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.

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.

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 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.

Perceptions of tissue storage in a dementia population among spouses and offspring.

Cognitively impaired patients with dementia often rely on health advocates or guardians, such as spouses or adult offspring, to consent for medical procedures. These family members may also decide whether an autopsy is performed after death or whether their family member donates tissues. However, spouses are not genetically related to the patient and may have different perspectives than genetically related family members when making medical decisions with genetic implications, such as participation in a tissue repository (biobank). Interviews were conducted with spouses and adult offspring of individuals with a progressive dementing disease. Both spouses and offspring were supportive of the patient with dementia to participate in tissue storage. The top perceived benefits of tissue storage in both offspring and spouses were future value for family members and advancement of medical knowledge. Concerns included misuse of the tissue and insurance discrimination. Although the personal genetic implications differ between spouses and offspring, they share similar attitudes about the importance of tissue banking for the individual with a dementing disease.

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.

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.

Organization of food protection behavior is differentially influenced by 192 IgG-saporin lesions of either the medial septum or the nucleus basalis magnocellularis.

Converging lines of evidence have supported a role for the nucleus basalis magnocellularis (NB) in attentional mechanisms; however, debate continues regarding the role of the medial septum in behavior (MS). Recent studies have supported a role for the septohippocampal system in the online processing of internally generated cues. The current study was designed to investigate a possible double dissociation in rat food protection behavior, a natural behavior that has been shown to depend on external and internal sources of information. The study examined the effects of intraparenchymal injections of 192 IgG-saporin into either the MS or NB on the organization of food protection behavior. NB cholinergic lesions reduced the number of successful food protection behaviors while sparing the temporal organization of food protection behavior. In contrast, MS cholinergic lesions disrupted the temporal organization of food protection behavior while sparing the ability to successfully protect food items. These observations are consistent with a double dissociation of NB and MS cholinergic systems' contributions to processing external and internal sources of information and provide further evidence for the septohippocampal system's involvement in processing internally generated cues.

Fractionating dead reckoning: role of the compass, odometer, logbook, and home base establishment in spatial orientation.

Rats use multiple sources of information to maintain spatial orientation. Although previous work has focused on rats' use of environmental cues, a growing number of studies have demonstrated that rats also use self-movement cues to organize navigation. This review examines the extent that kinematic analysis of naturally occurring behavior has provided insight into processes that mediate dead-reckoning-based navigation. This work supports a role for separate systems in processing self-movement cues that converge on the hippocampus. The compass system is involved in deriving directional information from self-movement cues; whereas, the odometer system is involved in deriving distance information from self-movement cues. The hippocampus functions similar to a logbook in that outward path unique information from the compass and odometer is used to derive the direction and distance of a path to the point at which movement was initiated. Finally, home base establishment may function to reset this system after each excursion and anchor environmental cues to self-movement cues. The combination of natural behaviors and kinematic analysis has proven to be a robust paradigm to investigate the neural basis of spatial orientation.

Selective hippocampal cholinergic deafferentation impairs self-movement cue use during a food hoarding task.

Investigations using selective lesion techniques suggest that the septohippocampal cholinergic system may not be critical for spatial orientation. These studies employ spatial tasks that provide the animal with access to both environmental and self-movement cues; therefore, intact performance may reflect spared spatial orientation or compensatory mechanisms associated with one class of spatial cues. The present study investigated the contribution of the septohippocampal cholinergic system to spatial behavior by examining performance in foraging tasks in which cue availability was manipulated. Thirteen female Long-Evans rats received selective lesions of the medial septum/vertical band with 192 IgG saporin, and 11 received sham surgeries. Rats were trained to forage for hazelnuts in an environment with access to both environmental and self-movement cues (cued condition). Manipulations include altering availability of environmental cues associated with the refuge (uncued probe), removing all visual environmental cues (dark probe), and placing environmental and self-movement cues into conflict (reversal probe). Medial septum lesions disrupted homeward segment topography only under conditions in which self-movement cues were critical for organizing food hoarding behavior (dark and reversal). These results are consistent with medial septum lesions producing a selective impairment in self-movement cue processing and suggest that these rats were able to compensate for deficits in self-movement cue processing when provided access to environmental cues.

Medial septum lesions disrupt exploratory trip organization: evidence for septohippocampal involvement in dead reckoning.

Rats organize their open field behavior into a series of exploratory trips focused around a central location or home base. In addition, differences in movement kinematics have been used to fractionate the exploratory trip into tour (i.e., sequences of linear movement or progressions punctuated by stops) and homeward (i.e., single progression direct to the home base) segments. The observation of these characteristics independent of environmental familiarity and visual cue availability has suggested a role for self-movement information or dead reckoning in organizing exploratory behavior. Although previous work has implicated a role for the septohippocampal system in dead reckoning based navigation, as of yet, no studies have investigated the contribution of the medial septum to dead reckoning. First, the present study examined the organization of exploratory behavior under dark and light conditions in control rats and rats receiving either electrolytic or sham medial septum lesions. Medial septum lesions produced a significant increase in homeward segment path circuity and variability of temporal pacing of linear speeds. Second, as an independent assessment of the effectiveness of the medial septum lesions, rats were trained to locate a hidden platform in the standard water maze procedure. Consistent with previous research, medial septum lesions attenuated learning the location of the hidden platform. These results demonstrate a role for the medial septum in organizing exploratory behavior and provide further support for the role of the septohippocampal system in dead reckoning based navigation.

Comparative analysis of movement characteristics during dead-reckoning-based navigation in humans and rats.

Human and rat movement organization was investigated as they searched for randomly located rewards without access to visual information. Under dark conditions, rats foraged for randomly located food pellets (Experiment 1). Blindfolded humans were instructed to search for an ostensible hidden coin using a metal detector (Experiment 2). After locating the food pellet, rats carried it back to the refuge, and after a designated searching time, humans were instructed to return to the start location. Although both species exhibited a high degree of similarity in searching path movement organization and ability to return to the start location, disruption of human searching path organization was associated with impairments in returning to the start location. These results support the vestibular "gain" account of movement organization during dead-reckoning-based navigation.