Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries.

Balanced chromosomal abnormalities (BCAs) represent a relatively untapped reservoir of single-gene disruptions in neurodevelopmental disorders (NDDs). We sequenced BCAs in patients with autism or related NDDs, revealing disruption of 33 loci in four general categories: (1) genes previously associated with abnormal neurodevelopment (e.g., AUTS2, FOXP1, and CDKL5), (2) single-gene contributors to microdeletion syndromes (MBD5, SATB2, EHMT1, and SNURF-SNRPN), (3) novel risk loci (e.g., CHD8, KIRREL3, and ZNF507), and (4) genes associated with later-onset psychiatric disorders (e.g., TCF4, ZNF804A, PDE10A, GRIN2B, and ANK3). We also discovered among neurodevelopmental cases a profoundly increased burden of copy-number variants from these 33 loci and a significant enrichment of polygenic risk alleles from genome-wide association studies of autism and schizophrenia. Our findings suggest a polygenic risk model of autism and reveal that some neurodevelopmental genes are sensitive to perturbation by multiple mutational mechanisms, leading to variable phenotypic outcomes that manifest at different life stages.

Subsequent Forearm Fractures Following Initial Surgical Fixation.

INTRODUCTION: Forearm fractures are a common pediatric injury. Currently, there is no consensus on treatment for fractures that recur following initial surgical fixation. The objective of this study was to investigate the subsequent fracture rate and patterns and describe the treatment of these forearm fractures. METHODS: We retrospectively identified patients who underwent surgical treatment for an initial forearm fracture at our institution between 2011 and 2019. Patients were included if they sustained a diaphyseal or metadiaphyseal forearm fracture that was initially treated surgically with a plate and screw construct (plate) or elastic stable intramedullary nail (ESIN), and if they subsequently sustained another fracture that was treated at our institution. RESULTS: A total of 349 forearm fractures were treated surgically with ESIN or a plate fixation. Of these, 24 sustained another fracture, yielding a subsequent fracture rate of 10.9% for the plate cohort and 5.1% for the ESIN cohort ( P =0.056). The majority of plate refractures (90%) occurred at the proximal or distal plate edge, while 79% of the fractures treated previously with ESINs occurred at the initial fracture site ( P <0.001). Ninety percent of plate refractures required revision surgery, with 50% underwent plate removal and conversion to ESIN, and 40% underwent revision plating. Within the ESIN cohort, 64% were treated nonsurgically, 21% underwent revision ESINs, and 14% underwent revision plating. Tourniquet time for revision surgeries were shorter for the ESIN cohort (46 vs. 92 min; P =0.012). In both cohorts, all revision surgeries had no complications and healed with evidence of radiographic union. However, 9 patients (37.5%) underwent implant removal (3 plates and 6 ESINs) after subsequent fracture healing. CONCLUSIONS: This is the first study to characterize subsequent forearm fractures following both ESIN and plate fixation and to describe and compare treatment options. Consistent with the literature, refractures following surgical fixation of pediatric forearm fractures may occur at a rate ranging from 5% to 11%. ESINs are both less invasive at the time of initial surgery and can often be treated nonoperatively if there is a subsequent fracture, while plate refractures are more likely to be treated with a second surgery and have a longer average surgery time. LEVEL OF EVIDENCE: Level IV-retrospective case series.

Exonic deletions in AUTS2 cause a syndromic form of intellectual disability and suggest a critical role for the C terminus.

Genomic rearrangements involving AUTS2 (7q11.22) are associated with autism and intellectual disability (ID), although evidence for causality is limited. By combining the results of diagnostic testing of 49,684 individuals, we identified 24 microdeletions that affect at least one exon of AUTS2, as well as one translocation and one inversion each with a breakpoint within the AUTS2 locus. Comparison of 17 well-characterized individuals enabled identification of a variable syndromic phenotype including ID, autism, short stature, microcephaly, cerebral palsy, and facial dysmorphisms. The dysmorphic features were more pronounced in persons with 3'AUTS2 deletions. This part of the gene is shown to encode a C-terminal isoform (with an alternative transcription start site) expressed in the human brain. Consistent with our genetic data, suppression of auts2 in zebrafish embryos caused microcephaly that could be rescued by either the full-length or the C-terminal isoform of AUTS2. Our observations demonstrate a causal role of AUTS2 in neurocognitive disorders, establish a hitherto unappreciated syndromic phenotype at this locus, and show how transcriptional complexity can underpin human pathology. The zebrafish model provides a valuable tool for investigating the etiology of AUTS2 syndrome and facilitating gene-function analysis in the future.

