Carrie Hanscom

Assessment of 2q23.1 microdeletion syndrome implicates MBD5 as a single causal locus of intellectual disability, epilepsy, and autism spectrum disorder

Persons with neurodevelopmental disorders or autism spectrum disorder (ASD) often harbor chromosomal microdeletions, yet the individual genetic contributors within these regions have not been systematically evaluated. We established a consortium of clinical diagnostic and research laboratories to accumulate a large cohort with genetic alterations of chromosomal region 2q23.1 and acquired 65 subjects with microdeletion or translocation. We sequenced translocation breakpoints; aligned microdeletions to determine the critical region; assessed effects on mRNA expression; and examined medical records, photos, and clinical evaluations. We identified a single gene, methyl-CpG-binding domain 5 (MBD5), as the only locus that defined the critical region. Partial or complete deletion of MBD5 was associated with haploinsufficiency of mRNA expression, intellectual disability, epilepsy, and autistic features. Fourteen alterations, including partial deletions of noncoding regions not typically captured or considered pathogenic by current diagnostic screening, disrupted MBD5 alone. Expression profiles and clinical characteristics were largely indistinguishable between MBD5-specific alteration and deletion of the entire 2q23.1 interval. No copy-number alterations of MBD5 were observed in 7878 controls, suggesting MBD5 alterations are highly penetrant. We surveyed MBD5 coding variations among 747 ASD subjects compared to 2043 non-ASD subjects analyzed by whole-exome sequencing and detected an association with a highly conserved methyl-CpG-binding domain missense variant, p.79Gly>Glu (c.236G>A) (p = 0.012). These results suggest that genetic alterations of MBD5 cause features of 2q23.1 microdeletion syndrome and that this epigenetic regulator significantly contributes to ASD risk, warranting further consideration in research and clinical diagnostic screening and highlighting the importance of chromatin remodeling in the etiology of these complex disorders.

Next-Generation Sequencing Strategies Enable Routine Detection of Balanced Chromosome Rearrangements for Clinical Diagnostics and Genetic Research

The contribution of balanced chromosomal rearrangements to complex disorders remains unclear because they are not detected routinely by genome-wide microarrays and clinical localization is imprecise. Failure to consider these events bypasses a potentially powerful complement to single nucleotide polymorphism and copy-number association approaches to complex disorders, where much of the heritability remains unexplained. To capitalize on this genetic resource, we have applied optimized sequencing and analysis strategies to test whether these potentially high-impact variants can be mapped at reasonable cost and throughput. By using a whole-genome multiplexing strategy, rearrangement breakpoints could be delineated at a fraction of the cost of standard sequencing. For rearrangements already mapped regionally by karyotyping and fluorescence in situ hybridization, a targeted approach enabled capture and sequencing of multiple breakpoints simultaneously. Importantly, this strategy permitted capture and unique alignment of up to 97% of repeat-masked sequences in the targeted regions. Genome-wide analyses estimate that only 3.7% of bases should be routinely omitted from genomic DNA capture experiments. Illustrating the power of these approaches, the rearrangement breakpoints were rapidly defined to base pair resolution and revealed unexpected sequence complexity, such as co-occurrence of inversion and translocation as an underlying feature of karyotypically balanced alterations. These findings have implications ranging from genome annotation to de novo assemblies and could enable sequencing screens for structural variations at a cost comparable to that of microarrays in standard clinical practice.

Disruption of a Large Intergenic Noncoding RNA in Subjects with Neurodevelopmental Disabilities

Large intergenic noncoding (linc) RNAs represent a newly described class of ribonucleic acid whose importance in human disease remains undefined. We identified a severely developmentally delayed 16-year-old female with karyotype 46,XX,t(2;11)(p25.1;p15.1)dn in the absence of clinically significant copy number variants (CNVs). DNA capture followed by next-generation sequencing of the translocation breakpoints revealed disruption of a single noncoding gene on chromosome 2, LINC00299, whose RNA product is expressed in all tissues measured, but most abundantly in brain. Among a series of additional, unrelated subjects referred for clinical diagnostic testing who showed CNV affecting this locus, we identified four with exon-crossing deletions in association with neurodevelopmental abnormalities. No disruption of the LINC00299 coding sequence was seen in almost 14,000 control subjects. Together, these subjects with disruption of LINC00299 implicate this particular noncoding RNA in brain development and raise the possibility that, as a class, abnormalities of lincRNAs may play a significant role in human developmental disorders.

