Optical genome mapping enables constitutional chromosomal aberration detection.

Chromosomal aberrations including structural variations (SVs) are a major cause of human genetic diseases. Their detection in clinical routine still relies on standard cytogenetics. Drawbacks of these tests are a very low resolution (karyotyping) and the inability to detect balanced SVs or indicate the genomic localization and orientation of duplicated segments or insertions (copy number variant [CNV] microarrays). Here, we investigated the ability of optical genome mapping (OGM) to detect known constitutional chromosomal aberrations. Ultra-high-molecular-weight DNA was isolated from 85 blood or cultured cells and processed via OGM. A de novo genome assembly was performed followed by structural variant and CNV calling and annotation, and results were compared to known aberrations from standard-of-care tests (karyotype, FISH, and/or CNV microarray). In total, we analyzed 99 chromosomal aberrations, including seven aneuploidies, 19 deletions, 20 duplications, 34 translocations, six inversions, two insertions, six isochromosomes, one ring chromosome, and four complex rearrangements. Several of these variants encompass complex regions of the human genome involved in repeat-mediated microdeletion/microduplication syndromes. High-resolution OGM reached 100% concordance compared to standard assays for all aberrations with non-centromeric breakpoints. This proof-of-principle study demonstrates the ability of OGM to detect nearly all types of chromosomal aberrations. We also suggest suited filtering strategies to prioritize clinically relevant aberrations and discuss future improvements. These results highlight the potential for OGM to provide a cost-effective and easy-to-use alternative that would allow comprehensive detection of chromosomal aberrations and structural variants, which could give rise to an era of "next-generation cytogenetics."

16p13.11 microduplication in 45 new patients: refined clinical significance and genotype-phenotype correlations.

BACKGROUND: The clinical significance of 16p13.11 duplications remains controversial while frequently detected in patients with developmental delay (DD), intellectual deficiency (ID) or autism spectrum disorder (ASD). Previously reported patients were not or poorly characterised. The absence of consensual recommendations leads to interpretation discrepancy and makes genetic counselling challenging. This study aims to decipher the genotype-phenotype correlations to improve genetic counselling and patients' medical care. METHODS: We retrospectively analysed data from 16 013 patients referred to 12 genetic centers for DD, ID or ASD, and who had a chromosomal microarray analysis. The referring geneticists of patients for whom a 16p13.11 duplication was detected were asked to complete a questionnaire for detailed clinical and genetic data for the patients and their parents. RESULTS: Clinical features are mainly speech delay and learning disabilities followed by ASD. A significant risk of cardiovascular disease was noted. About 90% of the patients inherited the duplication from a parent. At least one out of four parents carrying the duplication displayed a similar phenotype to the propositus. Genotype-phenotype correlations show no impact of the size of the duplicated segment on the severity of the phenotype. However, NDE1 and miR-484 seem to have an essential role in the neurocognitive phenotype. CONCLUSION: Our study shows that 16p13.11 microduplications are likely pathogenic when detected in the context of DD/ID/ASD and supports an essential role of NDE1 and miR-484 in the neurocognitive phenotype. Moreover, it suggests the need for cardiac evaluation and follow-up and a large study to evaluate the aortic disease risk.

Whole genome paired-end sequencing elucidates functional and phenotypic consequences of balanced chromosomal rearrangement in patients with developmental disorders.

BACKGROUND: Balanced chromosomal rearrangements associated with abnormal phenotype are rare events, but may be challenging for genetic counselling, since molecular characterisation of breakpoints is not performed routinely. We used next-generation sequencing to characterise breakpoints of balanced chromosomal rearrangements at the molecular level in patients with intellectual disability and/or congenital anomalies. METHODS: Breakpoints were characterised by a paired-end low depth whole genome sequencing (WGS) strategy and validated by Sanger sequencing. Expression study of disrupted and neighbouring genes was performed by RT-qPCR from blood or lymphoblastoid cell line RNA. RESULTS: Among the 55 patients included (41 reciprocal translocations, 4 inversions, 2 insertions and 8 complex chromosomal rearrangements), we were able to detect 89% of chromosomal rearrangements (49/55). Molecular signatures at the breakpoints suggested that DNA breaks arose randomly and that there was no major influence of repeated elements. Non-homologous end-joining appeared as the main mechanism of repair (55% of rearrangements). A diagnosis could be established in 22/49 patients (44.8%), 15 by gene disruption (KANSL1, FOXP1, SPRED1, TLK2, MBD5, DMD, AUTS2, MEIS2, MEF2C, NRXN1, NFIX, SYNGAP1, GHR, ZMIZ1) and 7 by position effect (DLX5, MEF2C, BCL11B, SATB2, ZMIZ1). In addition, 16 new candidate genes were identified. Systematic gene expression studies further supported these results. We also showed the contribution of topologically associated domain maps to WGS data interpretation. CONCLUSION: Paired-end WGS is a valid strategy and may be used for structural variation characterisation in a clinical setting.

