An investigation into the sensory properties of luffa [Luffa cylindrica (L.)] sap.

The seeds and sap of luffa [Luffa cylindrica (L.)] are usually discarded as waste. As such, this study aimed to identify the sensory properties of luffa sap (aqueous solution) and if it can be incorporated into a food item (orange juice) for added nutritional benefits and as an alternative sweetener. A sensory trial (n = 94) asked participants to evaluate a luffa sap sample and five different orange juice samples with increasing amounts of luffa sap (control [0%], 5%, 7.5%, 10%, 12.5%). The participants evaluated the samples using 9-point hedonic scales, check-all-that-apply, and an open-ended comment question. The luffa sap was described as having a mild flavor as well as woody, earthy, and floral attributes and an aftertaste. The liking scores for the orange juice with the 5% luffa sap did not significantly differ from the control. However, as the amount of luffa sap increased above 5%, the liking scores decreased and were significantly different from the control. The orange juice with luffa sap samples (7.5% and above) was associated with off-flavors, while the orange juice with 5% luffa sap and the control were associated with the attributes (sweet, fruity, orange, tropical, citrus) that increased the participants liking. Future studies should continue to investigate the sensory properties of luffa sap and its incorporation into different food products. PRACTICAL APPLICATION: This is one of the first studies to investigate the sensory properties of luffa sap with participants residing in the Western world. The luffa sap was found to be woody, earthy, bitter, and floral. It was acceptable to add luffa sap to orange juice up to 5% by volume. However, it did not increase the sweetness perception of the orange juice. At a 7.5% luffa sap addition and higher levels, off-flavors were observed in the orange juice.

Coevolutionary analysis of Pseudomonas syringae-phage interactions to help with rational design of phage treatments.

Treating plant bacterial diseases is notoriously difficult because of the lack of available antimicrobials. Pseudomonas syringae pathovar syringae (Pss) is a major pathogen of cherry (Prunus avium) causing bacterial canker of the stem, leaf and fruit, impacting productivity and leading to a loss of trees. In an attempt to find a treatment for this disease, naturally occurring bacteriophage (phage) that specifically target Pss is being investigated as a biocontrol strategy. However, before using them as a biocontrol treatment, it is important to both understand their efficacy in reducing the bacterial population and determine if the bacterial pathogens can evolve resistance to evade phage infection. To investigate this, killing curve assays of five MR phages targeting Pss showed that phage resistance rapidly emerges in vitro, even when using a cocktail of the five phages together. To gain insight to the changes occurring, Pss colonies were collected three times during a 66-h killing curve assay and separately, Pss and phage were also coevolved over 10 generations, enabling the measurement of genomic and fitness changes in bacterial populations. Pss evolved resistance to phages through modifications in lipopolysaccharide (LPS) synthesis pathways. Bacterial fitness (growth) and virulence were affected in only a few mutants. Deletion of LPS-associated genes suggested that LPS was the main target receptor for all five MR phages. Later generations of coevolved phages from the coevolution experiment were more potent at reducing the bacterial density and when used with wild-type phages could reduce the emergence of phage-resistant mutants. This study shows that understanding the genetic mechanisms of bacterial pathogen resistance to phages is important for helping to design a more effective approach to kill the bacteria while minimizing the opportunity for phage resistance to manifest.

Investigation into the Sensory Properties of Plant-Based Eggs, as Well as Acceptance, Emotional Response, and Use.

Consumers have become interested in plant-based alternatives to animal-based products. One of the under-studied alternatives is plant-based eggs (PBEs). This research investigated PBEs relative to conventional eggs and tofu scramble-another plant-based alternative. Firstly, participants (n = 93) completed a word association task asking them about PBEs. Participants then evaluated the different food samples using hedonic scales, check-all-that-apply (CATA), and temporal check-all-that-apply (TCATA), as well as identified their emotional response and proposed use for PBEs. Participants were interested in plant-based alternatives, including PBEs, but they were concerned about the sensory properties. When they evaluated the different samples, the flavour and texture of the PBEs were disliked in comparison to the eggs. This result may be due to the beany, bitterness, and off-flavour attributes associated with the PBEs. Participants also associated the PBEs with negative emotions. The liking of tofu scramble was not significantly different from the eggs, and the eggs and tofu scramble were mainly associated with positive emotions. During the TCATA evaluation, the participants focused on the flavour attributes of PBEs, while their evaluation of the eggs was dominated by the textural attributes. Whether following a plant-based diet or not, consumers are interested in PBEs, but the sensory properties of PBEs need to be improved before they are willing to adopt them into their diet. This study is one of the first to evaluate the sensory properties of PBEs, as well as consumers' emotional response to them and their attitudes about PBEs.