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.

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.

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.

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.

Treatment outcomes of hips in patients with epiphyseal dysplasia.

Hip containment surgeries in multiple epiphyseal and spondyloepiphyseal dysplasia (MED/SED) patients aim to improve the mechanical environment of the hip joint. The purpose of this study was to determine if surgical intervention to improve femoral head coverage improved radiographic and clinical outcomes. A retrospective study identified patients with MED/SED seen in clinic between May 2000 and September 2017, with a minimum of 2-year follow-up. Patient charts/radiographs were reviewed for radiographic hip measurements, pain, and gait. Sixty-nine hips in 35 patients were identified. Forty-four hips were treated nonoperatively and 25 were treated surgically. The mean age at diagnosis was 6.2 years. The mean follow-up was 7.7 years for the surgical group and 7.1 years for the nonsurgical group. The mean postoperative follow-up was 5.4 years. Acetabular index decreased from initial to final visit by 9.0° in the surgical group and 1.6° in the nonsurgical group. Tonnis angle decreased by 13.5° in the surgical group and 1.5° in the nonsurgical group. Center edge angle increased by 19.0° in the surgical group and 7.1° in the nonsurgical group. Hips in the surgical group were 6.1 times more likely to experience an improvement in pain compared with hips in the nonsurgical group. Gait at the final follow-up was similar among the two groups. In this study cohort, containment surgery provided increased femoral head coverage; however, there was equal femoral head deformation despite intervention. Hips treated surgically were more likely to experience an improvement in pain; however, gait alterations did not improve.

Preoperative factors associated with optimal outcomes of selective thoracic fusion at 5 years.

PURPOSE: Prior work identified optimal outcomes at 2 years following selective thoracic fusion (STF) for adolescent idiopathic scoliosis (AIS) but it is unclear whether these published predictors represent what is required to achieve an optimal outcome with further time and potential growth. The purpose of this study was to determine the preoperative factors associated with optimal outcome of STF for AIS at 5 years. METHODS: Patients with primary thoracic AIS (Lenke 1-4C curves) who underwent a STF and had minimum 5 years of follow-up were included. Optimal postoperative outcomes for a STF included a deformity-flexibility quotient (DFQ) < 4, lumbar curve < 26°, lumbar correction > 37%, C7-CSVL < 2 cm, lumbar prominence < 5°, and trunk shift < 1.5 cm. These outcomes were used to determine whether adhering to published recommendations for STF increased the likelihood of obtaining an optimal outcome at 5 years, which included: preoperative lumbar curve < 45°, lumbar bend < 25°, apical vertebral translation ratio > 1.2, and thoracic/thoracolumbar Cobb ratio > 1.2. RESULTS: 127 patients met inclusion. A preoperative lumbar curve < 45° was associated with an increased likelihood of achieving three of the optimal outcomes: DFQ < 4, lumbar curve < 26°, and lumbar prominence < 5°. Following the 25° bend rule resulted in two optimal outcomes, while Cobb ratio > 1.2 was only associated with one optimal outcome. AVT > 1.2 was not significantly associated with any optimal outcome measures. CONCLUSION: This study found that at 5 years, performing a STF when there is a preoperative lumbar Cobb < 45° remained the best guideline for increasing the likelihood of an optimal outcome.

Quality improvement in post-operative opioid and benzodiazepine regimen in adolescent patients after posterior spinal fusion.

