Tracy Brandt

Clinical application of whole-exome sequencing across clinical indications

Purpose:We report the diagnostic yield of whole-exome sequencing (WES) in 3,040 consecutive cases at a single clinical laboratory.Methods:WES was performed for many different clinical indications and included the proband plus two or more family members in 76% of cases.Results:The overall diagnostic yield of WES was 28.8%. The diagnostic yield was 23.6% in proband-only cases and 31.0% when three family members were analyzed. The highest yield was for patients who had disorders involving hearing (55%, N = 11), vision (47%, N = 60), the skeletal muscle system (40%, N = 43), the skeletal system (39%, N = 54), multiple congenital anomalies (36%, N = 729), skin (32%, N = 31), the central nervous system (31%, N = 1,082), and the cardiovascular system (28%, N = 54). Of 2,091 cases in which secondary findings were analyzed for 56 American College of Medical Genetics and Genomics–recommended genes, 6.2% (N = 129) had reportable pathogenic variants. In addition to cases with a definitive diagnosis, in 24.2% of cases a candidate gene was reported that may later be reclassified as being associated with a definitive diagnosis.Conclusion:Our experience with our first 3,040 WES cases suggests that analysis of trios significantly improves the diagnostic yield compared with proband-only testing for genetically heterogeneous disorders and facilitates identification of novel candidate genes.Genet Med 18 7, 696–704.

Detection of low-level mosaicism and placental mosaicism by oligonucleotide array comparative genomic hybridization.

Purpose: To determine the sensitivity of whole-genome oligonucleotide array comparative genomic hybridization for the detection of mosaic cytogenetic abnormalities.Methods: Mosaicism sensitivity was evaluated by testing artificially derived whole chromosome and segmental aneuploidies ranging from 0% to 100% abnormal and additional naturally occurring mosaic specimens.Results: Using combined dye-reversed replicates and an unfiltered analysis, oligonucleotide array comparative genomic hybridization detected as low as 10% and 20–30% mosaicism from whole chromosome and segmental aneuploidies, respectively. To investigate discrepancies between cultured and uncultured specimens, array comparative genomic hybridization was performed on DNA from additional direct product of conception specimens with abnormal karyotypes in culture. Interestingly, 5 of 10 product of conception specimens with double trisomies on cultured cell analysis had only a single trisomy by array comparative genomic hybridization and quantitative polymerase chain reaction on DNA from the uncultured direct specimen, and all harbored the more commonly observed trisomy. Thus, oligonucleotide array comparative genomic hybridization revealed previously unidentified placental mosaicism in half of the products of conception with double-aneuploid conventional karyotypes.Conclusion: Oligonucleotide array comparative genomic hybridization can detect low-level mosaicism for whole chromosome (∼10%) and segmental (∼20–30%) aneuploidies when using specific detection criteria. In addition, careful interpretation is required when performing array comparative genomic hybridization on DNA isolated from direct specimens as the results may differ from the cultured chromosome analysis.

Effect of Copy Number Variants on Outcomes for Infants With Single Ventricle Heart Defects

Background—Human genomes harbor copy number variants (CNVs), which are regions of DNA gains or losses. Although pathogenic CNVs are associated with congenital heart disease (CHD), their effect on clinical outcomes is unknown. This study sought to determine whether pathogenic CNVs among infants with single ventricle physiology were associated with inferior neurocognitive and somatic growth outcomes. Methods and Results—Genomic DNAs from 223 subjects of 2 National Heart, Lung, and Blood Institute–sponsored randomized clinical trials in infants with single ventricle CHD and 270 controls from The Cancer Genome Atlas project were analyzed for rare CNVs >300 kb using array comparative genomic hybridization. Neurocognitive and growth outcomes at 14 months from the CHD trials were compared among subjects with and without pathogenic CNVs. Putatively pathogenic CNVs, comprising 25 duplications and 6 deletions, had a prevalence of 13.9%, significantly greater than the 4.4% rate of such CNVs among controls. CNVs associated with genomic disorders were found in 13 cases but not in controls. Several CNVs likely to be causative of single ventricle CHD were observed, including aberrations altering the dosage of GATA4, MYH11, and GJA5. Subjects with pathogenic CNVs had worse linear growth, and those with CNVs associated with known genomic disorders had the poorest neurocognitive and growth outcomes. A minority of children with pathogenic CNVs were noted to be dysmorphic on clinical genetics examination. Conclusions—Pathogenic CNVs seem to contribute to the cause of single ventricle forms of CHD in ≥10% of cases and are clinically subtle but adversely affect outcomes in children harboring them.

