Alina Khromykh

Utility of whole-genome sequencing for detection of newborn screening disorders in a population cohort of 1,696 neonates

Purpose:To assess the potential of whole-genome sequencing (WGS) to replicate and augment results from conventional blood-based newborn screening (NBS).Methods:Research-generated WGS data from an ancestrally diverse cohort of 1,696 infants and both parents of each infant were analyzed for variants in 163 genes involved in disorders included or under discussion for inclusion in US NBS programs. WGS results were compared with results from state NBS and related follow-up testing.Results:NBS genes are generally well covered by WGS. There is a median of one (range: 0–6) database-annotated pathogenic variant in the NBS genes per infant. Results of WGS and NBS in detecting 28 state-screened disorders and four hemoglobin traits were concordant for 88.6% of true positives (n = 35) and 98.9% of true negatives (n = 45,757). Of the five infants affected with a state-screened disorder, WGS identified two whereas NBS detected four. WGS yielded fewer false positives than NBS (0.037 vs. 0.17%) but more results of uncertain significance (0.90 vs. 0.013%).Conclusion:WGS may help rule in and rule out NBS disorders, pinpoint molecular diagnoses, and detect conditions not amenable to current NBS assays.Genet Med 18 3, 221–230.

Expanding the phenotypic spectrum in EP300-related Rubinstein-Taybi syndrome.

Rubinstein–Taybi syndrome (RSTS) can be caused by heterozygous mutations or deletions involving CREBBP or, less commonly, EP300. To date, only 15 patients with EP300 mutations have been clinically described. Frequently reported manifestations in these patients include characteristic facial and limb features, varying degrees of neurocognitive dysfunction, and maternal preeclampsia. Other congenital anomalies are less frequently reported. We describe a child found to have a de novo EP300 mutation (c.4933C>T, predicted to result in p.Arg1645X) through research‐based whole‐genome sequencing of the family trio. The childs presentation involved dysmorphic features as well as unilateral renal agenesis, a myelomeningocele, and minor genitourinary anomalies. The involvement of congenital anomalies in all 16 clinically described patients with EP300 mutations (25% of which have been identified by “hypothesis free” methods, including microarray, exome, and whole‐genome sequencing) is reviewed. In summary, genitourinary anomalies have been identified in 38%, cardiovascular anomalies in 25%, spinal/vertebral anomalies in 19%, other skeletal anomalies in 19%, brain anomalies in 13%, and renal anomalies in 6%. Our patient expands the phenotypic spectrum in EP300‐related RSTS; this case demonstrates the evolving practice of clinical genomics related to increasing availability of genomic sequencing methods.

Diagnosis of an imprinted-gene syndrome by a novel bioinformatics analysis of whole-genome sequences from a family trio

Whole‐genome sequencing and whole‐exome sequencing are becoming more widely applied in clinical medicine to help diagnose rare genetic diseases. Identification of the underlying causative mutations by genome‐wide sequencing is greatly facilitated by concurrent analysis of multiple family members, most often the mother–father–proband trio, using bioinformatics pipelines that filter genetic variants by mode of inheritance. However, current pipelines are limited to Mendelian inheritance patterns and do not specifically address disorders caused by mutations in imprinted genes, such as forms of Angelman syndrome and Beckwith–Wiedemann syndrome. Using publicly available tools, we implemented a genetic inheritance search mode to identify imprinted‐gene mutations. Application of this search mode to whole‐genome sequences from a family trio led to a diagnosis for a proband for whom extensive clinical testing and Mendelian inheritance‐based sequence analysis were nondiagnostic. The condition in this patient, IMAGe syndrome, is likely caused by the heterozygous mutation c.832A>G (p.Lys278Glu) in the imprinted gene CDKN1C. The genotypes and disease status of six members of the family are consistent with maternal expression of the gene, and allele‐biased expression was confirmed by RNA‐Seq for the heterozygotes. This analysis demonstrates that an imprinted‐gene search mode is a valuable addition to genome sequence analysis pipelines for identifying disease‐causative variants.

Utilization of genomic sequencing for population screening of immunodeficiencies in the newborn

PurposeImmunodeficiency screening has been added to many state-directed newborn screening programs. The current methodology is limited to screening for severe T-cell lymphopenia disorders. We evaluated the potential of genomic sequencing to augment current newborn screening for immunodeficiency, including identification of non–T cell disorders.MethodsWe analyzed whole-genome sequencing (WGS) and clinical data from a cohort of 1,349 newborn–parent trios by genotype-first and phenotype-first approaches. For the genotype-first approach, we analyzed predicted protein-impacting variants in 329 immunodeficiency-related genes in the WGS data. As a phenotype-first approach, electronic health records were used to identify children with clinical features suggestive of immunodeficiency. Genomes of these children and their parents were analyzed using a separate pipeline for identification of candidate pathogenic variants for rare Mendelian disorders.ResultsWGS provides adequate coverage for most known immunodeficiency-related genes. 13,476 distinct variants and 8,502 distinct predicted protein-impacting variants were identified in this cohort; five individuals carried potentially pathogenic variants requiring expert clinical correlation. One clinically asymptomatic individual was found genomically to have complement component 9 deficiency. Of the symptomatic children, one was molecularly identified as having an immunodeficiency condition and two were found to have other molecular diagnoses.ConclusionNeonatal genomic sequencing can potentially augment newborn screening for immunodeficiency.

