Victor Wei Zhang

Targeted polymerase chain reaction-based enrichment and next generation sequencing for diagnostic testing of congenital disorders of glycosylation.

Purpose: Congenital disorders of glycosylation are a heterogeneous group of disorders caused by deficient glycosylation, primarily affecting the N-linked pathway. It is estimated that more than 40% of congenital disorders of glycosylation patients lack a confirmatory molecular diagnosis. The purpose of this study was to improve molecular diagnosis for congenital disorders of glycosylation by developing and validating a next generation sequencing panel for comprehensive mutation detection in 24 genes known to cause congenital disorders of glycosylation.Methods: Next generation sequencing validation was performed on 12 positive control congenital disorders of glycosylation patients. These samples were blinded as to the disease-causing mutations. Both RainDance and Fluidigm platforms were used for sequence enrichment and targeted amplification. The SOLiD platform was used for sequencing the amplified products. Bioinformatic analysis was performed using NextGENe® software.Results: The disease-causing mutations were identified by next generation sequencing for all 12 positive controls. Additional variants were also detected in three controls that are known or predicted to impair gene function and may contribute to the clinical phenotype.Conclusions: We conclude that development of next generation sequencing panels in the diagnostic laboratory where multiple genes are implicated in a disorder is more cost-effective and will result in improved and faster patient diagnosis compared with a gene-by-gene approach. Recommendations are also provided for data analysis from the next generation sequencing-derived data in the clinical laboratory, which will be important for the widespread use of this technology.

Clinical application of massively parallel sequencing in the molecular diagnosis of glycogen storage diseases of genetically heterogeneous origin

Purpose:Glycogen storage diseases are a group of inborn errors of glycogen synthesis or catabolism. The outcome for untreated patients can be devastating. Given the genetic heterogeneity and the limited availability of enzyme study data, the definitive diagnosis of glycogen storage diseases is made on the basis of sequence analysis of selected potentially causative genes.Methods:A massively parallel sequencing test was developed for simultaneous sequencing of 16 genes known to cause muscle and liver forms of glycogen storage diseases: GYS2, GYS1, G6PC, SLC37A4, GAA, AGL, GBE1, PYGM, PYGL, PFKM, PHKA2, PHKB, PHKG2, PHKA1, PGAM2, and PGM1. All the nucleotides in the coding regions of these 16 genes have been enriched with sufficient coverage in an unbiased manner.Results:Massively parallel sequencing demonstrated 100% sensitivity and specificity as compared with Sanger sequencing. Massively parallel sequencing correctly identified all types of mutations, including single-nucleotide substitutions, small deletions and duplications, and large deletions involving one or more exons. In addition, we have confirmed the molecular diagnosis in 11 of 17 patients in whom glycogen storage diseases were suspected.Conclusion:This report demonstrates the clinical utility of massively parallel sequencing technology in the diagnostic testing of a group of clinically and genetically heterogeneous disorders such as glycogen storage diseases, in a cost- and time-efficient manner.Genet Med 2013:15(2):106–114

Primary immunodeficiency diseases: Genomic approaches delineate heterogeneous Mendelian disorders

Background: Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes can overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. Objective: We sought to investigate the ability of whole‐exome screening methods to detect disease‐causing variants in patients with PIDDs. Methods: Patients with PIDDs from 278 families from 22 countries were investigated by using whole‐exome sequencing. Computational copy number variant (CNV) prediction pipelines and an exome‐tiling chromosomal microarray were also applied to identify intragenic CNVs. Analytic approaches initially focused on 475 known or candidate PIDD genes but were nonexclusive and further tailored based on clinical data, family history, and immunophenotyping. Results: A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/110) and management was directly altered in nearly a quarter (26/110) of families based on molecular findings. Twelve PIDD‐causing CNVs were detected, including 7 smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. Conclusion: This high‐throughput genomic approach enabled detection of disease‐related variants in unexpected genes; permitted detection of low‐grade constitutional, somatic, and revertant mosaicism; and provided evidence of a mutational burden in mixed PIDD immunophenotypes.