A large-scale survey of the novel 15q24 microdeletion syndrome in autism spectrum disorders identifies an atypical deletion that narrows the critical region

BackgroundThe 15q24 microdeletion syndrome has been recently described as a recurrent, submicroscopic genomic imbalance found in individuals with intellectual disability, typical facial appearance, hypotonia, and digital and genital abnormalities. Gene dosage abnormalities, including copy number variations (CNVs), have been identified in a significant fraction of individuals with autism spectrum disorders (ASDs). In this study we surveyed two ASD cohorts for 15q24 abnormalities to assess the frequency of genomic imbalances in this interval.MethodsWe screened 173 unrelated subjects with ASD from the Central Valley of Costa Rica and 1336 subjects with ASD from 785 independent families registered with the Autism Genetic Resource Exchange (AGRE) for CNVs across 15q24 using oligonucleotide arrays. Rearrangements were confirmed by array comparative genomic hybridization and quantitative PCR.ResultsAmong the patients from Costa Rica, an atypical de novo deletion of 3.06 Mb in 15q23-q24.1 was detected in a boy with autism sharing many features with the other 13 subjects with the 15q24 microdeletion syndrome described to date. He exhibited intellectual disability, constant smiling, characteristic facial features (high anterior hairline, broad medial eyebrows, epicanthal folds, hypertelorism, full lower lip and protuberant, posteriorly rotated ears), single palmar crease, toe syndactyly and congenital nystagmus. The deletion breakpoints are atypical and lie outside previously characterized low copy repeats (69,838-72,897 Mb). Genotyping data revealed that the deletion had occurred in the paternal chromosome. Among the AGRE families, no large 15q24 deletions were observed.ConclusionsFrom the current and previous studies, deletions in the 15q24 region represent rare causes of ASDs with an estimated frequency of 0.1 to 0.2% in individuals ascertained for ASDs, although the proportion might be higher in sporadic cases. These rates compare with a frequency of about 0.3% in patients ascertained for unexplained intellectual disability and congenital anomalies. This atypical deletion reduces the minimal interval for the syndrome from 1.75 Mb to 766 kb, implicating a reduced number of genes (15 versus 38). Sequencing of genes in the 15q24 interval in large ASD and intellectual disability samples may identify mutations of etiologic importance in the development of these disorders.

Large inverted repeats within Xp11.2 are present at the breakpoints of isodicentric X chromosomes in Turner syndrome

Turner syndrome (TS) results from whole or partial monosomy X and is mediated by haploinsufficiency of genes that normally escape X-inactivation. Although a 45,X karyotype is observed in half of all TS cases, the most frequent variant TS karyotype includes the isodicentric X chromosome alone [46,X,idic(X)(p11)] or as a mosaic [46,X,idic(X)(p11)/45,X]. Given the mechanism of idic(X)(p11) rearrangement is poorly understood and breakpoint sequence information is unknown, this study sought to investigate the molecular mechanism of idic(X)(p11) formation by determining their precise breakpoint intervals. Karyotype analysis and fluorescence in situ hybridization mapping of eight idic(X)(p11) cell lines and three unbalanced Xp11.2 translocation lines identified the majority of breakpoints within a 5 Mb region, from approximately 53 to 58 Mb, in Xp11.1-p11.22, clustering into four regions. To further refine the breakpoints, a high-resolution oligonucleotide microarray (average of approximately 350 bp) was designed and array-based comparative genomic hybridization (aCGH) was performed on all 11 idic(X)(p11) and Xp11.2 translocation lines. aCGH analyses identified all breakpoint regions, including an idic(X)(p11) line with two potential breakpoints, one breakpoint shared between two idic(X)(p11) lines and two Xp translocations that shared breakpoints with idic(X)(p11) lines. Four of the breakpoint regions included large inverted repeats composed of repetitive gene clusters and segmental duplications, which corresponded to regions of copy-number variation. These data indicate that the rearrangement sites on Xp11.2 that lead to isodicentric chromosome formation and translocations are probably not random and suggest that the complex repetitive architecture of this region predisposes it to rearrangements, some of which are recurrent.

Complex autism spectrum disorder in a patient with a 17q12 microduplication.

Autism spectrum disorders (ASDs) are phenotypically complex developmental neuropsychiatric disorders affecting approximately 0.6% of the population. About 30–70% of affected children are also considered to have intellectual disability (ID). The underlying genetic causes of ASDs are diverse with a defined etiology in 16–20%. Array comparative genomic hybridization (aCGH) has proven useful in identifying sub‐microscopic chromosome aberrations in a subset of patients, some of which have been shown to be recurrent. One such aberration is the 1.4 Mb microdeletion at chromosome 17q12, which has been reported to be associated with renal disease, growth restriction, diabetes, cognitive impairment, seizures, and in some cases an ASD. Patients with the reciprocal chromosome 17q12 microduplication typically have also been identified with ID and in some cases seizures and behavioral abnormalities. Here we report a patient with a de novo, 1.4 Mb microduplication diagnosed with significant ID involving complex deficits and autism. To our knowledge, this is the first report of a patient with the 17q12 microduplication and a complex ASD phenotype.

