Christine Tyson

Submicroscopic deletions and duplications in individuals with intellectual disability detected by array‐CGH

Intellectual disability (ID) affects about 3% of the population (IQ < 70), and in about 40% of moderate (IQ 35–49) to severe ID (IQ < 34), and 70% of cases of mild ID (IQ 50–70), the etiology of the disease remains unknown. It has long been suspected that chromosomal gains and losses undetectable by routine cytogenetic analysis (i.e., less than 5–10 Mb in size) are implicated in ID of unknown etiology. Array CGH has recently been used to perform a genome‐wide screen for submicroscopic gains and losses in individuals with a normal karyotype but with features suggestive of a chromosome abnormality. In two recent studies, the technique has demonstrated a ∼15% detection rate for de novo copy number changes of individual clones or groups of clones. Here, we describe a study of 22 individuals with mild to moderate ID and nonsyndromic pattern of dysmorphic features suspicious of an underlying chromosome abnormality, using the 3 Mb and 1 Mb commercial arrays (Spectral Genomics). Deletions and duplications of 16 clones, previously described to show copy number variability in normal individuals [Iafrate et al., 2004 ; Lapierre et al., 2004 ; Schoumans et al., 2004 ; Vermeesch et al., 2005 ] were seen in 21/22 subjects and were considered polymorphisms. In addition, three subjects showed submicroscopic deletions and duplications not previously reported as normal variants. Two of these submicroscopic changes were of de novo origin (microdeletions at 7q36.3 and a microduplication at 11q12.3‐13.1) and one was of unknown origin as parental testing of origin could not be performed (microduplication of Xp22.3). The clinical description of the three subjects with submicroscopic chromosomal changes at 7q36.3, 11q12.3‐13.1, Xp22.3 is provided.

Identification of copy number variants in miscarriages from couples with idiopathic recurrent pregnancy loss

BACKGROUND Recurrent pregnancy loss (RPL), defined as two or more miscarriages, affects 3-5% of couples trying to establish a family. Despite extensive evaluation, no factor is identified in ∼40% of cases. In this study, we investigated the possibility that submicroscopic chromosomal changes, not detectable by conventional cytogenetic analysis, exist in miscarriages with normal karyotypes (46,XY or 46,XX) from couples with idiopathic RPL. METHODS Array comparative genomic hybridization (array-CGH) was used to assess for DNA copy number variants (CNVs) in 26 miscarriages with normal karyotypes. Parental array-CGH analysis was performed to determine if miscarriage CNVs were de novo or inherited. RESULTS There were 11 unique (previously not described) CNVs, all inherited, identified in 13 miscarriages from 8 couples. The maternal origin of two CNVs was of interest as they involved the imprinted genes TIMP2 and CTNNA3, which are only normally expressed from the maternal copy in the placenta. Two additional cohorts, consisting of 282 women with recurrent miscarriage (RM) and 61 fertile women, were screened for these two CNVs using a Quantitative Multiplex Fluorescent PCR of Short Fragments assay. One woman with RM, but none of the fertile women, carried the CTNNA3-associated CNV. CONCLUSIONS This preliminary study shows that array-CGH is useful for detecting CNVs in cases of RPL. Further investigations of CNVs, particularly those involving genes that are imprinted in placenta, in women with RPL could be worthwhile.

