Rett syndrome diagnostic odyssey: Limitations of NextGen sequencing.

Typical (or classic) Rett syndrome (RTT) is an X-linked neurodevelopmental disorder characterized by a period of regression, partial or complete loss of purposeful hand movements, and acquired speech, impaired gait, and stereotyped hand movements. In over 95% of typical RTT, a pathogenic variant is found in the methyl-CPG binding protein 2 gene (MECP2). Here, we describe a young woman with clinically diagnosed typical RTT syndrome who lacked a genetic diagnosis despite 20 years of investigation and multiple rounds of sequencing the MECP2 gene. Recently, additional genetic testing using next-generation sequencing was completed, which revealed a partial insertion of the BCL11A gene within exon 4 of MECP2, resulting in a small deletion in MECP2, causing likely disruption of MeCP2 function due to a frameshift. This case demonstrates the ever-changing limitations of genetic testing, as well as the importance of continual pursuit of a diagnosis as technologies improve and are more widely utilized.

Epilepsy panels in clinical practice: Yield, variants of uncertain significance, and treatment implications.

OBJECTIVE: There has been increasing utilization of genetic testing for pediatric epilepsy in recent years. Little systematic data is available examining how practice changes have impacted testing yields, diagnostic pace, incidence of variants of uncertain significance (VUSs), or therapeutic management. METHODS: A retrospective chart review was performed at Children's Hospital Colorado from February 2016 through February 2020. All patients under 18 years for whom an epilepsy gene panel was sent were included. RESULTS: A total of 761 epilepsy gene panels were sent over the study period. During the study period, there was a 292% increase in the average number of panels sent per month. The time from seizure onset to panel result decreased over the study period from a median of 2.9 years to 0.7 years. Despite the increase in testing, the percentage of panels yielding a disease-causing result remained stable at 11-13%. A total of 90 disease-causing results were identified, > 75% of which provided guidance in management. Children were more likely to have a disease-causing result if they were < 3 years old at seizure onset (OR 4.4, p < 0.001), had neurodevelopmental concerns (OR 2.2, p = 0.002), or had a developmentally abnormal MRI (OR 3.8, p < 0.001). A total of 1417 VUSs were identified, equating to 15.7 VUSs per disease-causing result. Non-Hispanic white patients had a lower average number of VUSs than patients of all other races/ethnicities (1.7 vs 2.1, p < 0.001). SIGNIFICANCE: Expansion in the volume of genetic testing corresponded to a decrease in the time from seizure onset to testing result. Diagnostic yield remained stable, resulting in an increase in the absolute number of disease-causing results annually-most of which have implications for management. However, there has also been an increase in total VUSs, which likely resulted in additional clinical time spent on VUS resolution.

Genetic Testing to Inform Epilepsy Treatment Management From an International Study of Clinical Practice.

Importance: It is currently unknown how often and in which ways a genetic diagnosis given to a patient with epilepsy is associated with clinical management and outcomes. Objective: To evaluate how genetic diagnoses in patients with epilepsy are associated with clinical management and outcomes. Design, Setting, and Participants: This was a retrospective cross-sectional study of patients referred for multigene panel testing between March 18, 2016, and August 3, 2020, with outcomes reported between May and November 2020. The study setting included a commercial genetic testing laboratory and multicenter clinical practices. Patients with epilepsy, regardless of sociodemographic features, who received a pathogenic/likely pathogenic (P/LP) variant were included in the study. Case report forms were completed by all health care professionals. Exposures: Genetic test results. Main Outcomes and Measures: Clinical management changes after a genetic diagnosis (ie, 1 P/LP variant in autosomal dominant and X-linked diseases; 2 P/LP variants in autosomal recessive diseases) and subsequent patient outcomes as reported by health care professionals on case report forms. Results: Among 418 patients, median (IQR) age at the time of testing was 4 (1-10) years, with an age range of 0 to 52 years, and 53.8% (n = 225) were female individuals. The mean (SD) time from a genetic test order to case report form completion was 595 (368) days (range, 27-1673 days). A genetic diagnosis was associated with changes in clinical management for 208 patients (49.8%) and usually (81.7% of the time) within 3 months of receiving the result. The most common clinical management changes were the addition of a new medication (78 [21.7%]), the initiation of medication (51 [14.2%]), the referral of a patient to a specialist (48 [13.4%]), vigilance for subclinical or extraneurological disease features (46 [12.8%]), and the cessation of a medication (42 [11.7%]). Among 167 patients with follow-up clinical information available (mean [SD] time, 584 [365] days), 125 (74.9%) reported positive outcomes, 108 (64.7%) reported reduction or elimination of seizures, 37 (22.2%) had decreases in the severity of other clinical signs, and 11 (6.6%) had reduced medication adverse effects. A few patients reported worsening of outcomes, including a decline in their condition (20 [12.0%]), increased seizure frequency (6 [3.6%]), and adverse medication effects (3 [1.8%]). No clinical management changes were reported for 178 patients (42.6%). Conclusions and Relevance: Results of this cross-sectional study suggest that genetic testing of individuals with epilepsy may be materially associated with clinical decision-making and improved patient outcomes.

