric aberration was identified in 14 patients (6.7%) (10 deletions and four duplications). Five deletions were de novo; four were inherited from phenotypically normal parents, suggesting that these were polymorphisms. For one deletion, DNA samples of the parents were not available. Two de novo submicroscopic duplications were detected (dup 5qter, dup 12pter), while the other duplications (dup 18qter and dup 22qter) were inherited from phenotypically similarly affected parents. All clinically relevant aberrations (de novo or inherited from similarly affected parents) occurred in patients with a clinical score of 3 using an established checklist for subtelomeric rearrangements. Testing of patients with a clinical score of 3 increased the diagnostic yield twofold to 12.4%. Abnormalities with clinical relevance occurred in 6.3%, 5.1%, and 1.7% of mildly, moderately, and severely retarded patients, respectively, indicating that testing for subtelomeric aberrations among mildly retarded individuals is necessary.

Conclusions: The value of MLPA is confirmed. Subtelomeric screening can be offered to all mentally retarded patients, although clinical preselection increases the percentage of chromosomal aberrations detected. Duplications may be a more common cause of mental retardation than has been appreciated.

     

Fine-mapping subtelomeric deletions and duplications by comparative genomic hybridization in 42 individuals

Human subtelomere regions contain numerous gene-rich segments and are susceptible to germline rearrangements. The availability of diagnostic test kits to detect subtelomeric rearrangements has resulted in the diagnosis of numerous abnormalities with clinical implications including congenital heart abnormalities and mental retardation. Several of these have been described as clinically recognizable syndromes (e.g., deletion of 1p, 3p, 5q, 6p, 9q, and 22q). Given this, fine-mapping of subtelomeric breakpoints is of increasing importance to the assessment of genotype-phenotype correlations in these recognized syndromes as well as to the identification of additional syndromes. We developed a BAC and cosmid-based DNA array (TEL array) with high-resolution coverage of 10 Mb-sized subtelomeric regions, and used it to analyze 42 samples from unrelated patients with subtelomeric rearrangements whose breakpoints were previously either unmapped or mapped at a lower resolution than that achievable with the TEL array. Six apparently recurrent subtelomeric breakpoint loci were localized to genomic regions containing segmental duplication, copy number variation, and sequence gaps. Small (1 Mb or less) candidate gene regions for clinical phenotypes in separate patients were identified for 3p, 6q, 9q, and 10p deletions as well as for a 19q duplication. In addition to fine-mapping nearly all of the expected breakpoints, several previously unidentified rearrangements were detected.     

The clinical utility of enhanced subtelomeric coverage in array CGH

Telomeric chromosome abnormalities are a substantial cause of mental retardation and birth defects. Although subtelomeric fluorescence in situ hybridization (FISH) probes have been widely used to identify submicroscopic telomeric rearrangements, array-based comparative genomic hybridization (array CGH) has emerged as a more efficient and comprehensive detection method. Due to the clinical relevance of telomeric abnormalities, it has been proposed that array CGH using panels of BAC clones that map to regularly spaced intervals along the length of each telomere could be used to characterize subtelomeric aberrations more precisely in a single experiment. We have added 1,120 FISH-mapped BAC clones to our microarray to enhance the coverage of the 41 unique human subtelomeric regions. Contigs of clones were selected in increments of approximately 0.5 Mb beginning with the most distal unique sequence for each subtelomere and extending on average approximately 5.7 Mb toward the centromere. We have used this microarray to characterize 169 clinically significant subtelomeric abnormalities identified out of nearly 7,000 consecutive clinical cases analyzed by array CGH in our diagnostic laboratory. The expanded telomere coverage was sufficient to define the breakpoints of over half (56%) of the chromosome abnormalities. However, 44% of the subtelomeric aberrations extended beyond the size of this expanded coverage suggesting that many subtelomeric abnormalities are >5 Mb in size and that greater representation may be of even more value. In addition to identifying 6 cases of complex rearrangements, we have identified 42 cases of interstitial deletions that would have been missed by subtelomere FISH panels that use a single clone to the most distal unique sequence for each region. Microarrays designed to investigate regions known to be involved in chromosome abnormalities will enhance the detection of cytogenetic abnormalities at unprecedented resolution and frequency.     