Disruption of MBD5 contributes to a spectrum of psychopathology and neurodevelopmental abnormalities

Microdeletions of chromosomal region 2q23.1 that disrupt MBD5 (methyl-CpG-binding domain protein 5) contribute to a spectrum of neurodevelopmental phenotypes; however, the impact of this locus on human psychopathology has not been fully explored. To characterize the structural variation landscape of MBD5 disruptions and the associated human psychopathology, 22 individuals with genomic disruption of MBD5 (translocation, point mutation and deletion) were identified through whole-genome sequencing or cytogenomic microarray at 11 molecular diagnostic centers. The genomic impact ranged from a single base pair to 5.4 Mb. Parents were available for 11 cases, all of which confirmed that the rearrangement arose de novo. Phenotypes were largely indistinguishable between patients with full-segment 2q23.1 deletions and those with intragenic MBD5 rearrangements, including alterations confined entirely to the 5′-untranslated region, confirming the critical impact of non-coding sequence at this locus. We identified heterogeneous, multisystem pathogenic effects of MBD5 disruption and characterized the associated spectrum of psychopathology, including the novel finding of anxiety and bipolar disorder in multiple patients. Importantly, one of the unique features of the oldest known patient was behavioral regression. Analyses also revealed phenotypes that distinguish MBD5 disruptions from seven well-established syndromes with significant diagnostic overlap. This study demonstrates that haploinsufficiency of MBD5 causes diverse phenotypes, yields insight into the spectrum of resulting neurodevelopmental and behavioral psychopathology and provides clinical context for interpretation of MBD5 structural variations. Empirical evidence also indicates that disruption of non-coding MBD5 regulatory regions is sufficient for clinical manifestation, highlighting the limitations of exon-focused assessments. These results suggest an ongoing perturbation of neurological function throughout the lifespan, including risks for neurobehavioral regression.

Haploinsufficiency of KDM6A is associated with severe psychomotor retardation, global growth restriction, seizures and cleft palate

We describe a female subject (DGAP100) with a 46,X,t(X;5)(p11.3;q35.3)inv(5)(q35.3q35.1)dn, severe psychomotor retardation with hypotonia, global postnatal growth restriction, microcephaly, globally reduced cerebral volume, seizures, facial dysmorphia and cleft palate. Fluorescence in situ hybridization and whole-genome sequencing demonstrated that the X chromosome breakpoint disrupts KDM6A in the second intron. No genes were directly disrupted on chromosome 5. KDM6A is a histone 3 lysine 27 demethylase and a histone 3 lysine 4 methyltransferase. Expression of KDM6A is significantly reduced in DGAP100 lymphoblastoid cells compared to control samples. We identified nine additional cases with neurodevelopmental delay and various other features consistent with the DGAP100 phenotype with copy number variation encompassing KDM6A from microarray databases. We evaluated haploinsufficiency of kdm6a in a zebrafish model. kdm6a is expressed in the pharyngeal arches and ethmoid plate of the developing zebrafish, while a kdm6a morpholino knockdown exhibited craniofacial defects. We conclude KDM6A dosage regulation is associated with severe and diverse structural defects and developmental abnormalities.

