NeuroSCORE is a genome-wide omics-based model that identifies candidate disease genes of the central nervous system.

To identify candidate disease genes of central nervous system (CNS) phenotypes, we created the Neurogenetic Systematic Correlation of Omics-Related Evidence (NeuroSCORE). We identified five genome-wide metrics highly associated with CNS phenotypes to score 19,601 protein-coding genes. Genes scored one point per metric (range: 0-5), identifying 8298 scored genes (scores ≥ 1) and 1601 "high scoring" genes (scores ≥ 3). Using logistic regression, we determined the odds ratio that genes with a NeuroSCORE from 1 to 5 would be associated with known CNS-related phenotypes compared to genes that scored zero. We tested NeuroSCORE using microarray copy number variants (CNVs) in case-control cohorts and aggregate mouse model data. High scoring genes are associated with CNS phenotypes (OR = 5.5, p < 2E-16), enriched in case CNVs, and mouse ortholog genes that cause behavioral and nervous system abnormalities. We identified 1058 high scoring genes with no disease association in OMIM. Transforming the logistic regression results indicates high scoring genes have an 84-92% chance of being associated with a CNS phenotype. Top scoring genes include GRIA1, MAP4K4, SF1, TNPO2, and ZSWIM8. Finally, we interrogated CNVs in the Clinical Genome Resource, finding the majority of clinically significant CNVs contain high scoring genes. These findings can direct future research and improve molecular diagnostics.

Limitations of exome sequencing in detecting rare and undiagnosed diseases.

While exome sequencing (ES) is commonly the final diagnostic step in clinical genetics, it may miss diagnoses. To clarify the limitations of ES, we investigated the diagnostic yield of genetic tests beyond ES in our Undiagnosed Diseases Network (UDN) participants. We reviewed the yield of additional genetic testing including genome sequencing (GS), copy number variant (CNV), noncoding variant (NCV), repeat expansion (RE), or methylation testing in UDN cases with nondiagnostic ES results. Overall, 36/54 (67%) of total diagnoses were based on clinical findings and coding variants found by ES and 3/54 (6%) were based on clinical findings only. The remaining 15/54 (28%) required testing beyond ES. Of these, 7/15 (47%) had NCV, 6/15 (40%) CNV, and 2/15 (13%) had a RE or a DNA methylation disorder. Thus 18/54 (33%) of diagnoses were not solved exclusively by ES. Several methods were needed to detect and/or confirm the functional effects of the variants missed by ES, and in some cases by GS. These results indicate that tests to detect elusive variants should be considered after nondiagnostic preliminary steps. Further studies are needed to determine the cost-effectiveness of tests beyond ES that provide diagnoses and insights to possible treatment.

The Temple Grandin Genome: Comprehensive Analysis in a Scientist with High-Functioning Autism.

Autism spectrum disorder (ASD) is a heterogeneous condition with a complex genetic etiology. The objective of this study is to identify the complex genetic factors that underlie the ASD phenotype and other clinical features of Professor Temple Grandin, an animal scientist and woman with high-functioning ASD. Identifying the underlying genetic cause for ASD can impact medical management, personalize services and treatment, and uncover other medical risks that are associated with the genetic diagnosis. Prof. Grandin underwent chromosomal microarray analysis, whole exome sequencing, and whole genome sequencing, as well as a comprehensive clinical and family history intake. The raw data were analyzed in order to identify possible genotype-phenotype correlations. Genetic testing identified variants in three genes (SHANK2, ALX1, and RELN) that are candidate risk factors for ASD. We identified variants in MEFV and WNT10A, reported to be disease-associated in previous studies, which are likely to contribute to some of her additional clinical features. Moreover, candidate variants in genes encoding metabolic enzymes and transporters were identified, some of which suggest potential therapies. This case report describes the genomic findings in Prof. Grandin and it serves as an example to discuss state-of-the-art clinical diagnostics for individuals with ASD, as well as the medical, logistical, and economic hurdles that are involved in clinical genetic testing for an individual on the autism spectrum.

Abnormally Methylated FMR1 in Absence of a Detectable Full Mutation in a U.S.A Patient Cohort Referred for Fragile X Testing.