46,XY disorder of sex development and developmental delay associated with a novel 9q33.3 microdeletion encompassing NR5A1

Steroidogenic factor 1 (SF1) is a nuclear receptor encoded by the NR5A1 gene. SF1 affects both sexual and adrenal development through the regulation of target gene expression. Genotypic male and female SF1 knockout mice have adrenal and gonadal agenesis with persistent Müllerian structures and early lethality. There have been several reports of NR5A1 mutations in individuals with 46,XY complete gonadal dysgenesis (CGD) or other disorders of sex development (DSD) with or without an adrenal phenotype. To date microdeletions involving NR5A1 have been reported in only two patients with DSDs. We report a novel microdeletion encompassing NR5A1 in a patient with 46,XY DSD and developmental delay. The phenotypically female patient initially presented with mild developmental delay and dysmorphisms. Chromosome analysis revealed a 46,XY karyotype. A 1.54 Mb microdeletion of chromosome 9q33.3 including NR5A1 was detected by array CGH and confirmed by FISH. Normal maternal FISH results indicated that this was most likely a de novo event. Since most NR5A1 mutations have been ascertained through gonadal or adrenal abnormalities, the additional findings of developmental delay and minor facial dysmorphisms are possibly related to haploinsufficiency of other genes within the 1.54 Mb deleted region. This report further confirms the role of NR5A1 deletions in 46,XY DSD and reinforces the utility of aCGH in the work up of DSDs of unclear etiology.

The Impact of CNVs on Outcomes for Infants with Single Ventricle Heart Defects

Background—Human genomes harbor copy number variants (CNVs), which are regions of DNA gains or losses. Although pathogenic CNVs are associated with congenital heart disease (CHD), their effect on clinical outcomes is unknown. This study sought to determine whether pathogenic CNVs among infants with single ventricle physiology were associated with inferior neurocognitive and somatic growth outcomes. Methods and Results—Genomic DNAs from 223 subjects of 2 National Heart, Lung, and Blood Institute–sponsored randomized clinical trials in infants with single ventricle CHD and 270 controls from The Cancer Genome Atlas project were analyzed for rare CNVs >300 kb using array comparative genomic hybridization. Neurocognitive and growth outcomes at 14 months from the CHD trials were compared among subjects with and without pathogenic CNVs. Putatively pathogenic CNVs, comprising 25 duplications and 6 deletions, had a prevalence of 13.9%, significantly greater than the 4.4% rate of such CNVs among controls. CNVs associated with genomic disorders were found in 13 cases but not in controls. Several CNVs likely to be causative of single ventricle CHD were observed, including aberrations altering the dosage of GATA4, MYH11, and GJA5. Subjects with pathogenic CNVs had worse linear growth, and those with CNVs associated with known genomic disorders had the poorest neurocognitive and growth outcomes. A minority of children with pathogenic CNVs were noted to be dysmorphic on clinical genetics examination. Conclusions—Pathogenic CNVs seem to contribute to the cause of single ventricle forms of CHD in ≥10% of cases and are clinically subtle but adversely affect outcomes in children harboring them.

Diagnostic outcomes for genetic testing of 70 genes in 8565 patients with epilepsy and neurodevelopmental disorders

We evaluated >8500 consecutive, unselected patients with epilepsy and neurodevelopmental disorders who underwent multigene panel testing to determine the average age at molecular diagnosis and diagnostic yield of 70 genes.

Effect of Copy Number Variants on Outcomes for Infants With Single Ventricle Heart DefectsClinical Perspective

Background—Human genomes harbor copy number variants (CNVs), which are regions of DNA gains or losses. Although pathogenic CNVs are associated with congenital heart disease (CHD), their effect on clinical outcomes is unknown. This study sought to determine whether pathogenic CNVs among infants with single ventricle physiology were associated with inferior neurocognitive and somatic growth outcomes. Methods and Results—Genomic DNAs from 223 subjects of 2 National Heart, Lung, and Blood Institute–sponsored randomized clinical trials in infants with single ventricle CHD and 270 controls from The Cancer Genome Atlas project were analyzed for rare CNVs >300 kb using array comparative genomic hybridization. Neurocognitive and growth outcomes at 14 months from the CHD trials were compared among subjects with and without pathogenic CNVs. Putatively pathogenic CNVs, comprising 25 duplications and 6 deletions, had a prevalence of 13.9%, significantly greater than the 4.4% rate of such CNVs among controls. CNVs associated with genomic disorders were found in 13 cases but not in controls. Several CNVs likely to be causative of single ventricle CHD were observed, including aberrations altering the dosage of GATA4, MYH11, and GJA5. Subjects with pathogenic CNVs had worse linear growth, and those with CNVs associated with known genomic disorders had the poorest neurocognitive and growth outcomes. A minority of children with pathogenic CNVs were noted to be dysmorphic on clinical genetics examination. Conclusions—Pathogenic CNVs seem to contribute to the cause of single ventricle forms of CHD in ≥10% of cases and are clinically subtle but adversely affect outcomes in children harboring them.