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.

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.

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.

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.

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.

MEF2C Haploinsufficiency features consistent hyperkinesis, variable epilepsy, and has a role in dorsal and ventral neuronal developmental pathways.

MEF2C haploinsufficiency syndrome is an emerging neurodevelopmental disorder associated with intellectual disability, autistic features, epilepsy, and abnormal movements. We report 16 new patients with MEF2C haploinsufficiency, including the oldest reported patient with MEF2C deletion at 5q14.3. We detail the neurobehavioral phenotype, epilepsy, and abnormal movements, and compare our subjects with those previously reported in the literature. We also investigate Mef2c expression in the developing mouse forebrain. A spectrum of neurofunctional deficits emerges, with hyperkinesis a consistent finding. Epilepsy varied from absent to severe, and included intractable myoclonic seizures and infantile spasms. Subjects with partial MEF2C deletion were statistically less likely to have epilepsy. Finally, we confirm that Mef2c is present both in dorsal primary neuroblasts and ventral gamma-aminobutyric acid(GABA)ergic interneurons in the forebrain of the developing mouse. Given interactions with several key neurodevelopmental genes such as ARX, FMR1, MECP2, and TBR1, it appears that MEF2C plays a role in several developmental stages of both dorsal and ventral neuronal cell types.

Glutaric acidemia type 1: outcomes before and after expanded newborn screening.

Glutaric acidemia type 1 (GA-1) is an autosomal recessive disorder of lysine, hydroxylysine, and tryptophan metabolism. Patients may present with brain atrophy, macrocephaly, and acute dystonia secondary to striatal degeneration typically triggered by an infection, fever, and/or dehydration. This disorder is identified on expanded newborn screening by increased glutarylcarnitine. We evaluated the outcome of 19 patients with GA-1. Ten patients were diagnosed by newborn screening and 9 were diagnosed clinically. DNA testing in 12 patients identified 15 different mutations in the glutaryl-CoA dehydrogenase gene. Plasma glutarylcarnitine and urinary 3-hydroxyglutaric acid were elevated in all patients. However, only 10 of 17 patients who underwent urine organic acid analysis were high excretors of glutaric acid. Levels of glutarylcarnitine in plasma correlated with the urinary excretion of glutaric and 3-hydroxyglutaric acid, but not with clinical outcome. Plasma lysine was also significantly correlated with urinary glutaric acid, but not with urinary 3-hydroxyglutaric acid. Brain magnetic resonance imaging in all patients showed wide Sylvian fissures before treatment, which normalized by 4 years of age in treated patients. The occurrence of three adverse outcomes (oral motor function, ambulatory capability, and dystonic movements) was on average reduced by 75% (relative risk 0.25 to 0.28) in patients identified by newborn screening compared to patients diagnosed before newborn screening (Fisher's exact test; p=0.0055 for oral motor function and ambulatory capability; p=0.023 for dystonic movements). Newborn screening is effective in the prevention of complications in patients with GA-1 when coupled with treatment strategies.

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.

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.

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.

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.

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.