Prenatal diagnosis of chromothripsis, with nine breaks characterized by karyotyping, FISH, microarray and whole‐genome sequencing

Detection of de novo complex chromosomal rearrangements (CCR) in prenatal testing is extremely rare. CCRs are defined as constitutional structural rearrangements involving three or more chromosomes or more than three breakpoints. A survey of 269,371 prenatal studies1 detected only 0.03% complex rearrangements out of 246 that were determined to be de novo. Recent whole-genome sequencing studies using large-insert jumping libraries have found that cryptic complexity, particularly cryptic inversions, often occurs at the breakpoints and in some cases can introduce a degree of complexity as significant as ‘chromothripsis’ to events that appear to be canonical rearrangements at karyotypic resolution2. The term chromothripsis was initially coined by Stephens et al.3 to explain the mechanism involved in massive chromosomal rearrangements in cancers. Once defined, the concept was widely adopted to help explain complex rearrangements in the germline 4,2. All reported chromothripsis rearrangements share several features in common, including: a) The occurrence of a single catastrophic genomic event resulting in chromosome shattering. The shattered pieces contain double stranded breaks that are reassembled into mosaic chromosomes. b) The reassembly of the majority of fragments of DNA in what appears to be random fashion with little sequence homology at the breakpoints. c) Unique to congenital chromothripsis, the lack of major duplications and deletions during reassembly. Most genomic changes detected in the germline are copy neutral or span only a few base pairs, lacking the frequent larger deletions and duplications observed in cancer chromothripsis. This is likely due to selection in these cases for fetal viability. There is also growing evidence that chromothripsis occurs mainly in spermatogenesis. For reviews, see Kloosterman et. al. 20114, 20125, 20136 and Pellestor, 20147. We report a prenatal case initially diagnosed by karyotyping as a CCR with 6 breakpoints, 5 chromosomes involved in a four-way translocation and a separate two-way translocation. The p and q arms of the same chromosome 18 were involved in distinct translocations. Further analysis by whole-genome sequencing showed that two of the breakpoints were more complex than seen by karyotype, giving a total of 9 breakpoints in 5 chromosomes. Small < 1000 bp deletions or duplications were detected at these breakpoints, which interrupted 7 genes. We believe this case fits the criteria for chromothripsis. A 28 year old woman in her first pregnancy presented for amniocentesis sampling at 21 weeks gestation. Ultrasound and MRI revealed bilateral ventriculomegaly (13mm and 15mm) and colpocephaly, with partial agenesis of the corpus callosum. The prior family history was unremarkable with no unusual environmental exposures known to the mother or father upon questioning. The initial FISH analysis with AneuVysion (Abbott), suggested a normal female. However, the pregnancy was terminated at 22 weeks due to the ultrasound findings. Cytogenetic and FISH analysis with telomere probes on amniocytes harvested post termination revealed a 46,XX,t(3;18;5;7)(p25;p11.2;q13.3;q32),t(9;18)(p22;q21) karyotype in all cells examined. SNP oligonucleotide microarray analysis (Affymetrix Cytoscan HD) on fetal DNA showed no loss or gain of chromosomal material at any of the breakpoints. This unusual complex karyotype was confirmed in fetal kidney cells. Chromosomes from both parents were normal. Fetal genomic DNA was accessioned in the Developmental Genome Anatomy Project as case DGAP259. Next generation sequencing of fetal genomic DNA using large-insert jumping libraries at ~3 kb resolution, followed by PCR and Sanger validation, resolved 5 of the putative breaks. In addition to the 6 visible breakpoints, a 184.5kb cryptic inversion at the chr3/chr18 junction on the p arm of the derivative 18 was identified and the sixth break point on the derivative 5 was found to be more complex, involving the insertion of small portions of chromosomes 3 and 7 at the chr5/chr18 junction (Figure 1). The breakpoints were refined to 3p24.3, 3p26.3; 5q14.3; 7q35, 7q36.3 and inv(18p11.31p11.31). The formula for the 4 chromosome translocation was thus revised as: t(3;18;5;7)(7qtel→7q36.3::3p24.3→3qter;3pter→3p26.3::18p11.31p11.31::18p11.31→18q2 