Understanding the impact of 1q21.1 copy number variant

Background1q21.1 Copy Number Variant (CNV) is associated with a highly variable phenotype ranging from congenital anomalies, learning deficits/intellectual disability (ID), to a normal phenotype. Hence, the clinical significance of this CNV can be difficult to evaluate. Here we described the consequences of the 1q21.1 CNV on genome-wide gene expression and function of selected candidate genes within 1q21.1 using cell lines from clinically well described subjects.Methods and ResultsEight subjects from 3 families were included in the study: six with a 1q21.1 deletion and two with a 1q21.1 duplication. High resolution Affymetrix 2.7M array was used to refine the 1q21.1 CNV breakpoints and exclude the presence of secondary CNVs of pathogenic relevance. Whole genome expression profiling, studied in lymphoblast cell lines (LBCs) from 5 subjects, showed enrichment of genes from 1q21.1 in the top 100 genes ranked based on correlation of expression with 1q21.1 copy number. The function of two top genes from 1q21.1, CHD1L/ALC1 and PRKAB2, was studied in detail in LBCs from a deletion and a duplication carrier. CHD1L/ALC1 is an enzyme with a role in chromatin modification and DNA damage response while PRKAB2 is a member of the AMP kinase complex, which senses and maintains systemic and cellular energy balance. The protein levels for CHD1L/ALC1 and PRKAB2 were changed in concordance with their copy number in both LBCs. A defect in chromatin remodeling was documented based on impaired decatenation (chromatid untangling) checkpoint (DCC) in both LBCs. This defect, reproduced by CHD1L/ALC1 siRNA, identifies a new role of CHD1L/ALC1 in DCC. Both LBCs also showed elevated levels of micronuclei following treatment with a Topoisomerase II inhibitor suggesting increased DNA breaks. AMP kinase function, specifically in the deletion containing LBCs, was attenuated.ConclusionOur studies are unique as they show for the first time that the 1q21.1 CNV not only causes changes in the expression of its key integral genes, associated with changes at the protein level, but also results in changes in their known function, in the case of AMPK, and newly identified function such as DCC activation in the case of CHD1L/ALC1. Our results support the use of patient lymphoblasts for dissecting the functional sequelae of genes integral to CNVs in carrier cell lines, ultimately enhancing understanding of biological processes which may contribute to the clinical phenotype.

Elucidation of a cryptic interstitial 7q31.3 deletion in a patient with a language disorder and mild mental retardation by array-CGH.

We report on a 14‐year‐old boy who presented with bilateral cleft lip and palate, hearing loss, a language processing disorder, and mild mental retardation (MR). G‐banded chromosome analysis of the patient and his family revealed he carried an apparently balanced de novo complex translocation involving chromosomes 5, 6, and 7. Chromosomal comparative genomic hybridization (CGH) was performed to investigate the possibility of any genomic imbalance as a result of the complex rearrangement. No abnormality was detected at any of the translocation breakpoint regions (5p13.2, 6p24, 7q21.1, and 7q21.3), nor was there any other imbalance which fell inside our significance level of 0.8–1.2. Array‐CGH analysis was initiated to perform a higher resolution search for gains and losses, and revealed a deletion of two adjacent clones, CTB‐133K23 and RP11‐112P4, mapping to 7q31.3, which are 4.4 Mb apart. Fluorescence in situ hybridization (FISH) using these two clones confirmed the deletion. 7q31 has frequently been implicated in the search for genes involved in speech and language disorders. The specific 7q31.3 region deleted in our patient has significant overlap with some such areas of the genome. These findings are, therefore, of value in identifying genes involved in the speech and language phenotypes. This study has shown the importance of array‐CGH in investigating patients who have clinical features suggestive of a chromosome abnormality, but with apparently balanced chromosome rearrangements. It has demonstrated that the array‐CGH technique provides a much greater insight into submicroscopic chromosome imbalances than conventional cytogenetic techniques.

Genomic changes detected by array CGH in human embryos with developmental defects

Developmental abnormalities of human embryos can be visualized in utero using embryoscopy. Our previous embryoscopic and genetic evaluations detected developmental abnormalities in the majority of both euploid (74%) and aneuploid or polyploid (90%) miscarriages. Since we found the pattern of morphological changes to be similar in euploid and non-euploid embryos, we proposed that lethal submicroscopic changes, not detected by standard chromosome testing, may be responsible for miscarriage of euploid embryos. Whole genome oligo and bacterial artificial chromosome array comparative genome hybridization (CGH) was used to screen for submicroscopic chromosomal changes (DNA copy number variants or CNVs) in 17 euploid embryonic miscarriages, with a range of developmental abnormalities documented by embryoscopy. The CNV breakpoints were refined using a custom array (Agilent) with high resolution coverage of the CNVs. Six unique CNVs, previously not reported, were identified in 5 of the 17 embryos (29% of all cases or 50% of cases studied with higher resolution arrays). All six unique CNVs were <250 kb in size. On the basis of parental array CGH analysis, a de novo origin of a CNV was determined for one embryo (at 13q32.1) and suspected for another (at 10p15.3). Three CNVs, at Xq28, 1q25.3 and 7p14.3, were inherited and a CNV at 17p13.1 was of unknown origin. The genes contained within these unique CNVs will be discussed, with specific reference to rearrangements of syntaxin and tryptophan-aspartic acid (WD) repeat genes. Our report describes for the first time, de novo and inherited unique CNVs in euploid human embryos with specific developmental defects.