Diagnosis of D-Bifunctional Protein Deficiency through Whole-Genome Sequencing: Implications for Cost-Effective Care

D-Bifunctional protein deficiency, caused by recessive mutations in HSD17B4, is a severe disorder of peroxisomal fatty acid oxidation. Nonspecific clinical features may contribute to diagnostic challenges. We describe a newborn female with infantile-onset seizures and nonspecific mild dysmorphisms who underwent extensive genetic workup that resulted in the detection of a novel homozygous mutation (c.302+1_4delGTGA) in the HSD17B4 gene, consistent with a diagnosis of D-bifunctional protein deficiency. By comparing the standard clinical workup to diagnostic analysis performed through research-based whole-genome sequencing (WGS), which independently identified the causative mutation, we demonstrated the ability of genomic sequencing to serve as a timely and cost-effective diagnostic tool for the molecular diagnosis of apparent and occult newborn diseases. As genomic sequencing becomes more available and affordable, we anticipate that WGS and related omics technologies will eventually replace the traditional tiered approach to newborn diagnostic workup.

Mutation in an alternative transcript of CDKL5 in a boy with early-onset seizures

Infantile-onset epilepsies are a set of severe, heterogeneous disorders for which clinical genetic testing yields causative mutations in ∼20%–50% of affected individuals. We report the case of a boy presenting with intractable seizures at 2 wk of age, for whom gene panel testing was unrevealing. Research-based whole-genome sequencing of the proband and four unaffected family members identified a de novo mutation, NM_001323289.1:c.2828_2829delGA in CDKL5, a gene associated with X-linked early infantile epileptic encephalopathy 2. CDKL5 has multiple alternative transcripts, and the mutation lies in an exon in the brain-expressed forms. The mutation was undetected by gene panel sequencing because of its intronic location in the CDKL5 transcript typically used to define the exons of this gene for clinical exon-based tests (NM_003159). This is the first report of a patient with a mutation in an alternative transcript of CDKL5. This finding suggests that incorporating alternative transcripts into the design and variant interpretation of exon-based tests, including gene panel and exome sequencing, could improve the diagnostic yield.

Genomic analysis of an infant with intractable diarrhea and dilated cardiomyopathy

We describe a case of an infant presenting with intractable diarrhea who subsequently developed dilated cardiomyopathy, for whom a diagnosis was not initially achieved despite extensive clinical testing, including panel-based genetic testing. Research-based whole-genome sequences of the proband and both parents were analyzed by the SAVANNA pipeline, a variant prioritization strategy integrating features of variants, genes, and phenotypes, which was implemented using publicly available tools. Although the intestinal morphological abnormalities characteristic of congenital tufting enteropathy (CTE) were not observed in the initial clinical gastrointestinal tract biopsies of the proband, an intronic variant, EPCAM c.556-14A>G, previously identified as pathogenic for CTE, was found in the homozygous state. A newborn cousin of the proband also presenting with intractable diarrhea was found to carry the same homozygous EPCAM variant, and clinical testing revealed intestinal tufting and loss of EPCAM staining. This variant, however, was considered nonexplanatory for the probands dilated cardiomyopathy, which could be a sequela of the childs condition and/or related to other genetic variants, which include de novo mutations in the genes NEDD4L and GSK3A and a maternally inherited SCN5A variant. This study illustrates three ways in which genomic sequencing can aid in the diagnosis of clinically challenging patients: differential diagnosis despite atypical clinical presentation, distinguishing the possibilities of a syndromic condition versus multiple conditions, and generating hypotheses for novel contributory genes.

Putting the Pieces Together: Clinically Relevant Genetic and Genomic Resources for Hospitalists and Neonatologists.

Genetic conditions are individually rare but are common in aggregate, and they often present in the neonatal and early pediatric periods. These conditions are often severe, can be difficult to diagnose and manage, and may heavily affect patients, families, health care systems, and society. Because of recent technological advances, the availability and uptake of genetic and genomic testing are increasing rapidly. However, there is a dearth of trained geneticists and genetic counselors to help guide and explain these conditions and relevant tests. To help hospitalists, neonatologists, and related practitioners navigate this complex and evolving field, we have compiled a list of free (mostly Web-based) resources relevant to the diagnosis and management of genetic conditions and related disorders. These resources, which we describe individually, can be useful for nongeneticist clinicians, and some also include material that can be used to explain concepts and conditions to patients or families. The resources presented are divided into the following categories (which overlap): general information, databases of genetic conditions, resources that can help generate differential diagnoses, databases of genetic testing laboratories (to help with logistics of ordering tests), information on newborn screening, and other resources. We also include a separate list of helpful textbooks and manuals. We conclude with 2 examples describing how some of these resources would be used by a pediatric hospitalist or neonatologist during the inpatient management of a child with a suspected genetic condition.

Experience with genomic sequencing in pediatric patients with congenital cardiac defects in a large community hospital

Congenital cardiac defects, whether isolated or as part of a larger syndrome, are the most common type of human birth defect occurring on average in about 1% of live births depending on the malformation. As there is an expanding understanding of the underlying molecular mechanisms by which a cardiac defect may occur, there is a need to assess the current rates of diagnosis of cardiac defects by molecular sequencing in a clinical setting.