Centralized industrialization of pork in Europe and America contributes to the global spread of Salmonella enterica.

Salmonella enterica causes severe food-borne infections through contamination of the food supply chain. Its evolution has been associated with human activities, especially animal husbandry. Advances in intensive farming and global transportation have substantially reshaped the pig industry, but their impact on the evolution of associated zoonotic pathogens such as S. enterica remains unresolved. Here we investigated the population fluctuation, accumulation of antimicrobial resistance genes and international serovar Choleraesuis transmission of nine pig-enriched S. enterica populations comprising more than 9,000 genomes. Most changes were found to be attributable to the developments of the modern pig industry. All pig-enriched salmonellae experienced host transfers in pigs and/or population expansions over the past century, with pigs and pork having become the main sources of S. enterica transmissions to other hosts. Overall, our analysis revealed strong associations between the transmission of pig-enriched salmonellae and the global pork trade.

Sensory properties of thickened and protein-enriched plant-based frozen desserts.

The number of consumers following plant-based diets has increased and in turn, the variety of plant-based foods available on the market has also increased. Many plant-based foods aim to mimic the functionality and sensory properties of conventional dairy products; however, they may not be suitable for specific populations. Dysphagia, for example, is a swallowing condition requiring texture-modified foods that meet specific criteria. While many conventional thickened products exist that are safe for individuals with dysphagia, the growing interest in plant-based eating alongside the increasing prevalence of dysphagia prompts a need for research on the use and safety of thickened plant-based alternatives. This study investigated the sensory properties of a thickened protein-enhanced ice cream (dairy and whey) compared to thickened protein-enhanced plant-based frozen desserts (cashew and pea, and coconut and pea). The formulations were evaluated using the International Dysphagia Diet Standardization (IDDSI) Spoon Tilt Test and a sensory trial (n = 104 participants, 47 flexitarians and 57 typical consumers) using static (hedonic scales and check-all-that-apply [CATA]), and dynamic (temporal check-all-that-apply (TCATA)) methods. The dairy and whey sample consistently passed the IDDSI test, while the plant-based samples did not. TCATA identified that the plant-based samples had an increased cohesiveness and adhesiveness, and decreased slipperiness when compared to the dairy and whey sample. The differences in textural properties may explain why the plant-based samples did not pass the IDDSI test. The study identified that although plant-based foods strive to mimic conventional dairy products, they have different textural and flavor properties.

Embryonic statistical analyses reveal 2 growth phenotypes in mouse models of Down syndrome.

