Greyson P Twist

Whole-genome sequencing for identification of Mendelian disorders in critically ill infants: a retrospective analysis of diagnostic and clinical findings

BACKGROUND Genetic disorders and congenital anomalies are the leading causes of infant mortality. Diagnosis of most genetic diseases in neonatal and paediatric intensive care units (NICU and PICU) is not sufficiently timely to guide acute clinical management. We used rapid whole-genome sequencing (STATseq) in a level 4 NICU and PICU to assess the rate and types of molecular diagnoses, and the prevalence, types, and effect of diagnoses that are likely to change medical management in critically ill infants. METHODS We did a retrospective comparison of STATseq and standard genetic testing in a case series from the NICU and PICU of a large childrens hospital between Nov 11, 2011, and Oct 1, 2014. The participants were families with an infant younger than 4 months with an acute illness of suspected genetic cause. The intervention was STATseq of trios (both parents and their affected infant). The main measures were the diagnostic rate, time to diagnosis, and rate of change in management after standard genetic testing and STATseq. FINDINGS 20 (57%) of 35 infants were diagnosed with a genetic disease by use of STATseq and three (9%) of 32 by use of standard genetic testing (p=0·0002). Median time to genome analysis was 5 days (range 3-153) and median time to STATseq report was 23 days (5-912). 13 (65%) of 20 STATseq diagnoses were associated with de-novo mutations. Acute clinical usefulness was noted in 13 (65%) of 20 infants with a STATseq diagnosis, four (20%) had diagnoses with strongly favourable effects on management, and six (30%) were started on palliative care. 120-day mortality was 57% (12 of 21) in infants with a genetic diagnosis. INTERPRETATION In selected acutely ill infants, STATseq had a high rate of diagnosis of genetic disorders. Most diagnoses altered the management of infants in the NICU or PICU. The very high infant mortality rate indicates a substantial need for rapid genomic diagnoses to be allied with a novel framework for precision medicine for infants in NICU and PICU who are diagnosed with genetic diseases to improve outcomes. FUNDING Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Human Genome Research Institute, and National Center for Advancing Translational Sciences.

CYP2D6, SULT1A1 and UGT2B17 copy number variation: quantitative detection by multiplex PCR.

AIM Among the genes of drug-metabolizing enzymes, CYP2D6 is notoriously difficult to characterize owing to the complexity of gene deletions, duplications, multiplications and the presence of hybrid genes composed of CYP2D6 and CYP2D7. For SULT1A1 up to five gene copies have been reported, while UGT2B17 is known for gene deletions only. Different platforms exist for copy number variation (CNV) detection; however, there are no gold standards. Robust methods are required that address specific challenges to accurately determine gene CNVs in complex gene loci. MATERIALS & METHODS Quantitative multiplex PCR amplification (MPA) was performed on a diverse set of genomic DNA samples. Resulting PCR fragments were separated on an ABI 3730 instrument and analyzed with GeneMapper. CYP2D6 was targeted at four different gene regions and either normalized against CYP2D8 or UGT2B15 and SULT1A2. Inconsistent observations and CNVs contrasting genotype data were further characterized by long-range PCR and/or DNA sequence analysis. UGT2B17 and SULT1A1 were normalized against UGT2B15 and SULT1A2, respectively. RESULTS MPA detected 0-5, 1-5 and 0-2 copies for CYP2D6, SULT1A1 and UGT2B17, respectively. The interrogation of four CYP2D6 regions resulted in robust copy number assignments that were in agreement with genotype, sequencing and extra long PCR-based data. Gene deletions, duplication, and multiplications among known and novel hybrid genes were reliably identified. Novel findings regarding allelic variation include nonfunctional CYP2D6/2D7 hybrids such as CYP2D6*4N and *68, which were consistently identified on a subset of CYP2D6*4 alleles. In addition, a novel variant, designated CYP2D6*83, was discovered. For SULT1A1, we report the first six-copy case and for UGT2B15 and UGT2B17 we have evidence for rare deletion and duplication events, respectively. CONCLUSION This MPA-based copy number platform not only allowed us to determine CNVs, but also served as a tool for allele discovery and characterization in a diverse panel of samples in a fast and reliable manner.

