Long-Read Sequencing Improves the Detection of Structural Variations Impacting Complex Non-Coding Elements of the Genome.

The advent of long-read sequencing offers a new assessment method of detecting genomic structural variation (SV) in numerous rare genetic diseases. For autism spectrum disorders (ASD) cases where pathogenic variants fail to be found in the protein-coding genic regions along chromosomes, we proposed a scalable workflow to characterize the risk factor of SVs impacting non-coding elements of the genome. We applied whole-genome sequencing on an Emirati family having three children with ASD using long and short-read sequencing technology. A series of analytical pipelines were established to identify a set of SVs with high sensitivity and specificity. At 15-fold coverage, we observed that long-read sequencing technology (987 variants) detected a significantly higher number of SVs when compared to variants detected using short-read technology (509 variants) (p-value < 1.1020 × 10-57). Further comparison showed 97.9% of long-read sequencing variants were spanning within the 1-100 kb size range (p-value < 9.080 × 10-67) and impacting over 5000 genes. Moreover, long-read variants detected 604 non-coding RNAs (p-value < 9.02 × 10-9), comprising 58% microRNA, 31.9% lncRNA, and 9.1% snoRNA. Even at low coverage, long-read sequencing has shown to be a reliable technology in detecting SVs impacting complex elements of the genome.

FARP-1 deletion is associated with lack of response to autism treatment by early start denver model in a multiplex family.

BACKGROUND: Children with autism spectrum disorder (ASD) display impressive clinical heterogeneity, also involving treatment response. Genetic variants can contribute to explain this large interindividual phenotypic variability. METHODS: Array-CGH (a-CGH) and whole genome sequencing (WGS) were performed on a multiplex family with two small children diagnosed with ASD at 17 and 18 months of age. Both brothers received the same naturalistic intervention for one year according to the Early Start Denver Model (ESDM), applied by the same therapists, yielding dramatically different treatment outcomes. RESULTS: The older sibling came out of the autism spectrum, while the younger sibling displayed very little, in any, improvement. This boy was subsequently treated applying a structured Early Intensive Behavioral Intervention paired with Augmentative Alternative Communication, which yielded a partial response within another year. The ESDM nonresponsive child carries a novel maternally inherited 65 Kb deletion at chr. 13q32.2 spanning FARP1. Farp1 is a synaptic scaffolding protein, which plays a significant role in neural plasticity. CONCLUSION: These results represent a paradigmatic example of the heuristic potential of genetic markers in predicting treatment response and possibly in supporting the targeted prescription of specific early intervention approaches.

A large data resource of genomic copy number variation across neurodevelopmental disorders.

Copy number variations (CNVs) are implicated across many neurodevelopmental disorders (NDDs) and contribute to their shared genetic etiology. Multiple studies have attempted to identify shared etiology among NDDs, but this is the first genome-wide CNV analysis across autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), schizophrenia (SCZ), and obsessive-compulsive disorder (OCD) at once. Using microarray (Affymetrix CytoScan HD), we genotyped 2,691 subjects diagnosed with an NDD (204 SCZ, 1,838 ASD, 427 ADHD and 222 OCD) and 1,769 family members, mainly parents. We identified rare CNVs, defined as those found in <0.1% of 10,851 population control samples. We found clinically relevant CNVs (broadly defined) in 284 (10.5%) of total subjects, including 22 (10.8%) among subjects with SCZ, 209 (11.4%) with ASD, 40 (9.4%) with ADHD, and 13 (5.6%) with OCD. Among all NDD subjects, we identified 17 (0.63%) with aneuploidies and 115 (4.3%) with known genomic disorder variants. We searched further for genes impacted by different CNVs in multiple disorders. Examples of NDD-associated genes linked across more than one disorder (listed in order of occurrence frequency) are NRXN1, SEH1L, LDLRAD4, GNAL, GNG13, MKRN1, DCTN2, KNDC1, PCMTD2, KIF5A, SYNM, and long non-coding RNAs: AK127244 and PTCHD1-AS. We demonstrated that CNVs impacting the same genes could potentially contribute to the etiology of multiple NDDs. The CNVs identified will serve as a useful resource for both research and diagnostic laboratories for prioritization of variants.

Pathogenic WDFY3 variants cause neurodevelopmental disorders and opposing effects on brain size.