BACKGROUND: Down syndrome is associated with several comorbidities, including intellectual disability, growth restriction, and congenital heart defects. The prevalence of Down syndrome-associated comorbidities is highly variable, and intellectual disability, although fully penetrant, ranges from mild to severe. Understanding the basis of this interindividual variability might identify predictive biomarkers of in utero and postnatal outcomes that could be used as endpoints to test the efficacy of future therapeutic interventions. OBJECTIVE: The main objective of this study was to examine if antenatal interindividual variability exists in mouse models of Down syndrome and whether applying statistical approaches to clinically relevant measurements (ie, the weights of the embryo, placenta, and brain) could define cutoffs that discriminate between subgroups of trisomic embryos. STUDY DESIGN: Three commonly used mouse models of Down syndrome (Dp(16)1/Yey, Ts65Dn, and Ts1Cje) and a new model (Ts66Yah) were used in this study. Trisomic and euploid littermate embryos were used from each model with total numbers of 102 for Ts66Yah, 118 for Dp(16)1/Yey, 92 for Ts65Dn, and 126 for Ts1Cje. Placental, embryonic, and brain weights and volumes at embryonic day 18.5 were compared between genotypes in each model. K-mean clustering analysis was applied to embryonic and brain weights to identify severity classes in trisomic embryos, and brain and placental volumetric measurements were compared between genotypes and classes for each strain. In addition, Ts66Yah embryos were examined for malformations because embryonic phenotypes have never been examined in this model. RESULTS: Reduced body and brain weights were present in Ts66Yah, Dp(16)1/Yey, and Ts65Dn embyos. Cluster analysis identified 2 severity classes in trisomic embryos-mild and severe-in all 4 models that were distinguishable using a putative embryonic weight cutoff of <0.5 standard deviation below the mean. Ts66Yah trisomic embryos develop congenital anomalies that are also found in humans with Down syndrome, including congenital heart defects and renal pelvis dilation. CONCLUSION: Statistical approaches applied to clinically relevant measurements revealed 2 classes of phenotypic severity in trisomic mouse models of Down syndrome. Analysis of severely affected trisomic animals may facilitate the identification of biomarkers and endpoints that can be used to prenatally predict outcomes and the efficacy of treatments.

PGF2α induces a pro-labour phenotypical switch in human myometrial cells that can be inhibited with PGF2α receptor antagonists.

Preterm birth is the leading cause of infant morbidity and mortality. There has been an interest in developing prostaglandin F2α (PGF2α) antagonists as a new treatment for preterm birth, although much of the rationale for their use is based on studies in rodents where PGF2α initiates labour by regressing the corpus luteum and reducing systemic progesterone concentrations. How PGF2α antagonism would act in humans who do not have a fall in systemic progesterone remains unclear. One possibility, in addition to an acute stimulation of contractions, is a direct alteration of the myometrial smooth muscle cell state towards a pro-labour phenotype. In this study, we developed an immortalised myometrial cell line, MYLA, derived from myometrial tissue obtained from a pregnant, non-labouring patient, as well as a novel class of PGF2α receptor (FP) antagonist. We verified the functionality of the cell line by stimulation with PGF2α, resulting in Gαq-specific coupling and Ca2+ release, which were inhibited by FP antagonism. Compared to four published FP receptor antagonists, the novel FP antagonist N582707 was the most potent compound [Fmax 7.67 ± 0.63 (IC50 21.26 nM), AUC 7.30 ± 0.32 (IC50 50.43 nM), and frequency of Ca2+ oscillations 7.66 ± 0.41 (IC50 22.15 nM)]. RNA-sequencing of the MYLA cell line at 1, 3, 6, 12, 24, and 48 h post PGF2α treatment revealed a transforming phenotype from a fibroblastic to smooth muscle mRNA profile. PGF2α treatment increased the expression of MYLK, CALD1, and CNN1 as well as the pro-labour genes OXTR, IL6, and IL11, which were inhibited by FP antagonism. Concomitant with the inhibition of a smooth muscle, pro-labour transition, FP antagonism increased the expression of the fibroblast marker genes DCN, FBLN1, and PDGFRA. Our findings suggest that in addition to the well-described acute contractile effect, PGF2α transforms myometrial smooth muscle cells from a myofibroblast to a smooth muscle, pro-labour-like state and that the novel compound N582707 has the potential for prophylactic use in preterm labour management beyond its use as an acute tocolytic drug.

Rhizobial nitrogen fixation efficiency shapes endosphere bacterial communities and Medicago truncatula host growth.