STUDY DESIGN: Prospective, quality-improvement. OBJECTIVES: To evaluate pain management following posterior spinal fusion (PSF) for adolescent idiopathic scoliosis (AIS) and Scheuermann's Kyphosis (SK) determine the optimal opioid and benzodiazepine prescription amounts, and implement a multimodal post-operative pain regimen. The incidence of prescription opioid abuse is increasing in the United States. Orthopedic spine surgeons often prescribe large quantities of opioids post-operatively for pain control. Previous efforts on pain control have focused on in-patient post-operative regimens after PSF. METHODS: Between 2/1/17 and 5/30/18 patients with AIS or SK were sent home with pain diaries after discharge to document daily narcotic, benzodiazepine, non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen and gabapentin use following PSF. Diaries were collected at the 4 week post-operative visit. Data from two cohorts were reviewed: pre-intervention and post-intervention. Our prescription intervention went into effect 9/1/17. RESULTS: Twenty-four (30%) patients returned pain diaries. The pre-intervention cohort consisted of 12 patients (7 female; 5 males; 14.9 years (range 12-19)). Patients were prescribed on average 80 × 5 mg tabs (26-140) of oxycodone but used on average 45 tabs (12-129) over an average of 17.5 days (9-33). They were prescribed an average of 30 × 2 mg tabs (0-150) of diazepam, used on average 4.8 (0-105) tabs over 12.5 (5-25) days. The post-intervention cohort consists of 12 patients (9 female; 3 male; 14.8 years (12-19)). They were prescribed on average 50 × 5 mg tabs (35-80) of oxycodone, used 20.5 (0-39.5) tabs over 8.5 days (3-16). They were prescribed on average 18 × 2 mg tabs of diazepam (0-43), used 5.4 tabs (0-19) over 10 days (5-14). CONCLUSIONS: This analysis has directly impacted clinical practice. Prescribed opioid and benzodiazepine doses have been decreased by over 50%, and more resources are being directed towards determining the disparity between the amount of medications prescribed and consumed in our post-operative patients.

Femoral derotational osteotomy level does not effect resulting torsion.

PURPOSE: The purpose of this study was to assess the effect on femoral torsion by rotational osteotomies at three different levels as measured in 3D using both the mechanical and the anatomic axes. METHODS: Ten cadaveric lower extremities underwent femoral osteotomies perpendicular to the anatomic axis (AA) at three levels: subtrochanteric, mid-diaphyseal and supracondylar. Parallel pins were placed, one in each femur segment. Computed tomography (CT) was acquired in post-osteotomies neutral position, then post-external rotation of the femur at each osteotomy level. Femurs were returned to neutral rotation between imaging exams. Using 3D CT reconstructions, custom software calculated femoral torsion (angle between the femoral neck axis and the posterior condylar axis in the transverse plane) and pin angle between segments, reoriented to both the mechanical axis (MA) and the AA. Pin angle and torsion change were compared for the three osteotomy locations (regression analysis and ANOVA performed). RESULTS: Two specimens were omitted (inadequate imaging); the remaining eight donors were 55-90 years old (mean: 64 ± 15 years), CT confirmed no bony defects. All three levels of osteotomy demonstrated significant correlations between the amount of rotation at the osteotomy (pin angle change) and the resulting change in femoral torsion (R square range 0.658-0.847). No significant differences were found between osteotomy level in torsion (MA:p = 0.285, AA:p = 0.156) or in pin angle (MA:p = 0.756, AA:p = 0.753). CONCLUSIONS: Performing a corrective rotational osteotomy orthogonal to the AA achieves the desired effect on MA regardless of location. This suggests that a surgeon's osteotomy level choice may be based on other risks/benefits of the various techniques.

Using population admixture to help complete maps of the human genome.

Tens of millions of base pairs of euchromatic human genome sequence, including many protein-coding genes, have no known location in the human genome. We describe an approach for localizing the human genome's missing pieces using the patterns of genome sequence variation created by population admixture. We mapped the locations of 70 scaffolds spanning 4 million base pairs of the human genome's unplaced euchromatic sequence, including more than a dozen protein-coding genes, and identified 8 new large interchromosomal segmental duplications. We find that most of these sequences are hidden in the genome's heterochromatin, particularly its pericentromeric regions. Many cryptic, pericentromeric genes are expressed at the RNA level and have been maintained intact for millions of years while their expression patterns diverged from those of paralogous genes elsewhere in the genome. We describe how knowledge of the locations of these sequences can inform disease association and genome biology studies.

Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration.

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically interpreted translocations and inversions. We confirm that the recently described phenomenon of 'chromothripsis' (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline, where it can resolve to a relatively balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign copy-number variants (CNVs). We compared these results to experimentally generated DNA breakage-repair by sequencing seven transgenic animals, revealing extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion was the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.