In 2016, Methylation-Specific Quantitative Melt Analysis (MS-QMA) on 3,340 male probands increased diagnostic yield from 1.60% to 1.84% for fragile X syndrome (FXS) using a pooling approach. In this study probands from Lineagen (UT, U.S.A.) of both sexes were screened using MS-QMA without sample pooling. The cohorts included: (i) 279 probands with no FXS full mutation (FM: CGG > 200) detected by AmplideX CGG sizing; (ii) 374 negative and 47 positive controls. MS-QMA sensitivity and specificity in controls approached 100% for both sexes. For male probands with no FM detected by standard testing (n = 189), MS-QMA identified abnormal DNA methylation (mDNA) in 4% normal size (NS: < 44 CGGs), 6% grey zone (CGG 45-54) and 12% premutation (CGG 54-199) alleles. The abnormal mDNA was confirmed by AmplideX methylation sensitive (m)PCR and EpiTYPER tests. In contrast, no abnormal mDNA was detected in 89 males with NS alleles from the general population. For females, 11% of 43 probands with NS alleles by the AmplideX sizing assay had abnormal mDNA by MS-QMA, with FM / NS mosaicism confirmed by AmplideX mPCR. FMR1 MS-QMA analysis can cost-effectively screen probands of both sexes for methylation and FM mosaicism that may be missed by standard testing.

Clinical significance of copy number variants involving KANK1 in patients with neurodevelopmental disorders.

Copy number variants (CNV)s involving KANK1 are generally classified as variants of unknown significance. Several clinical case reports suggest that the loss of KANK1 on chromosome 9p24.3 has potential impact on neurodevelopment. These case studies are inconsistent in terms of patient phenotype and suspected pattern of inheritance. Further complexities arise because these published reports utilize a variety of genetic testing platforms with varying resolution of the 9p region; this ultimately causes uncertainty about the impacted genomic coordinates and gene transcripts. Beyond these case reports, large case-control studies and publicly available databases statistically cast doubt as to whether variants of KANK1 are clinically significant. However, these large data sources are neither easily extracted nor uniformly applied to clinical interpretation. In this report we provide an updated analysis of the data on this locus and its potential clinical relevance. This is based on a review of the literature as well as 28 patients who harbor a single copy number variant involving KANK1 with or without DOCK8 (27 of whom are not published previously) identified by our clinical laboratory using an ultra-high resolution chromosomal microarray analysis. We note that 13 of 16 patients have a documented diagnosis of autism spectrum disorder (ASD) while only two, with documented perinatal complications, have a documented diagnosis of cerebral palsy (CP). A careful review of the CNVs suggests a transcript-specific effect. After evaluation of our case series and reconsideration of the literature, we propose that KANK1 aberrations do not frequently cause CP but cannot exclude that they represent a risk factor for ASD, especially when the coding region of the shorter, alternate KANK1 transcript (termed "transcript 4" in the UCSC Genome Browser) is impacted.

Critical exon indexing improves clinical interpretation of copy number variants in neurodevelopmental disorders.

OBJECTIVE: To evaluate a new tool to aid interpretation of copy number variants (CNVs) in individuals with neurodevelopmental disabilities. METHODS: Critical exon indexing (CEI) was used to identify genes with critical exons (CEGs) from clinically reported CNVs, which may contribute to neurodevelopmental disorders (NDDs). The 742 pathogenic CNVs and 1,363 variants of unknown significance (VUS) identified by chromosomal microarray analysis in 5,487 individuals with NDDs were subjected to CEI to identify CEGs. CEGs identified in a subsequent random series of VUS were evaluated for relevance to CNV interpretation. RESULTS: CEI identified a total of 2,492 unique CEGs in pathogenic CNVs and 953 in VUS compared with 259 CEGs in 6,965 CNVs from 873 controls. These differences are highly significant (p < 0.00001) whether compared as frequency, average, or normalized by CNV size. Twenty-one percent of VUS CEGs were not represented in Online Mendelian Inheritance in Man, highlighting limitations of existing resources for identifying potentially impactful genes within CNVs. CEGs were highly correlated with other indices and known pathways of relevance. Separately, 136 random VUS reports were reevaluated, and 76% of CEGs had not been commented on. In multiple cases, further investigation yielded additional relevant literature aiding interpretation. As one specific example, we discuss GTF2I as a CEG, which likely alters interpretation of several reported duplication VUS in the Williams-Beuren region. CONCLUSIONS: Application of CEI to CNVs in individuals with NDDs can identify genes of potential clinical relevance, aid laboratories in effectively searching the clinical literature, and support the clinical reporting of poorly annotated VUS.