A de novo 10p11.23-p12.1 deletion recapitulates the phenotype observed in WAC mutations and strengthens the role of WAC in intellectual disability and behavior disorders.

Chromosomal microarray analysis has become a powerful diagnostic tool in the investigation of patients with intellectual disability leading to the discovery of dosage sensitive genes implicated in the manifestation of various genomic disorders. Interstitial deletions of the short arm of chromosome 10 represent rare genetic abnormalities, especially those encompassing the chromosomal region 10p11-p12. To date, only 10 postnatal cases with microdeletion of this region have been described, and all patients shared a common phenotype, including intellectual disability, abnormal behavior, distinct dysmorphic features, visual impairment, and cardiac malformations. WAC was suggested to be the main candidate gene for intellectual disability associated with 10 p11-p12 deletion syndrome. Here, we describe a new case of de novo 10p11.23-p12.1 microdeletion in a patient with intellectual disability, abnormal behavior, and distinct dysmorphic features. Our observation allows us to redefine the smallest region of overlap among patients reported so far, with a size of 80 Kb and which contains only the WAC gene. These findings strengthen the hypothesis that haploinsufficency of WAC gene might be likely responsible for intellectual disability and behavior disorders. Our data also led us to propose a clinical pathway for patients with this recognizable genetic syndrome depending on the facial dysmorphisms. © 2016 Wiley Periodicals, Inc.

Clinical and molecular delineation of Tetrasomy 9p syndrome: report of 12 new cases and literature review.

Tetrasomy 9p is a generic term describing the presence of a supernumerary chromosome incorporating two copies of the 9p arm. Two varieties exist: isodicentric chromosome 9p (i(9p)), where the two 9p arms are linked by a single centromeric region, and pseudodicentric 9p (idic(9p)), where one active and one inactive centromere are linked together by a proximal segment of 9q that may incorporate euchromatic material. In living patients, i(9p) and idic(9p) are usually present in a mosaic state. Fifty-four cases, including fetuses, have been reported, of which only two have been molecularly characterized using array-CGH. Tetrasomy 9p leads to a variable phenotype ranging from multiple congenital anomalies with severe intellectual disability and growth delay to subnormal cognitive and physical developments. Hypertelorism, abnormal ears, microretrognathia and bulbous nose are the most common dysmorphic traits. Microcephaly, growth retardation, joint dislocation, scoliosis, cardiac and renal anomalies were reported in several cases. Those physical anomalies are often, but not universally, accompanied by intellectual disability. The most recurrent breakpoints, defined by conventional cytogenetics, are 9p10, 9q12 and 9q13. We report on 12 new patients with tetrasomy 9p (3 i(9p), 8 idic(9p) and one structurally uncharacterized), including the first case of parental germline mosaicism. All rearrangements have been characterized by DNA microarray. Based on our results and a review of the literature, we further delineate the prenatal and postnatal clinical spectrum of this imbalance. Our results show poor genotype-phenotype correlations and underline the need of precise molecular characterization of the supernumerary marker.

Dose dependent expression of HDAC4 causes variable expressivity in a novel inherited case of brachydactyly mental retardation syndrome.

Histone deacetylase 4 (HDAC4) serves important roles in multiple human systems, including neurological, cardiac, and skeletal functions. Mutation or deletion of HDAC4 causes brachydactyly mental retardation syndrome (BDMR), a disorder that includes intellectual disability, behavioral abnormalities, autism spectrum disorder, and craniofacial and skeletal anomalies, including brachydactyly type E. We present a case of familial BDMR, including a parent with mild symptoms of the disorder and a child exhibiting a more severe phenotype. Cytogenetic testing showed a cryptic balanced translocation in the mother that resulted in a 2q37.1 monosomy and a 10q26.1 trisomy in the son. Gene expression analyses demonstrated 67% HDAC4 expression in the mother and 23% HDAC4 expression in the son relative to normal controls, lending evidence to the hypothesis that HDAC4 modulates severity of this disorder in a dosage-dependent manner.