Constellation: a tool for rapid, automated phenotype assignment of a highly polymorphic pharmacogene, CYP2D6 , from whole-genome sequences

[This corrects the article DOI: 10.1038/npjgenmed.2015.7.].

Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation.

Although CYP2B6 catalyzes the biotransformation of many drugs used clinically for children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric, and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs), and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes such that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to postnatal samples and fetal HLMs did not appear to catalyze bupropion hydroxylation; however, fetal CYP2B6 protein levels were not significantly different from postnatal levels. Considerable interindividual variation in CYP2B6 mRNA expression, protein levels, and activity was observed in postnatal HLMs (mRNA, ∼40,000-fold; protein, ∼300-fold; activity, ∼600-fold). The extremely wide range of interindividual variability in CYP2B6 expression and activity was significantly associated with age (P < 0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 interindividual variability was not significantly associated with genetic variation in CYP2B6, nor was it associated with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.

A patient with polymerase E1 deficiency (POLE1): clinical features and overlap with DNA breakage/instability syndromes

BackgroundChromosome instability syndromes are a group of inherited conditions associated with chromosomal instability and breakage, often leading to immunodeficiency, growth retardation and increased risk of malignancy.Case presentationWe performed exome sequencing on a girl with a suspected chromosome instability syndrome that manifested as growth retardation, microcephaly, developmental delay, dysmorphic features, poikiloderma, immune deficiency with pancytopenia, and myelodysplasia. She was homozygous for a previously reported splice variant, c.4444 + 3A > G in the POLE1 gene, which encodes the catalytic subunit of DNA polymerase E.ConclusionThis is the second family with POLE1-deficency, with the affected individual demonstrating a more severe phenotype than previously described.

In vivo characterization of CYP2D6*12, *29 and *84 using dextromethorphan as a probe drug: a case report

CYP2D6*84 was first described in a Black South African subject, however, its function remains unknown. Astrolabe, a probabilistic scoring tool developed in our laboratory to call genotypes from whole genome sequence, identified CYP2D6*84 in a trio. The father presented with intermediate metabolism when challenged with the CYP2D6 probe drug dextromethorphan (DM/dextrorphan [DX] = 0.0839). Since his second allele, CYP2D6*12, is nonfunctional, the observed activity is derived by CYP2D6*84. This finding suggests that the alleles hallmark P267H causes decreased activity toward DM and that this allele should receive a value of 0.5 for Activity Score calculations. The mothers DM/DX of 0.0543 was consistent with the decreased activity classification of CYP2D6*29. The child, a critically ill neonate, was not phenotyped, but predicted to be a normal metabolizer.

The CMH Warehouse: A Catalog of Genetic Variation in Patients of a Children's Hospital

Advances in high-throughput DNA sequencing have enabled the comprehensive identification of individual genetic variation on an unprecedented scale, powering the diagnosis of disease and personalized treatment. As our ability to detect genetic variation has grown, clinicians and researchers struggle to interpret the functional significance of the millions of variants found in each individual genome. The Variant Warehouse at the Center for Pediatric Genomic Medicine (CPGM) at Childrens Mercy, Kansas City, is a resource containing a record of over 150 million genomic variants detected in more than 5700 patients sequenced by the Center since 2011. Each variant has been characterized by the CPGMs Rapid Understanding of Nucleotide Effect Software (RUNES) pipeline, which records database cross references and predicted functional consequences as generated by multiple in silico tools. Additionally, a local minor allele frequency is calculated for each variant every 6 hours enabling clinicians and researchers to rapidly identify rare disease causing mutations in patients. The CMH Variant Warehouse website is publicly accessible and has implemented the Beacon API developed by Global Alliance for Genomics and Health for data sharing.