Clinical utility of exome sequencing in individuals with large homozygous regions detected by chromosomal microarray analysis.

BACKGROUND: Chromosomal microarray analysis (CMA) is recommended as the first-tier clinical diagnostic test for individuals with developmental disabilities. In addition to detecting copy number variations, CMA platforms with single nucleotide polymorphism probes can detect large homozygous regions within the genome, which represent potential risk for recessively inherited disorders. METHODS: To determine the frequency in which pathogenic or likely pathogenic variants can be detected in these regions of homozygosity, we performed whole exome sequencing (WES) in 53 individuals where homozygosity was detected by CMA. These patients were referred to our clinical laboratory for a variety of neurodevelopmental conditions including autism spectrum disorder, developmental delay, epilepsy, intellectual disability and microcephaly. RESULTS: In 11.3% (6/53) of cases, the analysis of homozygous variants revealed pathogenic or likely pathogenic variants in GJB2, TPP1, SLC25A15, TYR, PCCB, and NDUFV2 which are implicated in a variety of diseases. The evaluation of heterozygous variants with autosomal dominant inheritance, compound heterozygotes and variants with X-linked inheritance revealed pathogenic or likely pathogenic variants in PNPLA4, CADM1, HBB, SOS1, SFTPC, OTC and ASMT in 15.1% (8/53) of cases. Two of these patients harbored both homozygous and heterozygous variants relevant to their phenotypes (TPP1 and OTC; GJB2 and ASMT). CONCLUSIONS: Our study highlights the clinical utility of WES in individuals whose CMA uncovers homozygosity. Importantly, we show that when the phenotype is complex and homozygosity levels are high, WES can identify a significant number of relevant variants that explain neurodevelopmental phenotypes, and these mutations may lie outside of the regions of homozygosity, suggesting that the appropriate follow up test is WES rather than targeted sequencing.

A Novel Partial Duplication of ZEB2 and Review of ZEB2 Involvement in Mowat-Wilson Syndrome.

Mowat-Wilson syndrome is a rare genetic condition characterized by intellectual disability, structural anomalies, and dysmorphic features. It is caused by haploinsufficiency of the ZEB2 gene in chromosome 2q22.3. Over 180 distinct mutations in ZEB2 have been reported, including nonsense and missense point mutations, deletions, and large chromosomal rearrangements. We report on a 14-year-old female with a clinical diagnosis of Mowat-Wilson syndrome. Chromosomal microarray identified a novel de novo 69-kb duplication containing exons 1 and 2 of the ZEB2 gene. Sequence analysis identified no other variants in this gene. This is the first report of a partial duplication of the ZEB2 gene resulting in Mowat-Wilson syndrome.

Analytical and Clinical Validity Study of FirstStepDx PLUS: A Chromosomal Microarray Optimized for Patients with Neurodevelopmental Conditions.

INTRODUCTION: Chromosomal microarray analysis (CMA) is recognized as the first-tier test in the genetic evaluation of children with developmental delays, intellectual disabilities, congenital anomalies and autism spectrum disorders of unknown etiology. ARRAY DESIGN: To optimize detection of clinically relevant copy number variants associated with these conditions, we designed a whole-genome microarray, FirstStepDx PLUS (FSDX). A set of 88,435 custom probes was added to the Affymetrix CytoScanHD platform targeting genomic regions strongly associated with these conditions. This combination of 2,784,985 total probes results in the highest probe coverage and clinical yield for these disorders. RESULTS AND DISCUSSION: Clinical testing of this patient population is validated on DNA from either non-invasive buccal swabs or traditional blood samples. In this report we provide data demonstrating the analytic and clinical validity of FSDX and provide an overview of results from the first 7,570 consecutive patients tested clinically. We further demonstrate that buccal sampling is an effective method of obtaining DNA samples, which may provide improved results compared to traditional blood sampling for patients with neurodevelopmental disorders who exhibit somatic mosaicism.