1.31::9p23→9qter;5pter→5q14.3::7q35→7q36.3::3p24.3→3p26.3::18p11.31→18pter;7pter →7q35::5q14.3-5qter). The two-chromosome translocation was rewritten as t(9;18)(p23;q21.31). Figure 1 A. G-banded karyotype from kidney culture of the terminated fetus. Arrows indicate the 6 visible breakpoints. B. Diagram of the complex rearrangement after information from whole genome sequencing. The arrows indicate areas of complexity in 19p and 5q ... Using the new nomenclature for sequenced breakpoints proposed by Ordulu et al.8, this would be written as: “46,XX,t(3;18;5;7)(p25;p11.2;q13.3;q32),t(9;18)(p22;q21)dn.seq[GRCh37/hg19](3,5,7,9,18)cx,der(3)(7qter->7q36.3(155,701,797)::3p24.3(17,392,144)->3qter) dn,der(5)(5pter->5q14.3(88,756,2{48-56})::7q35q36.3(147,718,91{1-9}-155,700,873)::AGAAC::3p24.3p26.3(17,392,136-1,408,99{6})::18p11.31(6,375,05{1})->1 8pter)dn,der(7)(7pter->7q35(147,718,90{7-8})::5q14.3(88,756,2{39-40})->5qte r)dn,der(9)(18qter->18q21.31(54,660,13{8})::9p23(9,646,47{5})->9qter)dn,der (18)(3pter->3p26.3(1,408,984)::18p11.31(6,559,611-6,375,0{52-48})::18p11.31 q21.31(6,559,{598-602}-54,660,136)::9p23(9,646,471)->9pter)dn” Seven OMIM annotated genes were disrupted at the breakpoints, with small base pair losses or gains in all 7 genes (Figure 2). On chromosome 3, CNTN6 (a neuronal membrane protein that functions as a cell adhesion molecule, believed to play a role in the development of the nervous system) and TBC1D5 (acts as a GTPase-activating protein for Rab family protein(s)) are interrupted. On chromosome 7, CNTNAP2 (a member of the neurexin family that acts as a cell adhesion molecule in the vertebrate nervous system and is implicated in numerous neurodevelopmental disorders) is disrupted. On chromosome 9, PTPRD (a signaling molecule regulating cell growth and development is disrupted. On chromosome 18, L3MBTL4 (a conserved gene down regulated or mutated in tumors, LOC100130480 (uncharacterized) and WDR7 (a gene possibly involved in cell cycle progression and gene regulation) are disrupted. The chromosome 5 breakpoint does not involve any gene disruption but does contain a gain of 18 bp. The 7q36.3 breakpoint does not involve a gene but resulted in a loss of 923 bp. This brings the total number of breaks in this chromosome complement to 9. Figure 2 Reassembly of all chromosomal regions that were involved in the translocations, according to HG19 (www.genome.ucsc.edu). At each breakpoint interrupted genes are shown above and bps of gain or deletion are shown below. The gains and losses were all well ... In our case the pregnancy was terminated because of the ultrasound abnormalities: a complete fetal autopsy was performed, which showed a very small brain for gestation (40 gm. vs. normal 75 gm.), the ventriculomegaly seen on fetal MRI, and an absent left kidney and small right kidney. The corpus callosum could not be visualized. All other structures were unremarkable. In the absence of USG detected anomalies, it will be very difficult to provide a risk for developmental abnormalities when chromothripsis is detected prenatally. Most reported cases with clinical data have been detected postnatally as apparently balanced rearrangements in patients with developmental delay. The spectrum of phenotype in individuals with chromothripsis “balanced” at the array level is yet to be determined, but will presumably reflect the nature of the disrupted genes. Chromothripsis seen prenatally is unlikely to contain major imbalances because of in utero selection for survival to the time of diagnosis. The characterization of this extremely complex abnormality illustrates the necessity of both cytogenetic and molecular testing. Chiang et al.2 sequenced 141 breakpoints from what were originally classified as cytogenetically balanced rearrangements and found that 19.2% of these fit the criteria for CCRs, a much higher percentage than previously believed1. The number of congenital cases showing chromothripsis suggests that all de novo balanced rearrangements detected prenatally by karyotyping in cases with ultrasound abnormalities should ideally be further analyzed by sequencing to determine possible undetected genetic changes. It is ironic that as molecular testing is becoming extremely sophisticated, chromosomal analysis is at present the only reliable method to initially detect rearrangements that would fit the criteria for chromothripsis.