Improved molecular diagnosis by the detection of exonic deletions with target gene capture and deep sequencing

Purpose:We aimed to demonstrate the detection of exonic deletions using target capture and deep sequencing data.Methods:Sequence data from target gene capture followed by massively parallel sequencing were analyzed for the detection of exonic deletions using the normalized mean coverage of individual exons. We compared the results with those obtained from high-density exon-targeted array comparative genomic hybridization and applied similar analysis to examine samples from patients with pathogenic exonic deletions.Results:Thirty-eight samples, each containing 2,134, 2,833, or 4,688 coding exons from different panels, with a total of 103,863 exons, were analyzed by capture–massively parallel sequencing and array comparative genomic hybridization. Ten deletions detected by array comparative genomic hybridization were all detected by massively parallel sequencing, whereas only two of three duplications were detected. We were able to detect all pathogenic exonic deletions in 11 positive cases. Thirty-one exonic copy number changes from nine perspective clinical samples were also identified.Conclusion:Our results demonstrated the feasibility of using the same set of sequence data to detect both point mutations and exonic deletions, thus improving the diagnostic power of massively parallel sequencing–based assays.Genet Med 17 2, 99–107.

Transition to Next Generation Analysis of the Whole Mitochondrial Genome: A Summary of Molecular Defects

The diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity. Conventional analysis of the mitochondrial genome often starts with a screening panel for common mitochondrial DNA (mtDNA) point mutations and large deletions (mtScreen). If negative, it has been traditionally followed by Sanger sequencing of the entire mitochondrial genome (mtWGS). The recently developed “Next‐Generation Sequencing” (NGS) technology offers a robust high‐throughput platform for comprehensive mtDNA analysis. Here, we summarize the results of the past 6 years of clinical practice using the mtScreen and mtWGS tests on 9,261 and 2,851 unrelated patients, respectively. A total of 344 patients (3.7%) had mutations identified by mtScreen and 99 (3.5%) had mtDNA mutations identified by mtWGS. The combinatorial analyses of mtDNA and POLG revealed a diagnostic yield of 6.7% in patients with suspected mitochondrial disorders but no recognizable syndromes. From the initial mtWGS–NGS cohort of 391 patients, 21 mutation‐positive cases (5.4%) have been identified. The mtWGS–NGS provides a one‐step approach to detect common and uncommon point mutations, as well as deletions. Additionally, NGS provides accurate, sensitive heteroplasmy measurement, and the ability to map deletion breakpoints. A new era of more efficient molecular diagnosis of mtDNA mutations has arrived.

Dependable and efficient clinical utility of target capture-based deep sequencing in molecular diagnosis of retinitis pigmentosa.

PURPOSE The purpose of this study was to establish a fully validated, high-throughput next-generation sequencing (NGS) approach for comprehensive, cost-effective, clinical molecular diagnosis of retinitis pigmentosa (RP). METHODS Target sequences of a panel of 66 genes known to cause all nonsyndromic and a few syndromic forms of RP were enriched by using custom-designed probe hybridization. A total of 939 coding exons and 20 bp of their flanking intron regions with a total of 202,800 bp of target sequences were captured, followed by massively parallel sequencing (MPS) on the Illumina HiSeq2000 device. RESULTS Twelve samples with known mutations were used for test validation. We achieved an average sequence depth of ∼1000× per base. Exons with <20× insufficient coverage were completed by PCR/Sanger sequencing to ensure 100% coverage. We analyzed DNA from 65 unrelated RP patients and detected deleterious mutations in 53 patients with a diagnostic yield of ∼82%. CONCLUSIONS Clinical validation and consistently deep coverage of individual exons allow for the accurate identification of all types of mutations including point mutations, exonic deletions, and large insertions. Our comprehensive MPS approach greatly improves diagnostic acumen for RP in a cost- and time-efficient manner.

Diagnostic approaches to apparent homozygosity

Purpose:Sanger sequencing is a mainstay for the identification of gene mutations used in molecular diagnostic laboratories. However, in autosomal recessive disorders, failure of allele amplification can occur for a variety of reasons, leading heterozygous mutations to appear homozygous. We sought to investigate the frequency at which apparently homozygous mutations detected by Sanger sequencing in our laboratory appeared homozygous due to other molecular etiologies.Methods:A review of 12,406 cases from 40 different genetic tests that were submitted to the Medical Genetics Laboratories at Baylor College of Medicine for Sanger sequence analysis was performed. The molecular status of apparently homozygous cases was further investigated by testing parents using various methods.Results:A total of 291 cases of apparent homozygosity were identified, ranging from 0 to 37% of the total per gene. One-third of the apparently homozygous cases were followed up by parental testing. Parental carrier status was confirmed in 88% of the cases. Of the cases in which parental carrier status could not be confirmed, deletions encompassing point mutations, allele dropout due to single-nucleotide polymorphisms at primer sites, and uniparental isodisomy were observed.Conclusion:For individuals with autosomal recessive disorders and apparently homozygous mutations, confirmation by parental testing can rule out other causes of apparent homozygosity, including allele dropout, copy number variations, and uniparental isodisomy.Genet Med 2012:14(10):877–882