Clinical application of 2.7M Cytogenetics array for CNV detection in subjects with idiopathic autism and/or intellectual disability

Qiao Y, Tyson C, Hrynchak M, Lopez‐Rangel E, Hildebrand J, Martell S, Fawcett C, Kasmara L, Calli K, Harvard C, Liu X, Holden JJA, Lewis SME, Rajcan‐Separovic E. Clinical application of 2.7M Cytogenetics array for CNV detection in subjects with idiopathic autism and/or intellectual disability.

Submicroscopic deletions of 11q24-25 in individuals without Jacobsen syndrome: re-examination of the critical region by high-resolution array-CGH.

BackgroundJacobsen syndrome is a rare contiguous gene disorder that results from a terminal deletion of the long arm of chromosome 11. It is typically characterized by intellectual disability, a variety of physical anomalies and a distinctive facial appearance. The 11q deletion has traditionally been identified by routine chromosome analysis. Array-based comparative genomic hybridization (array-CGH) has offered new opportunities to identify and refine chromosomal abnormalities in regions known to be associated with clinical syndromes.ResultsUsing the 1 Mb BAC array (Spectral Genomics), we screened 70 chromosomally normal children with idiopathic intellectual disability (ID) and congenital abnormalities, and identified five cases with submicroscopic abnormalities believed to contribute to their phenotypes. Here, we provide detailed molecular cytogenetic descriptions and clinical presentation of two unrelated subjects with de novo submicroscopic deletions within chromosome bands 11q24-25. In subject 1 the chromosome rearrangement consisted of a 6.18 Mb deletion (from 128.25–134.43 Mb) and an adjacent 5.04 Mb duplication (from 123.15–128.19 Mb), while in subject 2, a 4.74 Mb interstitial deletion was found (from 124.29–129.03 Mb). Higher resolution array analysis (385 K Nimblegen) was used to refine all breakpoints. Deletions of the 11q24-25 region are known to be associated with Jacobsen syndrome (JBS: OMIM 147791). However, neither of the subjects had the typical features of JBS (trigonocephaly, platelet disorder, heart abnormalities). Both subjects had ID, dysmorphic features and additional phenotypic abnormalities: subject 1 had a kidney abnormality, bilateral preauricular pits, pectus excavatum, mild to moderate conductive hearing loss and behavioral concerns; subject 2 had macrocephaly, an abnormal MRI with delayed myelination, fifth finger shortening and squaring of all fingertips, and sensorineural hearing loss.ConclusionTwo individuals with ID who did not have the typical clinical features of Jacobsen syndrome were found to have deletions within the JBS region at 11q24-25. Their rearrangements facilitate the refinement of the JBS critical region and suggest that a) deletion of at least 3 of the 4 platelet function critical genes (ETS-1, FLI-1 and NFRKB and JAM3) is necessary for thrombocytopenia; b) one of the critical regions for heart abnormalities (conotruncal heart defects) may lie within 129.03 – 130.6 Mb; c) deletions of KCNJ1 and ADAMTS15 may contribute to the renal anomalies in Jacobsen Syndrome; d) the critical region for MRI abnormalities involves a region from 124.6 – 129.03 Mb. Our results reiterate the benefits of array-CGH for description of new phenotype/genotype associations and refinement of previously established ones.