First reported case of interstitial 15 q15.3-q21.3 deletion diagnosed prenatally and characterized with array CGH in a fetus with an isolated short femur.

We report on a fetus with an isolated short femur detected by ultrasound and a de novo interstitial deletion of chromosome 15. The deletion was diagnosed prenatally by karyotype and further mapped by fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array-CGH) to bands 15q15.3 to 15q21.3 with a size of 11.11 Mb. Fetal autopsy showed characteristic minor anomalies, urinary abnormalities, and delayed bone maturation, but neither craniosynostosis, nor congenital heart defects as observed in previously reported cases. Despite the existence of ultrasound abnormalities, all five cases reported so far were diagnosed after birth. This is the first case of an interstitial deletion involving chromosomal band 15q15.3-q21.3 diagnosed prenatally and characterized at the molecular level. Our observation suggests the absence of imprinted genes in the area of 15q15-q22 and strengthens the hypothesis that a critical region for craniosynostosis may be mapped outside the deleted region in the present patient.

Atypical presentations of 22q11.2 deletion syndrome: explaining the genetic defects and genome architecture.

22q11.2 deletion syndrome, the most common microdeletion syndrome, exhibits a broad range of phenotypes, implying a cumbersome diagnosis due to atypical or paucisymptomatic presentations. We present two atypical cases of 22q11.2 deletion syndrome and suggest a preferential occurrence of the breakpoints in regions poor in repetitive elements of SINE/Alu family.

Amniocentesis performed for karyotyping after identified ultrasonographic abnormalities: what to expect?

OBJECTIVES: The aim of this study was to evaluate the relative risk of identifying fetal chromosomal anomalies after finding ultrasonographic (US) abnormalities in a high-risk population who underwent amniocentesis. METHODS: A retrospective review of a cohort of patients with single pregnancies who underwent genetic amniocentesis was undertaken. Univariate and multivariate analysis were used to determine the best correlations between US findings and chromosomal abnormalities. RESULTS: Overall, 191 chromosomal abnormalities were found in 5,604 fetuses (3.4%). Multivariate analysis showed chromosomal abnormalities were significant ly associated with anomalies of the central nervous system (OR = 4.4, 95% CI 2.2-8.7), face and neck (OR = 15.7, 95% CI 9.2-26.8), heart (OR = 5.4, 95% CI 2.6-11.2), abdomen (OR = 5.6, 95% CI 2.9-10.9), extremities (OR = 5.7, 95% CI 2.4-13.4), an increased nuchal fold (OR = 5.2, 95% CI 3.3-8.1), an intrauterine growth restriction (OR = 3.6, 95% CI 1.6-7.9) and a short femur (OR = 4.1, 95% CI 1.4-12.1). CONCLUSIONS: Our results confirm the validity of specific US markers in detecting chromosomal abnormalities in the fetus.

A Rare Chromosome Rearrangement Leading to de la Chapelle Syndrome with a Mosaic 45,X Cell Line: (46,X,psu dic(X;Y)(p22.13;q11.221)/45,X/45,psu dic(X;Y)(p22.13;q11.221).

Infertility affects about 15% of couples of childbearing age. About half of these cases can be attributed predominantly to a male factor, such as a quantitative or qualitative impairment in spermatogenesis. The first-line genetic screening for non-obstructive azoospermia is limited to karyotyping (to identify chromosome abnormalities) and Y chromosome microdeletions screening, with a view to explaining the spermatogenetic failure and evaluating the likelihood of sperm retrieval in a testicular biopsy. For patients with de la Chapelle syndrome (a 46,XX karyotype with the presence of SRY (Sex determining region Y) gene) and/or Y chromosome microdeletions, or sex chromosome mosaicism, sperm retrieval is usually unsuccessful. Here, we report a patient with de la Chapelle syndrome and a short stature caused by mosaicism and a very rare chromosome rearrangement: mos 46,X,psu dic(X;Y)/45,X/45,psu dic(X;Y). This case indicates that in de la Chapelle syndrome, X- and Y-chromosome breakpoint variability is high.

