Looking beyond the exome: a phenotype-first approach to molecular diagnostic resolution in rare and undiagnosed diseases.

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses

Characterization of an Orphan Diterpenoid Biosynthetic Operon from Salinispora arenicola

While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe–microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms

Oxidative cyclization of prodigiosin by an alkylglycerol monooxygenase-like enzyme

Prodiginines, which are tripyrrole alkaloids displaying a wide array of bioactivities, occur as linear and cyclic congeners. Identification of an unclustered biosynthetic gene led to the discovery of the enzyme responsible for catalyzing the regiospecific C-H activation and cyclization of prodigiosin to cycloprodigiosin in Pseudoalteromonas rubra. This enzyme is related to alkylglycerol monooxygenase and unrelated to RedG, the Rieske oxygenase that produces cyclized prodiginines in Streptomyces, implying convergent evolution

617 The impact of the spectral composition of long-wavelength ultraviolet A1 and visible light on cutaneous biologic effects

Background & Aim: Recent studies have demonstrated visible light and long-wavelength UVA1 (VL+UVA1, 370-700 nm) to cause erythema in light skin and synergistically increased pigmentation in dark skin subjects.1, 2 Spectral compositions of VL+UVA1 may further impact these biologic effects. Yet, no phototesting guidelines exist, thus hindering the development of reliable sunscreens protective against this part of sunlight. The objective of this study was to optimize the spectral output of VL+UVA1 as a step to standardize the assessment of protection from VL+UVA1. Methods: Four subjects with Fitzpatrick skin phototype (SPT) I-III were enrolled in this prospective pilot study. Two VL+UVA1 light sources were used: one with 2% UVA1 and another with 4% UVA1. to match more closely that measured in sunlight. Subjects were irradiated with each light source at 320 J/cm2. Clinical scoring, diffuse reflectance spectroscopy (DRS), and colorimetry were performed immediately, 24 hours, 7 days, and 14 days after irradiation. Results: In all subjects, irradiation with VL+ 4% UVA1 resulted in a stronger cutaneous response than that with VL+ 2% UVA1, showing an average 4-fold and 3-fold increase in immediate erythema and delayed pigmentation, respectively. These results were supported by colorimetry measured Δ a*, Δ ITA, and DRS measured relative dyschromia. Conclusion: These preliminary results indicate that the spectral composition of VL+UVA1 impact cutaneous responses and an output resembling sunlight should be strongly considered when standardizing sunscreen phototesting guidelines. This will enable a realistic and standardized design for the evaluation of sunscreen photoprotection within this spectrum

Sustained therapeutic response to riboflavin in a child with a progressive neurological condition, diagnosed by whole-exome sequencing

One of the most promising outcomes of whole-exome sequencing (WES) is the alteration of medical management following an accurate diagnosis in patients with previously unresolved disorders. Although case reports of targeted therapies resulting from WES have been published, there are few reports with long-term follow-up that confirm a sustained therapeutic response. Following a diagnosis by WES of Brown-Vialetto-Van Laere Syndrome 2 (BVVLS2), high-dose riboflavin therapy was instituted in a 20-mo-old child. An immediate clinical response with stabilization of signs and symptoms was noted over the first 2-4 wk. Subsequent clinical follow-up over the following 8 mo demonstrates not just stabilization, but continuing and sustained improvements in all manifestations of this usually fatal condition, which generally includes worsening motor weakness, sensory ataxia, hearing, and vision impairments. This case emphasizes that early application of WES can transform patient care, enabling therapy that in addition to being lifesaving can sometimes reverse the disabling disease processes in a progressive condition