Genome-wide screening using automated fluorescent genotyping to detect cryptic cytogenetic abnormalities in children with idiopathic syndromic mental retardation

Mental retardation (MR) is the most common developmental disability, affecting approximately 2% of the population. The causes of MR are diverse and poorly understood, but chromosomal rearrangements account for 4-28% of cases, and duplications/deletions smaller than 5 Mb are known to cause syndromic MR. We have previously developed a strategy based on automated fluorescent microsatellite genotyping to test for telomere integrity. This strategy detected about 10% of cryptic subtelomeric rearrangements in patients with idiopathic syndromic MR. Because telomere screening is a first step toward the goal of analyzing the entire genome for chromosomal rearrangements in MR, we have extended our strategy to 400 markers evenly distributed along the chromosomes to detect interstitial anomalies. Among 97 individuals tested, three anomalies were found: two deletions (one in three siblings) and one parental disomy. These results emphasize the value of a genome-wide microsatellite scan for the detection of interstitial aberrations and demonstrate that automated genotyping is a sensitive method that not only detects small interstitial rearrangements and their parental origin but also provides a unique opportunity to detect uniparental disomies. This study will hopefully contribute to the delineation of new contiguous gene syndromes and the identification of new imprinted regions.     

Diverse chromosome breakage mechanisms underlie subtelomeric rearrangements, a common cause of mental retardation

Subtelomeric rearrangements are an important cause of both isolated and familial idiopathic mental retardation. A variety of different rearrangements such as pure truncations, unbalanced translocations, interstitial deletions, and inverted duplications have been detected throughout various screening studies. The cause of these aberrations is poorly understood as only few of the breakpoints have been determined and studied. We molecularly characterized the breakpoints of three rearrangements including a 1p subtelomeric deletion, a 1q subtelomeric deletion, and an unbalanced translocation between chromosomes 11q and 20q; we propose that diverse chromosome breakage mechanisms underlie subtelomeric rearrangements. The breakpoint sequences suggest that unusual non-B-DNA structures including triplex, tetraplex, and hairpin structures may be involved. In addition, we saw that the seemingly pure truncations of chromosomes 1p and 1q were in fact more complex rearrangements as highly repetitive sequences were joined to the chromosome end at the site of breakage.     

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

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

Usefulness of high-resolution comparative genomic hybridization (CGH) for detecting and characterizing constitutional chromosome abnormalities

Comparative genomic hybridization (CGH) is a technique for detection of chromosomal imbalances in a genomic DNA sample. We here report the application of the recently developed method of high-resolution CGH on DNA samples from 66 children having various degrees of delayed psychomotor development with or without clear dysmorphic features and congenital malformations. In 5 of 50 patients with apparently normal karyotypes, a deletion or duplication was revealed by CGH. Only one of these cases had a subtelomeric rearrangement. In one of seven cases with a de novo apparently balanced translocation, deletions were found. In all nine cases where the origin of a marker chromosome or additional chromosomal material was difficult to determine, CGH gave a precise identification. The following findings were from cases having a deletion or duplication as the sole chromosomal imbalance; dup(2)(p16p21), del(4)(q21q21), del(6)(q14q15), del(6)(p12p12), dup(6)(q24qter), and dup(15)(q11q13). One case had dup(9)(p11pter) combined with a very small subtelomeric deletion on 6q. In our hands, CGH is highly useful not only for identifying known chromosomal imbalances, but also for finding elusive deletions or duplications in the large group of children with developmental delay with or without congenital abnormalities. In such cases, the diagnostic yield of CGH appears to be higher than what has been reported from subtelomeric FISH screening.     

Blepharophimosis and mental retardation (BMR) phenotypes caused by chromosomal rearrangements: description in a boy with partial trisomy 10q and monosomy 4q and review of the literature

Blepharophimosis is a rare congenital anomaly of the palpebral fissure which is often associated with mental retardation and additional malformations. We report on a boy with blepharophimosis, ptosis and severe mental retardation carrying an unbalanced 4;10 translocation with terminal duplication of 10q [dup(10)(q25.1-->qter)] and monosomy of a small terminal segment of chromosome 4q [del(4)(34.3-->qter)]. Detailed clinical examination and review of the literature showed that the phenotype of the patient was mainly determined by the dup(10q). This paper reviews the chromosomal aberrations associated with BMR (blepharophimosis mental retardation) phenotypes. Searching different databases and reviewing the literature revealed 14 microscopically visible aberrations (among them UPD(14)pat) and two submicroscopic rearrangements causing blepharophimosis and mental retardation (BMR) syndrome. Some of these rearrangements-like the terminal dup(10q) identified in our patient or interstitial del(2q)-are associated with clearly defined phenotypes and can be well distinguished from each other on basis of clinical examination. This paper should assist clinicians and cytogeneticists when evaluating patients with BMR syndrome.     