Clinical Utility of a Comprehensive, Whole Genome CMA Testing Platform in Pediatrics: A Prospective Randomized Controlled Trial of Simulated Patients in Physician Practices.

BACKGROUND: Developmental disorders (DD), including autism spectrum disorder (ASD) and intellectual disability (ID), are a common group of clinical manifestations caused by a variety of genetic abnormalities. Genetic testing, including chromosomal microarray (CMA), plays an important role in diagnosing these conditions, but CMA can be limited by incomplete coverage of genetic abnormalities and lack of guidance for conditions rarely seen by treating physicians. METHODS: We conducted a longitudinal, randomized controlled trial investigating the impact of a higher resolution 2.8 million (MM) probe-CMA test on the quality of care delivered by practicing general pediatricians and specialists. To overcome the twin problems of finding an adequate sample size of multiple rare conditions and under/incorrect diagnoses, we used standardized simulated patients known as CPVs. Physicians, randomized into control and intervention groups, cared for the CPV pediatric patients with DD/ASD/ID. Care responses were scored against evidence-based criteria. In round one, participants could order diagnostic tests including existing CMA tests. In round two, intervention physicians could order the 2.8MM probe-CMA test. Outcome measures included overall quality of care and quality of the diagnosis and treatment plan. RESULTS: Physicians ordering CMA testing had 5.43% (p<0.001) higher overall quality scores than those who did not. Intervention physicians ordering the 2.8MM probe-CMA test had 7.20% (p<0.001) higher overall quality scores. Use of the 2.8MM probe-CMA test led to a 10.9% (p<0.001) improvement in the diagnosis and treatment score. Introduction of the 2.8MM probe-CMA test led to significant improvements in condition-specific interventions including an 8.3% (p = 0.04) improvement in evaluation and therapy for gross motor delays caused by Hunter syndrome, a 27.5% (p = 0.03) increase in early cognitive intervention for FOXG1-related disorder, and an 18.2% (p<0.001) improvement in referrals to child neurology for Dravet syndrome. CONCLUSION: Physician use of the 2.8MM probe-CMA test significantly improves overall quality as well as diagnosis and treatment quality for simulated cases of pediatric DD/ASD/ID patients, and delivers additional clinical utility over existing CMA tests.

Clinical Performance of an Ultrahigh Resolution Chromosomal Microarray Optimized for Neurodevelopmental Disorders.

Copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) significantly contribute to the etiology of neurodevelopmental disorders, such as developmental delay (DD), intellectual disability (ID), and autism spectrum disorder (ASD). This study summarizes the results of 3.5 years of CMA testing by a CLIA-certified clinical testing laboratory 5487 patients with neurodevelopmental conditions were clinically evaluated for rare copy number variants using a 2.8-million probe custom CMA optimized for the detection of CNVs associated with neurodevelopmental disorders. We report an overall detection rate of 29.4% in our neurodevelopmental cohort, which rises to nearly 33% when cases with DD/ID and/or MCA only are considered. The detection rate for the ASD cohort is also significant, at 25%. Additionally, we find that detection rate and pathogenic yield of CMA vary significantly depending on the primary indications for testing, the age of the individuals tested, and the specialty of the ordering doctor. We also report a significant difference between the detection rate on the ultrahigh resolution optimized array in comparison to the array from which it originated. This increase in detection can significantly contribute to the efficient and effective medical management of neurodevelopmental conditions in the clinic.

Chromosomal Microarray Analysis of Consecutive Individuals with Autism Spectrum Disorders Using an Ultra-High Resolution Chromosomal Microarray Optimized for Neurodevelopmental Disorders.