BACKGROUND: Despite the knowledge that the soil-plant-microbiome nexus is shaped by interactions amongst its members, very little is known about how individual symbioses regulate this shaping. Even less is known about how the agriculturally important symbiosis of nitrogen-fixing rhizobia with legumes is impacted according to soil type, yet this knowledge is crucial if we are to harness or improve it. We asked how the plant, soil and microbiome are modulated by symbiosis between the model legume Medicago truncatula and different strains of Sinorhizobium meliloti or Sinorhizobium medicae whose nitrogen-fixing efficiency varies, in three distinct soil types that differ in nutrient fertility, to examine the role of the soil environment upon the plant-microbe interaction during nodulation. RESULTS: The outcome of symbiosis results in installment of a potentially beneficial microbiome that leads to increased nutrient uptake that is not simply proportional to soil nutrient abundance. A number of soil edaphic factors including Zn and Mo, and not just the classical N/P/K nutrients, group with microbial community changes, and alterations in the microbiome can be seen across different soil fertility types. Root endosphere emerged as the plant microhabitat more affected by this rhizobial efficiency-driven community reshaping, manifested by the accumulation of members of the phylum Actinobacteria. The plant in turn plays an active role in regulating its root community, including sanctioning low nitrogen efficiency rhizobial strains, leading to nodule senescence in particular plant-soil-rhizobia strain combinations. CONCLUSIONS: The microbiome-soil-rhizobial dynamic strongly influences plant nutrient uptake and growth, with the endosphere and rhizosphere shaped differentially according to plant-rhizobial interactions with strains that vary in nitrogen-fixing efficiency levels. These results open up the possibility to select inoculation partners best suited for plant, soil type and microbial community. Video Abstract.

Haplotype-based analysis resolves missing heritability in oculocutaneous albinism type 1B.

Oculocutaneous albinism (OCA) is a rare disorder of pigment production. Affected individuals have variably decreased global pigmentation and visual-developmental changes that lead to low vision. OCA is notable for significant missing heritability, particularly among individuals with residual pigmentation. Tyrosinase (TYR) is the rate-limiting enzyme in melanin pigment biosynthesis and mutations that decrease enzyme function are one of the most common causes of OCA. We present the analysis of high-depth short-read TYR sequencing data for a cohort of 352 OCA probands, ∼50% of whom were previously sequenced without yielding a definitive diagnostic result. Our analysis identified 66 TYR single-nucleotide variants (SNVs) and small insertion/deletions (indels), 3 structural variants, and a rare haplotype comprised of two common frequency variants (p.Ser192Tyr and p.Arg402Gln) in cis-orientation, present in 149/352 OCA probands. We further describe a detailed analysis of the disease-causing haplotype, p.[Ser192Tyr; Arg402Gln] ("cis-YQ"). Haplotype analysis suggests that the cis-YQ allele arose by recombination and that multiple cis-YQ haplotypes are segregating in OCA-affected individuals and control populations. The cis-YQ allele is the most common disease-causing allele in our cohort, representing 19.1% (57/298) of TYR pathogenic alleles in individuals with type 1 (TYR-associated) OCA. Finally, among the 66 TYR variants, we found several additional alleles defined by a cis-oriented combination of minor, potentially hypomorph-producing alleles at common variant sites plus a second, rare pathogenic variant. Together, these results suggest that identification of phased variants for the full TYR locus are required for an exhaustive assessment for potentially disease-causing alleles.

Highly Sensitive and Specific Detection of Bladder Cancer via Targeted Ultra-deep Sequencing of Urinary DNA.