Whole-exome sequencing and adrenocorticotropic hormone therapy in individuals with infantile spasms.

AIM: To identify additional genes associated with infantile spasms using a cohort with defined infantile spasms. METHOD: Whole-exome sequencing (WES) was performed on 21 consented individuals with infantile spasms and their unaffected parents (a trio-based study). Clinical history and imaging were reviewed. Potentially deleterious exonic variants were identified and segregated. To refine potential candidates, variants were further prioritized on the basis of evidence for relevance to disease phenotype or known associations with infantile spasms, epilepsy, or neurological disease. RESULTS: Likely pathogenic de novo variants were identified in NR2F1, GNB1, NEUROD2, GABRA2, and NDUFAF5. Suggestive dominant and recessive candidate variants were identified in PEMT, DYNC1I1, ASXL1, RALGAPB, and STRADA; further confirmation is required to support their relevance to disease etiology. INTERPRETATION: This study supports the utility of WES in uncovering the genetic etiology in undiagnosed individuals with infantile spasms with an overall yield of five out of 21. High-priority candidates were identified in an additional five individuals. WES provides additional support for previously described disease-associated genes and expands their already broad mutational and phenotypic spectrum.

CDKL5 deficiency disorder: Relationship between genotype, epilepsy, cortical visual impairment, and development.

OBJECTIVE: The cyclin-dependent kinase like 5 (CDKL5) gene is a known cause of early onset developmental and epileptic encephalopathy, also known as CDKL5 deficiency disorder (CDD). We sought to (1) provide a description of seizure types in patients with CDD, (2) provide an assessment of the frequency of seizure-free periods and cortical visual impairment (CVI), (3) correlate these features with genotype and gender, and (4) correlate these features with developmental milestones. METHODS: This is a cohort study of patients with CDD. Phenotypic features were explored and correlated with gene variant grouping and gender. A developmental score was created based on achieving seven primary milestones. Phenotypic variables were correlated with the developmental score to explore markers of better developmental outcomes. Multivariate linear regression was used to account for age at last visit. RESULTS: Ninety-two patients with CDD were seen during the enrollment period. Eighteen were male (19%); median age at last visit was 5 years (interquartile range = 2.0-11.0). Eighty-one percent of patients developed epileptic spasms, but only 47% of those also had hypsarrhythmia. Previously described hypermotor-tonic-spasms sequence was seen in only 24% of patients, but 56% of patients had seizures with multiple phases (often tonic and spasms). Forty-three percent of patients experienced a seizure-free period ranging from 1 to >12 months, but only 6% were still seizure-free at the last visit. CVI was present in 75% of all CDD patients. None of these features was associated with genotype group or gender. CVI was correlated with reduced milestone achievement after adjusting for age at last visit and a history of hypsarrhythmia. SIGNIFICANCE: The most common seizure types in CDD are epileptic spasms (often without hypsarrhythmia) and tonic seizures that may cluster together. CVI is a common feature in CDD and is correlated with achieving fewer milestones.

Genetic testing in a cohort of patients with potential epilepsy with myoclonic-atonic seizures.

Epilepsy with myoclonic-atonic seizures (EMAS) accounts for 1-2% of all childhood-onset epilepsies. EMAS has been shown to have an underlying genetic component, however the genetics of this disorder is not yet well understood. The purpose of this study was to review genetic testing results for a cohort of EMAS patients. A retrospective chart review was conducted for 77 patients evaluated at Children's Hospital Colorado with a potential diagnosis of EMAS. Genetic testing and biochemical testing was reviewed. Family history data was also collected. Seventy-seven percent of the cohort had at least one genetic test performed, and a molecular diagnosis was reached for six patients. Thirty-seven patients had a microarray, six of which identified a copy number variant. Only one was felt to contribute to the phenotype (2p16.3 deletion including NRXN1). Fifty-one patients had an epilepsy panel, two of which were positive (likely pathogenic variant in SCN1A, pathogenic variant in GABRG2). Of the six patients who had whole exome sequencing, two were negative, three were positive or likely positive, and one had multiple variants not felt to explain the phenotype. While EMAS is widely accepted to have a strong genetic component, the diagnostic yield of genetic testing remains low. This may be because several genes now thought to be associated with EMAS are not included on the more commonly ordered epilepsy panels, or have only recently been added to them.