Array-based comparative genomic hybridization identifies a high frequency of copy number variations in patients with syndromic overgrowth

Overgrowth syndromes are a heterogeneous group of conditions including endocrine hormone disorders, several genetic syndromes and other disorders with unknown etiopathogenesis. Among genetic causes, chromosomal deletions and duplications such as dup(4)(p16.3), dup(15)(q26qter), del(9)(q22.32q22.33), del(22)(q13) and del(5)(q35) have been identified in patients with overgrowth. Most of them, however, remain undetectable using banding karyotype analysis. In this study, we report on the analysis using a 1-Mb resolution array-based comparative genomic hybridization (CGH) of 93 patients with either a recognizable overgrowth condition (ie, Sotos syndrome or Weaver syndrome) or an unclassified overgrowth syndrome. Five clinically relevant imbalances (three duplications and two deletions) were identified and the pathogenicity of two additional anomalies (one duplication and one deletion) is discussed. Altered segments ranged in size from 0.32 to 18.2 Mb, and no recurrent abnormality was identified. These results show that array-CGH provides a high diagnostic yield in patients with overgrowth syndromes and point to novel chromosomal regions associated with these conditions. Although chromosomal deletions are usually associated with growth retardation, we found that the majority of the imbalances detected in our patients are duplications. Besides their importance for diagnosis and genetic counseling, our results may allow to delineate new contiguous gene syndromes associated with overgrowth, pointing to new genes, the deregulation of which may be responsible for growth defect.     

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.     

Subtelomeric 6p deletion: clinical, FISH, and array CGH characterization of two cases

Thirty patients have been described with cytogenetically visible deletion of the short arm of chromosome 6. However, subtelomeric 6p deletion detected by subtelomeric specific probes has been reported only twice. We report two new patients with terminal 6p deletion detected by subtelomeric screening using fluorescence in situ hybridization (FISH). The two patients exhibited mental retardation, ocular abnormalities, hearing loss, and a characteristic facial appearance. Detailed FISH analyses with probes covering the distal 6p25 region estimated the size of the terminal deletions to approximately 5.5 Mb and approximately 4.8 Mb. Array-based comparative genomic hybridization (array CGH) was used to confirm the cryptic deletions. Most patients with subtelomeric defects lack a characteristic phenotype. However, some of the subtelomeric deletions result in a specific phenotype, which can direct the clinician towards the diagnosis. Submicroscopic 6p deletion appears to be a recognizable clinical phenotype, and this region should be thoroughly investigated with FISH probes, including at least a subtelomeric 6p probe and a probe covering FOXC1, for patients presenting with a characteristic facial appearance, ocular abnormalities, predominantly anterior-chamber eye defects, hearing loss, and mental retardation.     

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.     

Pure subtelomeric microduplications as a cause of mental retardation

Submicroscopic subtelomeric aberrations are a common cause of mental retardation (MR). New molecular techniques allow the identification of subtelomeric microduplications, but their frequency and significance are largely unknown. We determined the frequency of subtelomeric, pure microduplications in a cohort of 624 patients with MR and/or multiple congenital anomalies using multiplex ligation dependent probe amplification (MLPA) and delineated the identified microduplications using array based comparative genomic hybridization (array CGH). In 11 patients, MLPA revealed a subtelomeric duplication without a concurrent deletion. Additional fluorescence in situ hybridization studies and parental analyses showed that three had occurred de novo: one duplication 5q34qter (12.7 Mb), one duplication 9q34.13qter (7.2 Mb) and one duplication 9p24.2pter (4.1 Mb). Five microduplications (9p, 11q, 12q, 15q and 16p) appeared to be inherited from an unaffected parent, while in three cases (9p, 12p and 17p) the parents were not available for testing. Based on our findings and data from the literature, the three de novo duplications were the only ones likely to be disease-causing, leading to a frequency of pathogenic subtelomeric, pure microduplications of 0.5%. Our study shows that subtelomeric microduplications are an infrequent cause of MR and that additional clinical and family studies are required to assess their clinical significance.     

Two 22q telomere deletions serendipitously detected by FISH.

Cryptic telomere deletions have been proposed to be a significant cause of idiopathic mental retardation. We present two unrelated subjects, with normal G banding analysis, in whom 22q telomere deletions were serendipitously detected at two different institutions using fluorescence in situ hybridisation (FISH). Both probands presented with several of the previously described features associated with 22q deletions, including hypotonia, developmental delay, and absence of speech. Our two cases increase the total number of reported 22q telomere deletions to 19, the majority of which were identified by cytogenetic banding analysis. With the limited sensitivity of routine cytogenetic studies (approximately 2-5 Mb), these two new cases suggest that the actual prevalence of 22q telomere deletions may be higher than currently documented. Of additional interest is the phenotypic overlap with Angelman syndrome (AS) as it raises the possibility of a 22q deletion in patients in whom AS has been ruled out. The use of telomeric probes as diagnostic reagents would be useful in determining an accurate prevalence of chromosome 22q deletions and could result in a significantly higher detection rate of subtelomeric rearrangements.