BACKGROUND: There is an unmet need for an accurate, validated, noninvasive test for diagnosing and monitoring bladder cancer (BC). Detection of BC-associated mutations in urinary DNA via targeted deep sequencing could meet this need. OBJECTIVE: To test the ability of mutational analysis of urinary DNA to noninvasively detect BC within the context of haematuria investigations and non-muscle-invasive BC (NMIBC) surveillance. DESIGN, SETTING, AND PARTICIPANTS: Capture-based ultra-deep sequencing was performed for 443 somatic mutations in 23 genes in 591 urine cell-pellet DNAs from haematuria clinic patients and 293 from NMIBC surveillance patients. Variant calling was optimised to minimise false positives using urine samples from 162 haematuria clinic patients without BC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The sensitivity and specificity for BC diagnosis were determined. RESULTS AND LIMITATIONS: Mutational analysis of urinary DNA detected 144 of the 165 haematuria patients diagnosed with incident BC from two independent cohorts, yielding overall sensitivity of 87.3% (95% confidence interval [CI] 81.2-92.0%) at specificity of 84.8% (95% CI 79.9-89.0%). The sensitivity was 97.4% for grade 3, 86.5% for grade 2, and 70.8% for grade 1 BC. Among NMIBC surveillance patients, 25 out of 29 recurrent BCs were detected, yielding sensitivity of 86.2% (95% CI 70.8-97.7%) at specificity of 62.5% (95% CI 56.1-68.0%); a positive urine mutation test in the absence of clinically detectable disease was associated with a 2.6-fold increase in the risk of future recurrence. The low number of recurrences in the NMIBC surveillance cohort and the lower sensitivity for detecting grade 1 pTa BC are limitations. CONCLUSIONS: Detection of mutations in a small panel of BC-associated genes could facilitate noninvasive BC testing and expedite haematuria investigations. Following further validation, the test could also play a role in NMIBC surveillance. PATIENT SUMMARY: Identification of alterations in genes that are frequently mutated in bladder cancer appears to be a promising strategy for detecting disease from urine samples and reducing reliance on examination of the bladder via a telescopic camera inserted through the urethra.

EnteroBase: hierarchical clustering of 100 000s of bacterial genomes into species/subspecies and populations.

The definition of bacterial species is traditionally a taxonomic issue while bacterial populations are identified by population genetics. These assignments are species specific, and depend on the practitioner. Legacy multilocus sequence typing is commonly used to identify sequence types (STs) and clusters (ST Complexes). However, these approaches are not adequate for the millions of genomic sequences from bacterial pathogens that have been generated since 2012. EnteroBase (http://enterobase.warwick.ac.uk) automatically clusters core genome MLST allelic profiles into hierarchical clusters (HierCC) after assembling annotated draft genomes from short-read sequences. HierCC clusters span core sequence diversity from the species level down to individual transmission chains. Here we evaluate HierCC's ability to correctly assign 100 000s of genomes to the species/subspecies and population levels for Salmonella, Escherichia, Clostridoides, Yersinia, Vibrio and Streptococcus. HierCC assignments were more consistent with maximum-likelihood super-trees of core SNPs or presence/absence of accessory genes than classical taxonomic assignments or 95% ANI. However, neither HierCC nor ANI were uniformly consistent with classical taxonomy of Streptococcus. HierCC was also consistent with legacy eBGs/ST Complexes in Salmonella or Escherichia and with O serogroups in Salmonella. Thus, EnteroBase HierCC supports the automated identification of and assignment to species/subspecies and populations for multiple genera. This article is part of a discussion meeting issue 'Genomic population structures of microbial pathogens'.

Correlation of age of onset and clinical severity in Niemann-Pick disease type C1 with lysosomal abnormalities and gene expression.

Niemann-Pick disease type C1 (NPC1) is a rare, prematurely fatal lysosomal storage disorder which exhibits highly variable severity and disease progression as well as a wide-ranging age of onset, from perinatal stages to adulthood. This heterogeneity has made it difficult to obtain prompt diagnosis and to predict disease course. In addition, small NPC1 patient sample sizes have been a limiting factor in acquiring genome-wide transcriptome data. In this study, primary fibroblasts from an extensive cohort of 41 NPC1 patients were used to validate our previous findings that the lysosomal quantitative probe LysoTracker can be used as a predictor for age of onset and disease severity. We also examined the correlation between these clinical parameters and RNA expression data from primary fibroblasts and identified a set of genes that were significantly associated with lysosomal defects or age of onset, in particular neurological symptom onset. Hierarchical clustering showed that these genes exhibited distinct expression patterns among patient subgroups. This study is the first to collect transcriptomic data on such a large scale in correlation with clinical and cellular phenotypes, providing a rich genomic resource to address NPC1 clinical heterogeneity and discover potential biomarkers, disease modifiers, or therapeutic targets.