The underpinnings of mild to moderate neurodevelopmental delay remain elusive, often leading to late diagnosis and interventions. Here, we present data on exome and genome sequencing as well as array analysis of 13 individuals that point to pathogenic, heterozygous, mostly de novo variants in WDFY3 (significant de novo enrichment P = 0.003) as a monogenic cause of mild and non-specific neurodevelopmental delay. Nine variants were protein-truncating and four missense. Overlapping symptoms included neurodevelopmental delay, intellectual disability, macrocephaly, and psychiatric disorders (autism spectrum disorders/attention deficit hyperactivity disorder). One proband presented with an opposing phenotype of microcephaly and the only missense-variant located in the PH-domain of WDFY3. Findings of this case are supported by previously published data, demonstrating that pathogenic PH-domain variants can lead to microcephaly via canonical Wnt-pathway upregulation. In a separate study, we reported that the autophagy scaffolding protein WDFY3 is required for cerebral cortical size regulation in mice, by controlling proper division of neural progenitors. Here, we show that proliferating cortical neural progenitors of human embryonic brains highly express WDFY3, further supporting a role for this molecule in the regulation of prenatal neurogenesis. We present data on Wnt-pathway dysregulation in Wdfy3-haploinsufficient mice, which display macrocephaly and deficits in motor coordination and associative learning, recapitulating the human phenotype. Consequently, we propose that in humans WDFY3 loss-of-function variants lead to macrocephaly via downregulation of the Wnt pathway. In summary, we present WDFY3 as a novel gene linked to mild to moderate neurodevelopmental delay and intellectual disability and conclude that variants putatively causing haploinsufficiency lead to macrocephaly, while an opposing pathomechanism due to variants in the PH-domain of WDFY3 leads to microcephaly.

Rare copy number variations affecting the synaptic gene DMXL2 in neurodevelopmental disorders.

BACKGROUND: Ultra-rare genetic variants, including non-recurrent copy number variations (CNVs) affecting important dosage-sensitive genes, are important contributors to the etiology of neurodevelopmental disorders (NDDs). Pairing family-based whole-genome sequencing (WGS) with detailed phenotype data can enable novel gene associations in NDDs. METHODS: We performed WGS of six members from a three-generation family, where three individuals each had a spectrum of features suggestive of a NDD. CNVs and sequence-level variants were identified and further investigated in disease and control databases. RESULTS: We identified a novel 252-kb deletion at 15q21 that overlaps the synaptic gene DMXL2 and the gene GLDN. The microdeletion segregated in NDD-affected individuals. Additional rare inherited and de novo sequence-level variants were found that may also be involved, including a missense change in GRIK5. Multiple CNVs and loss-of-function sequence variants affecting DMXL2 were discovered in additional unrelated individuals with a range of NDDs. CONCLUSIONS: Disruption of DMXL2 may predispose to NDDs including autism spectrum disorder. The robust interpretation of private variants requires a multifaceted approach that incorporates multigenerational pedigrees and genome-wide and population-scale data.

Genome-wide copy number variation analysis identifies novel candidate loci associated with pediatric obesity.

Obesity is a multifactorial condition that is highly heritable. There have been ~60 susceptibility loci identified, but they only account for a fraction of cases. As copy number variations (CNVs) have been implicated in the etiology of a multitude of human disorders including obesity, here, we investigated the contribution of rare (<1% population frequency) CNVs in pediatric cases of obesity. We genotyped 67 such individuals, including 22 with co-morbid developmental delay and prioritized rare CNVs at known obesity-associated loci, as well as, those impacting genes involved in energy homeostasis or related processes. We identified clinically relevant or potentially clinically relevant CNVs in 15% (10/67) of individuals. Of these, 4% (3/67) had 16p11.2 microdeletions encompassing the known obesity risk gene SH2B1. Notably, we identified two unrelated probands harboring different 6p22.2 microduplications encompassing SCGN, a potential novel candidate gene for obesity. Further, we identified other biologically relevant candidate genes for pediatric obesity including ARID5B, GPR39, PTPRN2, and HNF4G. We found previously reported candidate loci for obesity, and new ones, suggesting CNV analysis may assist in the diagnosis of pediatric obesity.

The Personal Genome Project Canada: findings from whole genome sequences of the inaugural 56 participants.

BACKGROUND: The Personal Genome Project Canada is a comprehensive public data resource that integrates whole genome sequencing data and health information. We describe genomic variation identified in the initial recruitment cohort of 56 volunteers. METHODS: Volunteers were screened for eligibility and provided informed consent for open data sharing. Using blood DNA, we performed whole genome sequencing and identified all possible classes of DNA variants. A genetic counsellor explained the implication of the results to each participant. RESULTS: Whole genome sequencing of the first 56 participants identified 207 662 805 sequence variants and 27 494 copy number variations. We analyzed a prioritized disease-associated data set (n = 1606 variants) according to standardized guidelines, and interpreted 19 variants in 14 participants (25%) as having obvious health implications. Six of these variants (e.g., in BRCA1 or mosaic loss of an X chromosome) were pathogenic or likely pathogenic. Seven were risk factors for cancer, cardiovascular or neurobehavioural conditions. Four other variants - associated with cancer, cardiac or neurodegenerative phenotypes - remained of uncertain significance because of discrepancies among databases. We also identified a large structural chromosome aberration and a likely pathogenic mitochondrial variant. There were 172 recessive disease alleles (e.g., 5 individuals carried mutations for cystic fibrosis). Pharmacogenomics analyses revealed another 3.9 potentially relevant genotypes per individual. INTERPRETATION: Our analyses identified a spectrum of genetic variants with potential health impact in 25% of participants. When also considering recessive alleles and variants with potential pharmacologic relevance, all 56 participants had medically relevant findings. Although access is mostly limited to research, whole genome sequencing can provide specific and novel information with the potential of major impact for health care.