Population-based characterization of the genetic landscape of chronic lymphocytic leukemia patients referred for cytogenetic testing in British Columbia, Canada: the role of provincial laboratory standardization

Detection of recurrent chromosome abnormalities by fluorescence in situ hybridization (FISH) is an essential component of care in chronic lymphocytic leukemia (CLL) patients. In the province of British Columbia (BC), Canada, population 4.6 million, CLL patients receive uniform evaluation and therapy with FISH testing performed in three jurisdictions. The aims of this study were to (i) validate CLL-FISH testing among the BC cytogenetic laboratories to ensure standardization of results and (ii) characterize population-level CLL-FISH abnormalities by pooling provincial data. From 2004 to 2011, 585 consecutive patients underwent pretreatment CLL-FISH testing at laboratory A (50.1%), laboratory B (32.3%), or laboratory C (17.6%). For validation purposes, 26 CLL-FISH abnormalities were tested by each laboratorys protocol, with 91% result concordance. Discordant results involved percent abnormalities at or near cutoff values; therefore, a 10% universal cutoff was established when pooling results. Applying the universal cutoff to the provincial cohort, CLL-FISH abnormalities were detected in 74.9%: 54.9% 13q-, 18.8% +12, 8.5% 11q-, and 7.7% 17p-. In this large population-based cohort of patients referred for CLL-FISH testing, frequencies of abnormalities detected by FISH analysis were highly consistent with those reported in single-institution and clinical trial populations. Provinces or districts that work together to care for CLL patients can effectively pool data with appropriate laboratory validation to ensure standardization of results.

Genotype-phenotype analysis of 18q12.1-q12.2 copy number variation in autism

Autism Spectrum Disorders (ASD) are complex neurodevelopmental conditions characterized by delays in social interactions and communication as well as displays of restrictive/repetitive interests. DNA copy number variants have been identified as a genomic susceptibility factor in ASDs and imply significant genetic heterogeneity. We report a 7-year-old female with ADOS-G and ADI-R confirmed autistic disorder harbouring a de novo 4 Mb duplication (18q12.1). Our subject displays severely deficient expressive language, stereotypic and repetitive behaviours, mild intellectual disability (ID), focal epilepsy, short stature and absence of significant dysmorphic features. Search of the PubMed literature and DECIPHER database identified 4 additional cases involving 18q12.1 associated with autism and/or ID that overlap our case: one duplication, two deletions and one balanced translocation. Notably, autism and ID are seen with genomic gain or loss at 18q12.1, plus epilepsy and short stature in duplication cases, and hypotonia and tall stature in deletion cases. No consistent dysmorphic features were noted amongst the reviewed cases. We review prospective ASD/ID candidate genes integral to 18q12.1, including those coding for the desmocollin/desmoglein cluster, ring finger proteins 125 and 138, trafficking protein particle complex 8 and dystrobrevin-alpha. The collective clinical and molecular features common to microduplication 18q12.1 suggest that dosage-sensitive, position or contiguous gene effects may be associated in the etiopathogenesis of this autism-ID-epilepsy syndrome.

Expansion of a 12-kb VNTR containing the REXO1L1 gene cluster underlies the microscopically visible euchromatic variant of 8q21.2

Copy number variants visible with the light microscope have been described as euchromatic variants (EVs) and EVs with extra G-light material at 8q21.2 have been reported only once before. We report four further patients with EVs of 8q21.2 ascertained for clinical (3) or reproductive reasons (1). Enhanced signal strength from two overlapping bacterial artificial chromosomes (BACs) and microarray analysis mapped the EV to a 284-kb interval in the reference genome. This interval consists of a sequence gap flanked by segmental duplications that contain the 12-kb components of one of the largest Variable Number Tandem Repeat arrays in the human genome. Using digital NanoString technology with a custom probe for the RNA exonuclease 1 homologue (S. cerevisiae)-like 1 (REXO1L1) gene within each 12-kb repeat, significantly enhanced diploid copy numbers of 270 and 265 were found in an EV family and a median diploid copy number of 166 copies in 216 controls. These 8q21.2 EVs are not thought to have clinical consequences as the phenotypes of the probands were inconsistent, those referred for reproductive reasons were otherwise phenotypically normal and the REXO1L1 gene has no known disease association. This EV was found in 4/3078 (1 in 770) consecutive referrals for chromosome analysis and needs to be distinguished from pathogenic imbalances of medial 8q. The REXO1L1 gene product is a marker of hepatitis C virus (HCV) infection and a possible association between REXO1L1 copy number and susceptibility to HCV infection, progression or response to treatment has not yet been excluded.