Plant circadian clock control of Medicago truncatula nodulation via regulation of nodule cysteine-rich peptides.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialized polyploid cells within root nodules, which undergo tightly regulated metabolic activity. By carrying out expression analysis of transcripts over time in Medicago truncatula nodules, we found that the circadian clock enables coordinated control of metabolic and regulatory processes linked to nitrogen fixation. This involves the circadian clock-associated transcription factor LATE ELONGATED HYPOCOTYL (LHY), with lhy mutants being affected in nodulation. Rhythmic transcripts in root nodules include a subset of nodule-specific cysteine-rich peptides (NCRs) that have the LHY-bound conserved evening element in their promoters. Until now, studies have suggested that NCRs act to regulate bacteroid differentiation and keep the rhizobial population in check. However, these conclusions came from the study of a few members of this very large gene family that has complex diversified spatio-temporal expression. We suggest that rhythmic expression of NCRs may be important for temporal coordination of bacterial activity with the rhythms of the plant host, in order to ensure optimal symbiosis.

Comparative Genomics across Three Ensifer Species Using a New Complete Genome Sequence of the Medicago Symbiont Sinorhizobium (Ensifer) meliloti WSM1022.

Here, we report an improved and complete genome sequence of Sinorhizobium (Ensifer) meliloti strain WSM1022, a microsymbiont of Medicago species, revealing its tripartite structure. This improved genome sequence was generated combining Illumina and Oxford nanopore sequencing technologies to better understand the symbiotic properties of the bacterium. The 6.75 Mb WSM1022 genome consists of three scaffolds, corresponding to a chromosome (3.70 Mb) and the pSymA (1.38 Mb) and pSymB (1.66 Mb) megaplasmids. The assembly has an average GC content of 62.2% and a mean coverage of 77X. Genome annotation of WSM1022 predicted 6058 protein coding sequences (CDSs), 202 pseudogenes, 9 rRNAs (3 each of 5S, 16S, and 23S), 55 tRNAs, and 4 ncRNAs. We compared the genome of WSM1022 to two other rhizobial strains, closely related Sinorhizobium (Ensifer) meliloti Sm1021 and Sinorhizobium (Ensifer) medicae WSM419. Both WSM1022 and WSM419 species are high-efficiency rhizobial strains when in symbiosis with Medicago truncatula, whereas Sm1021 is ineffective. Our findings report significant genomic differences across the three strains with some similarities between the meliloti strains and some others between the high efficiency strains WSM1022 and WSM419. The addition of this high-quality rhizobial genome sequence in conjunction with comparative analyses will help to unravel the features that make a rhizobial symbiont highly efficient for nitrogen fixation.

Improved systemic AAV gene therapy with a neurotrophic capsid in Niemann-Pick disease type C1 mice.

Niemann-Pick C1 disease (NPC1) is a rare, fatal neurodegenerative disease caused by mutations in NPC1, which encodes the lysosomal cholesterol transport protein NPC1. Disease pathology involves lysosomal accumulation of cholesterol and lipids, leading to neurological and visceral complications. Targeting the central nervous system (CNS) from systemic circulation complicates treatment of neurological diseases with gene transfer techniques. Selected and engineered capsids, for example, adeno-associated virus (AAV)-PHP.B facilitate peripheral-to-CNS transfer and hence greater CNS transduction than parental predecessors. We report that systemic delivery to Npc1 m1N/m1N mice using an AAV-PHP.B vector ubiquitously expressing NPC1 led to greater disease amelioration than an otherwise identical AAV9 vector. In addition, viral copy number and biodistribution of GFP-expressing reporters showed that AAV-PHP.B achieved more efficient, albeit variable, CNS transduction than AAV9 in Npc1 m1N/m1N mice. This variability was associated with segregation of two alleles of the putative AAV-PHP.B receptor Ly6a in Npc1 m1N/m1N mice. Our data suggest that robust improvements in NPC1 disease phenotypes occur even with modest CNS transduction and that improved neurotrophic capsids have the potential for superior NPC1 AAV gene therapy vectors.

Transcriptome of HPßCD-treated Niemann-Pick disease type C1 cells highlights GPNMB as a biomarker for therapeutics.