An integrated approach for classifying mitochondrial DNA variants: one clinical diagnostic laboratory’s experience

Purpose:The mitochondrial genome is highly polymorphic. A unique feature of deleterious mitochondrial DNA (mtDNA) mutations is heteroplasmy. Genetic background and variable penetrance also play roles in the pathogenicity for a mtDNA variant. Clinicians are increasingly interested in requesting mtDNA testing. However, interpretation of uncharacterized mtDNA variants is a great challenge. We suggest a stepwise interpretation procedure for clinical service.Methods:We describe the algorithms used to interpret novel and rare mtDNA variants. mtDNA databases and in silico predictive algorithms are used to evaluate the pathogenic potential of novel and/or rare mtDNA variants.Results:mtDNA variants can be classified into three categories: benign variants, unclassified variants, and deleterious mutations based on database search and in silico prediction. Targeted DNA sequence analysis of matrilineal relatives, heteroplasmy quantification, and functional studies are useful to classify mtDNA variants.Conclusion:Clinical significance of a novel or rare variant is critical in the diagnosis of the disease and counseling of the family. Based on the results from clinical, biochemical, and molecular genetic studies of multiple family members, proper interpretation of mtDNA variants is important for clinical laboratories and for patient care.Genet Med 2012:14(6):620–626

Vaccine-associated varicella and rubella infections in severe combined immunodeficiency with isolated CD4 lymphocytopenia and mutations in IL7R detected by tandem whole exome sequencing and chromosomal microarray

In areas without newborn screening for severe combined immunodeficiency (SCID), disease‐defining infections may lead to diagnosis, and in some cases, may not be identified prior to the first year of life. We describe a female infant who presented with disseminated vaccine‐acquired varicella (VZV) and vaccine‐acquired rubella infections at 13 months of age. Immunological evaluations demonstrated neutropenia, isolated CD4 lymphocytopenia, the presence of CD8+ T cells, poor lymphocyte proliferation, hypergammaglobulinaemia and poor specific antibody production to VZV infection and routine immunizations. A combination of whole exome sequencing and custom‐designed chromosomal microarray with exon coverage of primary immunodeficiency genes detected compound heterozygous mutations (one single nucleotide variant and one intragenic copy number variant involving one exon) within the IL7R gene. Mosaicism for wild‐type allele (20–30%) was detected in pretransplant blood and buccal DNA and maternal engraftment (5–10%) demonstrated in pretransplant blood DNA. This may be responsible for the patients unusual immunological phenotype compared to classical interleukin (IL)‐7Rα deficiency. Disseminated VZV was controlled with anti‐viral and immune‐based therapy, and umbilical cord blood stem cell transplantation was successful. Retrospectively performed T cell receptor excision circle (TREC) analyses completed on neonatal Guthrie cards identified absent TREC. This case emphasizes the danger of live viral vaccination in severe combined immunodeficiency (SCID) patients and the importance of newborn screening to identify patients prior to high‐risk exposures. It also illustrates the value of aggressive pathogen identification and treatment, the influence newborn screening can have on morbidity and mortality and the significant impact of newer genomic diagnostic tools in identifying the underlying genetic aetiology for SCID patients.

Biochemical, molecular, and clinical diagnoses of patients with cerebral creatine deficiency syndromes

Cerebral creatine deficiency syndromes (CCDS) are a group of inborn errors of creatine metabolism that involve AGAT and GAMT for creatine biosynthesis disorders and SLC6A8 for creatine transporter (CT1) deficiency. Deficiencies in the three enzymes can be distinguished by intermediate metabolite levels, and a definitive diagnosis relies on the presence of deleterious mutations in the causative genes. Mutations and unclassified variants were identified in 41 unrelated patients, and 22 of these mutations were novel. Correlation of sequencing and biochemical data reveals that using plasma guanidinoacetate (GAA) as a biomarker has 100% specificity for both AGAT and GAMT deficiencies, but AGAT deficiency has decreased sensitivity in this assay. Furthermore, the urine creatine:creatinine ratio is an effective screening test with 100% specificity in males suspected of having creatine transporter deficiency. This test has a high false-positive rate due to dietary factors or dilute urine samples and lacks sensitivity in females. We conclude that biochemical screening for plasma GAA and measuring of the urine creatine:creatinine ratio should be performed for suspected CCDS patients prior to sequencing. Also, based on the results of this study, we feel that sequencing should only be considered if a patient has abnormal biochemical results on repeat testing.