Molecular cytogenetic characterization of the first reported case of inv dup del 20p compatible with a U-type exchange model.

Inverted duplications with terminal deletions have been reported for an increasing number of chromosome ends. The best characterized and most frequent rearrangement reported involves the short arm of chromosome 8. It derives from non-allelic homologous recombination (NAHR) between two inverted LCRs (low copy repeats) of the olfactory receptor (OR) gene cluster during maternal meiosis. We report here on the cytogenetic characterization of the first inversion duplication deletion involving the short arm of chromosome 20 (inv dup del 20p) in an 18-month-old boy presenting with clinical signs consistent with 20p trisomy syndrome. This abnormality was suspected on karyotyping, but high-resolution molecular cytogenetic investigations were required to define the breakpoints of the rearrangement and to obtain insight into the mechanism underlying its formation. The duplicated region was estimated to be 18.16 Mb in size, extending from 20p13 to 20p11.22, and the size of the terminal deletion was estimated at 2.02 Mb in the 20p13 region. No single copy region was detected between the deleted and duplicated segments. As neither LCR nor inversion was identified in the 20p13 region, the inv dup del (20p) chromosome abnormality probably did not arise by NAHR. The most likely mechanism involves a break in the 20p13 region, leading to chromosome instability and reparation by U-type exchange or end-to-end fusion.

Two cases of mosaicism for complex chromosome rearrangements (CCRM) associated with secondary infertility.

Structural chromosomal abnormalities can be associated with infertility through meiosis impairment or the formation of unbalanced gametes. Among these structural abnormalities, complex chromosomal rearrangements (CCR) relate to situations with more than two breakpoints and/or more than two chromosomes involved. Mosaic balanced chromosomal rearrangements are very rare events usually ascertained through infertility, recurrent miscarriages or liveborn abnormal children. Mosaicism for complex chromosome rearrangements (CCRM) has never been described to date. Here we report on two patients with secondary infertility whose karyotype revealed mosaic partially cryptic CCRs, revealed by fluorescence in situ hybridization (FISH). To our knowledge, this is the first report of mosaicism for complex chromosomal rearrangements (CCRM). We discuss the importance of molecular cytogenetic characterization of structural rearrangements to assist in genetic counseling related to the possible use of assisted reproductive technology.

Pure familial 6q21q22.1 duplication in two generations.

Familial transmissions of unbalanced chromosomal abnormalities are rare. We report here the first case of a maternally inherited pure partial duplication of the long arm of chromosome 6 [46,XX,dup(6)(q21q22.1)mat]. The proband was referred for karyotyping as she presented intrauterine growth retardation (IUGR), moderate mental retardation and facial dysmorphism. Molecular cytogenetics analysis with various BACs showed a duplication of 5-10 Mb between 6q21 and 6q22.1. The proband's mother was found to have the same chromosome abnormality and a similar phenotype, but less severe dysmorphism. This variability in clinical findings between generations may have several causes, including attenuation with aging, imprinting or mosaicism. Only three other cases of pure partial 6q duplication similar to that of our case have been reported. The available information for all four cases was used to refine the karyotype-phenotype correlations for duplications of the 6q21q22 segment.

Neonatal Silver-Russell syndrome with maternal uniparental heterodisomy, trisomy 7 mosaicism, and dysplasia of the cerebellum.

OBJECTIVES: We report here the unusual association of Silver-Russell syndrome (SRS) and cerebellar dysplasia with trisomy 7 mosaicism and maternal uniparental disomy of chromosome 7 [UPD(7)m]. METHODS: Low-level trisomy 7 mosaicism was diagnosed prenatally on amniocytes, and UPD(7)m was confirmed after birth. RESULTS: Medical examination at birth showed dysmorphic facial features of SRS. Cytogenetic analysis on several tissues and cells confirmed mosaic trisomy 7. Unusual severe psychomotor retardation, hypotonia, and choreoathetoid movement were noted at 6 months. Brain magnetic resonance imaging showed both cerebellar hypoplasia and dysplasia. CONCLUSIONS: This unusual association of SRS and dysplasia of the cerebellum might be related to the presence of the trisomy 7 mosaicism on the cerebellum. Our observation strengthens the hypothesis that the phenotype observed in patients with SRS with UPD(7)m might also result from an undetected low level of trisomy 7 mosaicism that could best be revealed by performing cytogenetic investigations.