A de novo deletion in a boy with cerebral palsy suggests a refined critical region for the 4q21.22 microdeletion syndrome.

We present an 18-year-old boy with cerebral palsy, intellectual disability, speech delay, and seizures. He carries a likely pathogenic 1.3 Mb de novo heterozygous deletion in the 4q21.22 microdeletion syndrome region. He also carries a 436 kb maternally-inherited duplication impacting the first three exons of CHRNA7. The majority of previously published cases with 4q21.22 syndrome shared common features including growth restriction, muscular hypotonia, and absent or severely delayed speech. Using copy number variation (CNV) data available for other subjects, we defined a minimal critical region of 170.8 kb within the syndromic region, encompassing HNRNPD. We also identified a larger 2 Mb critical region encompassing ten protein-coding genes, of which six (PRKG2, RASGEF1B, HNRNPDL, HNRNPD, LIN54, COPS4) have a significantly low number of truncating loss-of-function mutations. Long-range chromatin interaction data suggest that this deletion may alter chromatin interactions at the 4q21.22 microdeletion region. We suggest that the deletion or misregulation of these genes is likely to contribute to the neurodevelopmental and neuromuscular abnormalities in 4q21.22 syndrome.

Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder.

We are performing whole-genome sequencing of families with autism spectrum disorder (ASD) to build a resource (MSSNG) for subcategorizing the phenotypes and underlying genetic factors involved. Here we report sequencing of 5,205 samples from families with ASD, accompanied by clinical information, creating a database accessible on a cloud platform and through a controlled-access internet portal. We found an average of 73.8 de novo single nucleotide variants and 12.6 de novo insertions and deletions or copy number variations per ASD subject. We identified 18 new candidate ASD-risk genes and found that participants bearing mutations in susceptibility genes had significantly lower adaptive ability (P = 6 × 10-4). In 294 of 2,620 (11.2%) of ASD cases, a molecular basis could be determined and 7.2% of these carried copy number variations and/or chromosomal abnormalities, emphasizing the importance of detecting all forms of genetic variation as diagnostic and therapeutic targets in ASD.

Using extended pedigrees to identify novel autism spectrum disorder (ASD) candidate genes.

Copy number variation has emerged as an important cause of phenotypic variation, particularly in relation to some complex disorders. Autism spectrum disorder (ASD) is one such disorder, in which evidence is emerging for an etiological role for some rare penetrant de novo and rare inherited copy number variants (CNVs). De novo variation, however, does not always explain the familial nature of ASD, leaving a gap in our knowledge concerning the heritable genetic causes of this disorder. Extended pedigrees, in which several members have ASD, provide an opportunity to investigate inherited genetic risk factors. In this current study, we recruited 19 extended ASD pedigrees, and, using the Illumina HumanOmni2.5 BeadChip, conducted genome-wide CNV interrogation. We found no definitive evidence of an etiological role for segregating CNVs in these pedigrees, and no evidence that linkage signals in these pedigrees are explained by segregating CNVs. However, a small number of putative de novo variants were transmitted from BAP parents to their ASD offspring, and evidence emerged for a rare duplication CNV at 11p13.3 harboring two putative 'developmental/neuropsychiatric' susceptibility gene(s), GSTP1 and NDUFV1.

Effectiveness of an acoustical product in reducing high-frequency sound within unoccupied incubators.

Few noise measurement studies in the neonatal intensive care unit have reported sound frequencies within incubators. Sound frequencies within incubators are markedly different from sound frequencies within the gravid uterus. This article reports the results of sound spectral analysis (SSA) within unoccupied incubators under control and treatment conditions. SSA indicated that acoustical foam panels (treatment condition) markedly reduced sound frequencies > or =500 Hz when compared with the control condition. The main findings of this study (a) illustrate the need to monitor high-frequency sound within incubators and (b) indicate one method to reduce atypical sound exposure within incubators.

Sound spectral analysis in the intensive care nursery: measuring high-frequency sound.

Uterine structures protect the fetus from high-frequency sounds (i.e., >500 Hz). Preterm infants are not protected from sound >500 Hz. It is necessary to assess sound frequencies to protect preterm infants from high-frequency sound. However, no noise measurement study has been reported using sound spectral analysis (SSA). This article reports the results of an SSA in two Level III neonatal intensive care units (NICUs). The results of the SSA indicated that significant high-frequency sound existed within the infant's immediate care environment. Results of SSA assessments may lend evidentiary support to clinical managers seeking to redesign traditional NICUs.