Design of Large‐Insert Jumping Libraries for Structural Variant Detection Using Illumina Sequencing

Next‐generation sequencing is an important and efficient tool for the identification of structural variation, particularly balanced chromosomal rearrangements, because such events are not routinely detected by microarray and localization of altered regions by karyotype is imprecise. Indeed, the degree of resolution that can be obtained through next‐generation technologies enables elucidation of precise breakpoints and has facilitated the discovery of numerous pathogenic loci in human disease and congenital anomalies. The protocol described here explains one type of large‐insert “jumping library” and the steps required to generate such a library for multiplexed sequencing using Illumina sequencing technology. This approach allows for cost‐efficient multiplexing of samples and provides a very high yield of fragments with large inserts, or “jumping” fragments. Curr. Protoc. Hum. Genet. 80:7.22.1‐7.22.9.

Implication of LRRC4C and DPP6 in neurodevelopmental disorders

We performed whole‐genome sequencing on an individual from a family with variable psychiatric phenotypes that had a sensory processing disorder, apraxia, and autism. The proband harbored a maternally inherited balanced translocation (46,XY,t(11;14)(p12;p12)mat) that disrupted LRRC4C, a member of the highly specialized netrin G family of axon guidance molecules. The proband also inherited a paternally derived chromosomal inversion that disrupted DPP6, a potassium channel interacting protein. Copy Number (CN) analysis in 14,077 cases with neurodevelopmental disorders and 8,960 control subjects revealed that 60% of cases with exonic deletions in LRRC4C had a second clinically recognizable syndrome associated with variable clinical phenotypes, including 16p11.2, 1q44, and 2q33.1 CN syndromes, suggesting LRRC4C deletion variants may be modifiers of neurodevelopmental disorders. In vitro, functional assessments modeling patient deletions in LRRC4C suggest a negative regulatory role of these exons found in the untranslated region of LRRC4C, which has a single, terminal coding exon. These data suggest that the probands autism may be due to the inheritance of disruptions in both DPP6 and LRRC4C, and may highlight the importance of the netrin G family and potassium channel interacting molecules in neurodevelopmental disorders.

Potential molecular consequences of transgene integration: The R6/2 mouse example

Integration of exogenous DNA into a host genome represents an important route to generate animal and cellular models for exploration into human disease and therapeutic development. In most models, little is known concerning structural integrity of the transgene, precise site of integration, or its impact on the host genome. We previously used whole-genome and targeted sequencing approaches to reconstruct transgene structure and integration sites in models of Huntington’s disease, revealing complex structural rearrangements that can result from transgenesis. Here, we demonstrate in the R6/2 mouse, a widely used Huntington’s disease model, that integration of a rearranged transgene with coincident deletion of 5,444 bp of host genome within the gene Gm12695 has striking molecular consequences. Gm12695, the function of which is unknown, is normally expressed at negligible levels in mouse brain, but transgene integration has resulted in cortical expression of a partial fragment (exons 8–11) 3’ to the transgene integration site in R6/2. This transcript shows significant expression among the extensive network of differentially expressed genes associated with this model, including synaptic transmission, cell signalling and transcription. These data illustrate the value of sequence-level resolution of transgene insertions and transcription analysis to inform phenotypic characterization of transgenic models utilized in therapeutic research.

Estrogen-related receptor gamma implicated in a phenotype including hearing loss and mild developmental delay

Analysis of chromosomal rearrangements has been highly successful in identifying genes involved in many congenital abnormalities including hearing loss. Herein, we report a subject, designated DGAP242, with congenital hearing loss (HL) and a de novo balanced translocation 46,XX,t(1;5)(q32;q15)dn. Using multiple next-generation sequencing techniques, we obtained high resolution of the breakpoints. This revealed disruption of the orphan receptor ESRRG on chromosome 1, which is differentially expressed in inner ear hair cells and has previously been implicated in HL, and disruption of KIAA0825 on chromosome 5. Given the translocation breakpoints and supporting literature, disruption of ESRRG is the most likely cause for DGAP242’s phenotype and implicates ESRRG in a monogenic form of congenital HL, although a putative contributory role for KIAA0825 in the subject’s disorder cannot be excluded.