Factors Affecting Migration to GRCh38 in Laboratories Performing Clinical Next-Generation Sequencing.

The most recent build of the human reference genome, GRCh38, was released in 2013. However, many laboratories performing next-generation sequencing (NGS) continue to align to GRCh37. Our aim was to assess the number of clinical diagnostic laboratories that have migrated to GRCh38 and discern factors impeding migration for those still using GRCh37. A brief, five-question survey was electronically administered to 71 clinical laboratories offering constitutional NGS-based testing and analyzed categorically. Twenty-eight responses meeting inclusion criteria were collected from 24 academic and four commercial diagnostic laboratories. Most of these (14; 50%) reported volumes of <500 NGS-based tests in 2019. Only two respondents (7%) had already migrated entirely to GRCh38; most laboratories (15; 54%) had no plans to migrate. The two prevailing reasons for not yet migrating were as follows: laboratories did not feel the benefits outweighed the time and monetary costs (14; 50%); and laboratories had insufficient staff to facilitate the migration (12; 43%). These data, although limited, suggest most clinical molecular laboratories are reluctant to migrate to GRCh38, and there appear to be multiple obstacles to overcome before GRCh38 is widely adopted.

Pathogenic variants in KPTN gene identified by clinical whole-genome sequencing.

Status epilepticus is not rare in critically ill intensive care unit patients, but its diagnosis is often delayed or missed. The mortality for convulsive status epilepticus is dependent on the underlying aetiologies and the age of the patients and thus varies from study to study. In this context, effective molecular diagnosis in a pediatric patient with a genetically heterogeneous phenotype is essential. Homozygous or compound heterozygous variants in KPTN have been recently associated with a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. We describe a comprehensive investigation of a 9-yr-old male patient who was admitted to the intensive care unit, with focal epilepsy, static encephalopathy, autism spectrum disorder, and macrocephaly of unknown etiology, who died of status epilepticus. Clinical whole-genome sequencing revealed compound heterozygous variants in the KPTN gene. The first variant is a previously characterized 18-bp in-frame duplication (c.714_731dup) in exon 8, resulting in the protein change p.Met241_Gln246dup. The second variant, c.394 + 1G > A, affects the splice junction of exon 3. These results are consistent with a diagnosis of autosomal recessive KPTN-related disease. This is the fourth clinical report for KPTN deficiency, providing further evidence of a wider range of severity.

Clinical genome sequencing in an unbiased pediatric cohort.

PURPOSE: We report for the first time, the use of clinical genome sequencing (GS) in an unbiased pediatric cohort. We describe the clinical validation, patient metrics, ordering patterns, results, reimbursement, and physician retrieval of results for the first consecutive 80 cases. METHODS: Clinical GS was performed for both inpatients and outpatients undergoing etiologic evaluations. Results were reported in the electronic medical record. Evidence of report retrieval by clinicians and whether interpretation was concordant with laboratory report was obtained through retrospective chart review. RESULTS: Twenty definitive diagnoses were made in 19 patients (24%; n = 80). Except for two partial gene deletions, all diagnostic variants would have been detectable by our exome methods. Surprisingly, there was no documentation of communication of results to the family in the medical record for 17.5% of patients, and in 7.5%, physician and laboratory interpretations were discordant. Average insurance reimbursement was 30.2%, with yield for commercial payers significantly higher, at 54.1%. CONCLUSIONS: The detection rate of GS is equivalent and potentially superior to exome sequencing (ES). Reimbursement rates were variable but overall satisfactory for commercial insurers, and poor for government entities. In addition, we identify opportunities for improvement in the communication of results to families, likely translatable to other tests and other institutions.

Novel heterozygous pathogenic variants in CHUK in a patient with AEC-like phenotype, immune deficiencies and 1q21.1 microdeletion syndrome: a case report.

BACKGROUND: Ectodermal dysplasias (ED) are a group of diseases that affects the development or function of the teeth, hair, nails and exocrine and sebaceous glands. One type of ED, ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC or Hay-Wells syndrome), is an autosomal dominant disease characterized by the presence of skin erosions affecting the palms, soles and scalp. Other clinical manifestations include ankyloblepharon filiforme adnatum, cleft lip, cleft palate, craniofacial abnormalities and ectodermal defects such as sparse wiry hair, nail changes, dental changes, and subjective hypohydrosis. CASE PRESENTATION: We describe a patient presenting clinical features reminiscent of AEC syndrome in addition to recurrent infections suggestive of immune deficiency. Genetic testing for TP63, IRF6 and RIPK4 was negative. Microarray analysis revealed a 2 MB deletion on chromosome 1 (1q21.1q21.2). Clinical exome sequencing uncovered compound heterozygous variants in CHUK; a maternally-inherited frameshift variant (c.1365del, p.Arg457Aspfs*6) and a de novo missense variant (c.1388C > A, p.Thr463Lys) on the paternal allele. CONCLUSIONS: To our knowledge, this is the fourth family reported with CHUK-deficiency and the second patient with immune abnormalities. This is the first case of CHUK-deficiency with compound heterozygous pathogenic variants, including one variant that arose de novo. In comparison to cases found in the literature, this patient demonstrates a less severe phenotype than previously described.

The NSIGHT1-randomized controlled trial: rapid whole-genome sequencing for accelerated etiologic diagnosis in critically ill infants.