Copy number variants (CNVs) detected by chromosomal microarray analysis (CMA) significantly contribute to understanding the etiology of autism spectrum disorder (ASD) and other related conditions. In recognition of the value of CMA testing and its impact on medical management, CMA is in medical guidelines as a first-tier test in the evaluation of children with these disorders. As CMA becomes adopted into routine care for these patients, it becomes increasingly important to report these clinical findings. This study summarizes the results of over 4 years of CMA testing by a CLIA-certified clinical testing laboratory. Using a 2.8 million probe microarray optimized for the detection of CNVs associated with neurodevelopmental disorders, we report an overall CNV detection rate of 28.1% in 10,351 consecutive patients, which rises to nearly 33% in cases without ASD, with only developmental delay/intellectual disability (DD/ID) and/or multiple congenital anomalies (MCA). The overall detection rate for individuals with ASD is also significant at 24.4%. The detection rate and pathogenic yield of CMA vary significantly with the indications for testing, age, and gender, as well as the specialty of the ordering doctor. We note discrete differences in the most common recurrent CNVs found in individuals with or without a diagnosis of ASD.

Chromosomal microarray testing identifies a 4p terminal region associated with seizures in Wolf-Hirschhorn syndrome.

BACKGROUND: Wolf-Hirschhorn syndrome (WHS) is a contiguous gene deletion syndrome involving variable size deletions of the 4p16.3 region. Seizures are frequently, but not always, associated with WHS. We hypothesised that the size and location of the deleted region may correlate with seizure presentation. METHODS: Using chromosomal microarray analysis, we finely mapped the breakpoints of copy number variants (CNVs) in 48 individuals with WHS. Seizure phenotype data were collected through parent-reported answers to a comprehensive questionnaire and supplemented with available medical records. RESULTS: We observed a significant correlation between the presence of an interstitial 4p deletion and lack of a seizure phenotype (Fisher's exact test p=3.59e-6). In our cohort, there were five individuals with interstitial deletions with a distal breakpoint at least 751 kbp proximal to the 4p terminus. Four of these individuals have never had an observable seizure, and the fifth individual had a single febrile seizure at the age of 1.5 years. All other individuals in our cohort whose deletions encompass the terminal 751 kbp region report having seizures typical of WHS. Additional examples from the literature corroborate these observations and further refine the candidate seizure susceptibility region to a region 197 kbp in size, starting 368 kbp from the terminus of chromosome 4. CONCLUSIONS: We identify a small terminal region of chromosome 4p that represents a seizure susceptibility region. Deletion of this region in the context of WHS is sufficient for seizure occurrence.

Mosaic deletion of 20pter due to rescue by somatic recombination.

We report on a unique case of a mosaic 20pter-p13 deletion due to a somatic repair event identified by allele differentiating single nucleotide polymorphism (SNP) probes on chromosomal microarray. Small terminal deletions of 20p have been reported in a few individuals and appear to result in a variable phenotype. This patient was a 24-month-old female who presented with failure to thrive and speech delay. Chromosomal microarray analysis (CMA) performed on peripheral blood showed a 1.6 Mb deletion involving the terminus of 20p (20pter-20p13). This deletion appeared mosaic by CMA and this suspicion was confirmed by fluorescence in situ hybridization (FISH) analysis. Additionally, the deletion interval at 20p was directly adjacent to 15 Mb of mosaic copy-neutral loss of heterozygosity (LOH). The pattern of SNP probes was highly suggestive of a somatic repair event that resulted in rescue of the deleted region using the non-deleted homologue as a template. Structural mosaicism is rare and most often believed to be due to a postzygotic mechanism. This case demonstrates the additional utility of allele patterns to help distinguish mechanisms and in this case identified the possibility of either a post-zygotic repair of a germline deletion or a post-zygotic deletion with somatic recombination repair in a single step.

Variants of unknown significance on chromosomal microarray analysis: parental perspectives.