Diagnosis switching and outcomes in a cohort of patients with potential epilepsy with myoclonic-atonic seizures.

INTRODUCTION: There is overlap in the electroclinical features of many childhood epilepsy syndromes, especially those presenting with multiple seizure types, such as epilepsy with myoclonic-atonic seizures (EMAS) and Lennox-Gastaut syndrome (LGS). This study aimed to determine the frequency of diagnosis switching and the factors influencing epilepsy syndrome diagnosis in a cohort of children with possible EMAS, as well as to explore the relationship between epilepsy syndrome diagnoses, key electroclinical features, and clinically relevant outcomes. METHODS: This is a cross-sectional retrospective chart review of children treated at the Children's Hospital of Colorado with a potential diagnosis of EMAS. RESULTS: There were 77 patients that met eligibility criteria, including 39% (n = 30) with an initial diagnosis of EMAS and 74% (n = 57) with a final diagnosis of EMAS. On average, for the 65% of patients who received more than one epilepsy diagnosis, the first, second, and third diagnoses were received within one year, three years, and ten years after epilepsy onset, respectively. Final diagnosis was significantly related to obtaining at least a six-month period of seizure freedom, p = 0.03. Classic LGS traits, including paroxysmal fast activity, slow spike-and-wave, and tonic seizures were present in 50% of the overall cohort, although a minority of these patients had a final diagnosis of LGS. However, the presence of more LGS traits was associated with a higher likelihood of ongoing seizures. Adjusted for age of epilepsy onset, seizure freedom was half as likely for every additional LGS trait observed (0.49[0.31, 0.77], p = 0.002). CONCLUSION: Current epilepsy syndrome classification has reduced applicability due to overlapping features. This results in diagnosis switching and limited prognostic value for patients with an overlapping clinical phenotype. Future studies should attempt to stratify patients based not only on epilepsy syndrome diagnosis, but also on the presence of various electroclinical traits to more accurately predict outcome.

Familial transmission of 5p13.2 duplication due to maternal der(X)ins(X;5).

Submicroscopic duplications of 5p13 have been recently reported in several cases, warranting the description of a new clinical entity (Chromosome 5p13 Duplication Syndrome; MIM 613174). These microduplications, while variable in size, all contain at least part of the NIPBL gene. Patients with duplications in this region present with intellectual disability/developmental delay (ID/DD) and dysmorphic facies. In addition, skeletal and brain abnormalities have been variably reported, as well as propensity for obesity in adulthood and hypotonia. We report a family with two affected sons and two affected daughters, each carrying a duplication at 5p13.2 encompassing the 3' portion of SLC1A3 and the 5' portion of NIPBL. Upon confirming the SNP microarray finding by FISH in the proband, it was discovered that the 5p13.2 duplication was located on the short arm of the X chromosome. Further FISH studies on the family demonstrated that all affected children and their mother carried a derivative X chromosome with insertion of material from 5p13.2 into the intermediate region of Xp [der(X)ins(X;5)(p2?2.1;p13.2p13.2)]. To our knowledge, this is the first report of an inherited duplication of 5p13.2 with multiple affected family members. This family underscores the need to confirm array findings by FISH, both in the proband and family members, to discern implications for pathogenicity and more accurately define the recurrence risk.

Identification of Critical Region Responsible for Split Hand/Foot Malformation Type 3 (SHFM3) Phenotype through Systematic Review of Literature and Mapping of Breakpoints Using Microarray Data.

Split hand/foot malformation (SHFM) is a limb malformation with underdeveloped or absent central digital rays, clefts of hands and feet, and variable syndactyly of the remaining digits. There are six types of SHFM. Here, we report a boy with SHFM type 3 having normal 4th and 5th digits, absent 2nd and 3rd digits, and a 4th finger flexion deformity, as well as absent 2nd, 3rd and 4th toes bilaterally. His father, two paternal uncles, and two paternal first cousins have similar phenotype. Chromosome analysis showed a normal male karyotype. A 514 kb gain at 10q24.31-q24.32 (chr10:102,962,134-103,476,346, hg19) was identified using 6.0 Single nucleotide polymorphism (SNP) microarray, resulting in the duplication of nine genes, including BTRC and FBXW4. A detailed systematic review of literature and mapping of breakpoints using microarray data from all reported cases in PubMed and DECIPHER were conducted, and exon 1 of BTRC gene was identified as the critical region responsible for the SHFM3 phenotype. The potential mechanism and future studies of this critical region causing the SHFM3 phenotype are discussed.