Genetic disorders are a leading cause of morbidity and mortality in infants in neonatal and pediatric intensive care units (NICU/PICU). While genomic sequencing is useful for genetic disease diagnosis, results are usually reported too late to guide inpatient management. We performed an investigator-initiated, partially blinded, pragmatic, randomized, controlled trial to test the hypothesis that rapid whole-genome sequencing (rWGS) increased the proportion of NICU/PICU infants receiving a genetic diagnosis within 28 days. The participants were families with infants aged <4 months in a regional NICU and PICU, with illnesses of unknown etiology. The intervention was trio rWGS. Enrollment from October 2014 to June 2016, and follow-up until November 2016. Of all, 26 female infants, 37 male infants, and 2 infants of undetermined sex were randomized to receive rWGS plus standard genetic tests (n = 32, cases) or standard genetic tests alone (n = 33, controls). The study was terminated early due to loss of equipoise: 73% (24) controls received genomic sequencing as standard tests, and 15% (five) controls underwent compassionate cross-over to receive rWGS. Nevertheless, intention to treat analysis showed the rate of genetic diagnosis within 28 days of enrollment (the primary end-point) to be higher in cases (31%, 10 of 32) than controls (3%, 1 of 33; difference, 28% [95% CI, 10-46%]; p = 0.003). Among infants enrolled in the first 25 days of life, the rate of neonatal diagnosis was higher in cases (32%, 7 of 22) than controls (0%, 0 of 23; difference, 32% [95% CI, 11-53%];p = 0.004). Median age at diagnosis (25 days [range 14-90] in cases vs. 130 days [range 37-451] in controls) and median time to diagnosis (13 days [range 1-84] in cases, vs. 107 days [range 21-429] in controls) were significantly less in cases than controls (p = 0.04). In conclusion, rWGS increased the proportion of NICU/PICU infants who received timely diagnoses of genetic diseases.

Hypotonia and intellectual disability without dysmorphic features in a patient with PIGN-related disease.

BACKGROUND: Defects in the human glycosylphosphatidylinositol anchor biosynthetic pathway are associated with inherited glycosylphosphatidylinositol (GPI)-deficiencies characterized by a broad range of clinical phenotypes including multiple congenital anomalies, dysmorphic faces, developmental delay, hypotonia, and epilepsy. Biallelic variants in PIGN, encoding phosphatidylinositol-glycan biosynthesis class N have been recently associated with multiple congenital anomalies hypotonia seizure syndrome. CASE PRESENTATION: Our patient is a 2 year old male with hypotonia, global developmental delay, and focal epilepsy. Trio whole-exome sequencing revealed heterozygous variants in PIGN, c.181G > T (p.Glu61*) and c.284G > A (p.Arg95Gln). Analysis of FLAER and anti-CD59 by flow-cytometry demonstrated a shift in this patient's granulocytes, confirming a glycosylphosphatidylinositol-biosynthesis defect, consistent with PIGN-related disease. CONCLUSIONS: To date, a total of 18 patients have been reported, all but 2 of whom have congenital anomalies and/or obvious dysmorphic features. Our patient has no significant dysmorphic features or multiple congenital anomalies, which is consistent with recent reports linking non-truncating variants with a milder phenotype, highlighting the importance of functional studies in interpreting sequence variants.

PCDH19-related epileptic encephalopathy in a male mosaic for a truncating variant.

Variants in the X-linked gene PCDH19 are associated with early infantile epileptic encephalopathy-9. This unusual condition spares hemizygous males except for psychiatric and behavioral abnormalities, and for this reason is also known as female limited epilepsy. Some cases are due to de novo PCDH19 variants, but may also be paternally inherited. Our patient is a 6-year-old male with epileptic encephalopathy. Exome sequencing revealed apparent heterozygosity in PCDH19 for a novel nonsense variant, c.605C>A (p.Ser202*), inconsistent with expectations for a male. Testing of other tissues revealed a mixture of mutant and normal alleles. These results are consistent with somatic mosaicism for p.Ser202*. This is the second male with somatic mosaicism for PCDH19 deficiency, providing further support for cellular interference as the pathogenic mechanism for this condition, which leads to this unusual mode of inheritance in which females are more severely affected than males. © 2016 Wiley Periodicals, Inc.

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 children's 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.

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

BACKGROUND: Chromosome 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 PRESENTATION: We 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. CONCLUSION: This is the second family with POLE1-deficency, with the affected individual demonstrating a more severe phenotype than previously described.

Effectiveness of exome and genome sequencing guided by acuity of illness for diagnosis of neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) affect more than 3% of children and are attributable to single-gene mutations at more than 1000 loci. Traditional methods yield molecular diagnoses in less than one-half of children with NDD. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can enable diagnosis of NDD, but their clinical and cost-effectiveness are unknown. One hundred families with 119 children affected by NDD received diagnostic WGS and/or WES of parent-child trios, wherein the sequencing approach was guided by acuity of illness. Forty-five percent received molecular diagnoses. An accelerated sequencing modality, rapid WGS, yielded diagnoses in 73% of families with acutely ill children (11 of 15). Forty percent of families with children with nonacute NDD, followed in ambulatory care clinics (34 of 85), received diagnoses: 33 by WES and 1 by staged WES then WGS. The cost of prior negative tests in the nonacute patients was $19,100 per family, suggesting sequencing to be cost-effective at up to $7640 per family. A change in clinical care or impression of the pathophysiology was reported in 49% of newly diagnosed families. If WES or WGS had been performed at symptom onset, genomic diagnoses may have been made 77 months earlier than occurred in this study. It is suggested that initial diagnostic evaluation of children with NDD should include trio WGS or WES, with extension of accelerated sequencing modalities to high-acuity patients.