Sperm FISH analysis in two healthy infertile brothers with t(15;18) unbalanced translocation: Implications for genetic counselling and reproductive management.

Numerous studies have shown that balanced reciprocal or Robertsonian translocations and inversions are associated with reduced or absent sperm production. In contrast, a similar association has been rarely reported for unbalanced translocations. An unbalanced translocation, 45,XY,-15,der(18)t(15;18)(q11.2;q23), was found in two healthy infertile brothers who were referred to our hospital together with their partners for infertility. At least two routine semen analyses and karyotyping were done for each of the brothers. Sperm meiotic segregation was studied for both with a three-color FISH assay using locus-specific probes. Semen analyses showed a severe oligo-astheno-teratozoospermia with remarkably similar profiles in the two brothers. The unbalanced translocation had a deletion of 15pter-15q11.2 as well as a deletion of 18q23-18qter. The meiotic segregation was similar in the two brothers with a prevalence of alternate segregation mode. However, no phenotypic effect in the offspring can be expected only if the normal chromosomes 15 and 18 are transmitted to progeny. According to the sperm FISH results, the theoretical probability of this happening is about 25%. Based on the overall results, genetic and reproductive counselling was offered to both couples. Finally, both couples chose the alternative of donor insemination rather than preimplantation genetic diagnosis. The present study helps delineating a phenotypically silent CNV at the distal part of chromosome 18 long arm and illustrates the advantages of an integrated multidisciplinary genetic, reproductive and psychological approach to give the best possible assistance to couples who are faced with a complex and distressing genetic cause of infertility.

Optimized criteria for using fluorescence in situ hybridization in the prenatal diagnosis of common aneuploidies.

OBJECTIVE: To evaluate the medical and economic performance of three strategies for selecting patients eligible for interphase FISH in the prenatal diagnosis of common aneuploidies. METHODS: We evaluated three protocols on the same population that was referred for prenatal diagnosis between June 2001 and December 2006. The number of aneuploidies detected by FISH and the relative cost (reagent and technical staff cost) are reported for each strategy. RESULTS: 2707 women were referred for prenatal diagnosis either because of advanced maternal age over 38 (48%), abnormal maternal serum screening (35%) or prenatal ultrasound anomalies (17%). A total of 4.8% chromosomal anomalies (balanced and unbalanced) were diagnosed after karyotyping. Theoretically, interphase FISH should have detected 79.4% of the unbalanced anomalies. We observed a significant improvement in the trisomy 21 detection by selecting the probes according to the reason for referral. The last protocol adopted, which offers a rapid test to 57% of women undergoing amniocentesis, presents the best aneuploidy detection rate (68% of total aneuploidies, 87% of trisomy 21). CONCLUSION: Selecting probes according to medical criteria patients combined with a technical procedure modification allows medico-economic improvement of interphase FISH in routine diagnosis.

First reported case of intrachromosomal cryptic inv dup del Xp in a boy with developmental retardation.

We report here on a 6-year-old boy referred to the laboratory for karyotyping and SHOX microdeletion testing. The most significant clinical findings in this boy were small stature, Madelung deformity, facial dysmorphism, mild mental retardation and behavioral problems. R-, G- and RTBG-banding chromosome analysis showed a normal male karyotype. Fine molecular characterization, by FISH, of terminal Xp microdeletion revealed an associated partial duplication. Further refinement of the molecular analysis indicated an inverted duplication of the Xp22.31-Xp22.32 (13.7 Mb) region including the STS, VCX-A and KAL1 genes, associated with a terminal Xp deletion Xp22.33-Xpter (3.6 Mb) encompassing the SHOX and ARSE genes. Such rearrangements have been characterized for other chromosomal pairs, but this is the first reported male patient involving the short arm of the X chromosome. Molecular analysis of the maternal and patient's microsatellite markers showed interchromatid mispairing leading to non-allelic homologous recombination to be the most likely mechanism underlying this rearrangement. This case highlights the importance of clinically driven FISH investigations in order to uncover cryptic micro-rearrangements.