Chromosomal microarray is the recommended first-tier genetic test when a child presents with idiopathic developmental delay (DD), intellectual disability (ID), and/or autism spectrum disorder (ASD). Microarray may discover variants of unknown clinical significance (VUS) and been suggested to cause parental stress and anxiety. A retrospective, mixed methods study investigated parental perceptions of chromosomal microarray results that contain VUS. Surveys were sent to parents of children with DD/ID/ASD following a VUS result to seek information regarding parental understanding of the result, perceived value, and perceptions of child vulnerability and parental stress. Parents reported that chromosomal microarray was important for understanding their child's diagnosis and they were satisfied with the information. A majority of parents reported high confidence in their ability to explain a VUS result to others. Of the parents who reported they received support, many reported that the support was from a genetic counselor. Based on these results, VUS results are important to parents of children with DD/ID/ASD and genetic counseling regarding VUS results contributes positively to both parental understanding and support.

Clinical utility of chromosomal microarray analysis of DNA from buccal cells: detection of mosaicism in three patients.

Mosaic chromosomal abnormalities are relatively common. However, mosaicism may be missed due to multiple factors including failure to recognize clinical indications and order appropriate testing, technical limitations of diagnostic assays, or sampling tissue (s) in which mosaicism is either not present, or present at very low levels. Blood leukocytes have long been the "gold standard" sample for cytogenetic analysis; however, the culturing process for routine chromosome analysis can complicate detection of mosaicism since the normal cell line may have a growth advantage in culture, or may not be present in the cells that produce metaphases (the lymphocytes). Buccal cells are becoming increasingly utilized for clinical analyses and are proving to have many advantages. Buccal swabs allow for simple and noninvasive DNA collection. When coupled with a chromosomal microarray that contains single nucleotide polymorphic probes, analysis of buccal cells can maximize a clinician's opportunity to detect cytogenetic mosaicism. We present three cases of improved diagnosis of mosaic aberrations using buccal specimens for chromosomal microarray analysis. In each case, the aberration was either undetectable in blood or present at such a low level it likely could have gone undetected. These cases highlight the limitations of certain laboratory methodologies for identifying mosaicism. We also present practice implications for genetic counselors, including clinic workflow changes and counseling approaches based on increasing use of buccal samples.

Academia, advocacy, and industry: a collaborative method for clinical research advancement.

Professionals who work in academia, advocacy, and industry often carry out mutually exclusive activities related to research and clinical care. However, there are several examples of collaboration among such professionals that ultimately allows for improved scientific and clinical understanding. This commentary recounts our particular experience (a collaboration between geneticists at the Universities of Minnesota and Utah, the 4p- Support Group, and Lineagen, Inc) and reviews other similar projects. We formally propose this collaborative method as a conduit for future clinical research programs. Specifically, we encourage academicians, directors of family/advocacy/support groups, and members of industry to establish partnerships and document their experiences. The medical community as a whole will benefit from such partnerships and, specifically, families will teach us lessons that could never be learned in a laboratory or textbook.

Familial KANK1 deletion that does not follow expected imprinting pattern.

Deletion of the KANK1 gene (also called ANKRD15), located at chromosome position 9p24.3, has been associated with neurodevelopmental disease including congenital cerebral palsy, hypotonia, quadriplegia, and intellectual disability in a four-generation family. The inheritance pattern in this family was suggested to be maternal imprinting, as all affected individuals inherited the deletion from their fathers and monoallelic protein expression was observed. We present a family in which the proband's phenotype, including autism spectrum disorder, motor delay, and intellectual disability, is consistent with this previous report of KANK1 deletions. However, a paternally inherited deletion in the proband's unaffected sibling did not support maternal imprinting. This family raises consideration of further complexity of the KANK1 locus, including variable expressivity, incomplete penetrance, and the additive effects of additional genomic variants or the potential benign nature of inherited copy number variations (CNVs). However, when considered with the previous publication, our case also suggests that KANK1 may be subject to random monoallelic expression as a possible mode of inheritance. It is also important to consider that KANK1 has two alternately spliced transcripts, A and B. These have differential tissue expression and thus potentially differential clinical significance. Based upon cases in the literature, the present case, and information in the Database of Genomic Variants, it is possible that only aberrations of variant A contribute to neurodevelopmental disease. The familial deletion in this present case does not support maternal imprinting as an inheritance pattern. We suggest that other inheritance patterns and caveats should be considered when evaluating KANK1 deletions, which may become increasingly recognized through whole genome microarray testing, whole genome sequencing, and whole exome sequencing techniques.