The rare, fatal neurodegenerative disorder Niemann-Pick disease type C1 (NPC1) arises from lysosomal accumulation of unesterified cholesterol and glycosphingolipids. These subcellular pathologies lead to phenotypes of hepatosplenomegaly, neurological degeneration and premature death. The timing and severity of NPC1 clinical presentation is extremely heterogeneous. This study analyzed RNA-Seq data from 42 NPC1 patient-derived, primary fibroblast cell lines to determine transcriptional changes induced by treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a compound currently under investigation in clinical trials. A total of 485 HPßCD-responsive genes were identified. Pathway enrichment analysis of these genes showed significant involvement in cholesterol and lipid biosynthesis. Furthermore, immunohistochemistry of the cerebellum as well as measurements of plasma from Npc1m1N null mice treated with HPßCD and adeno-associated virus gene therapy suggests that one of the identified genes, GPNMB, may serve as a useful biomarker of treatment response in NPC1 disease. Overall, this large NPC1 patient-derived dataset provides a comprehensive foundation for understanding the genomic response to HPßCD treatment.

A custom capture sequence approach for oculocutaneous albinism identifies structural variant alleles at the OCA2 locus.

Oculocutaneous albinism (OCA) is a heritable disorder of pigment production that manifests as hypopigmentation and altered eye development. Exon sequencing of known OCA genes is unsuccessful in producing a complete molecular diagnosis for a significant number of affected individuals. We sequenced the DNA of individuals with OCA using short-read custom capture sequencing that targeted coding, intronic, and noncoding regulatory regions of known OCA genes, and genome-wide association study-associated pigmentation loci. We identified an OCA2 complex structural variant (CxSV), defined by a 143 kb inverted segment reintroduced in intron 1, upstream of the native location. The corresponding CxSV junctions were observed in 11/390 probands screened. The 143 kb CxSV presents in one family as a copy number variant duplication for the 143 kb region. In the remaining 10/11 families, the 143 kb CxSV acquired an additional 184 kb deletion across the same region, restoring exons 3-19 of OCA2 to a copy-number neutral state. Allele-associated haplotype analysis found rare SNVs rs374519281 and rs139696407 are linked with the 143 kb CxSV in both OCA2 alleles. For individuals in which customary molecular evaluation does not reveal a biallelic OCA diagnosis, we recommend preliminary screening for these haplotype-associated rare variants, followed by junction-specific validation for the OCA2 143 kb CxSV.

Genetic background modifies phenotypic severity and longevity in a mouse model of Niemann-Pick disease type C1.

Niemann-Pick disease type C1 (NPC1) is a rare, fatal neurodegenerative disorder characterized by lysosomal accumulation of unesterified cholesterol and glycosphingolipids. These subcellular pathologies lead to phenotypes of hepatosplenomegaly, neurological degeneration and premature death. NPC1 is extremely heterogeneous in the timing of clinical presentation and is associated with a wide spectrum of causative NPC1 mutations. To study the genetic architecture of NPC1, we have generated a new NPC1 mouse model, Npc1em1PavNpc1em1Pav/em1Pav mutants showed notably reduced NPC1 protein compared to controls and displayed the pathological and biochemical hallmarks of NPC1. Interestingly, Npc1em1Pav/em1Pav mutants on a C57BL/6J genetic background showed more severe visceral pathology and a significantly shorter lifespan compared to Npc1em1Pav/em1Pav mutants on a BALB/cJ background, suggesting that strain-specific modifiers contribute to disease severity and survival. QTL analysis for lifespan of 202 backcross N2 mutants on a mixed C57BL/6J and BALB/cJ background detected significant linkage to markers on chromosomes 1 and 7. The discovery of these modifier regions demonstrates that mouse models are powerful tools for analyzing the genetics underlying rare human diseases, which can be used to improve understanding of the variability in NPC1 phenotypes and advance options for patient diagnosis and therapy.This article has an associated First Person interview with the first author of the paper.

NPC1 Deficiency in Mice is Associated with Fetal Growth Restriction, Neonatal Lethality and Abnormal Lung Pathology.

The rare lysosomal storage disorder Niemann-Pick disease type C1 (NPC1) arises from mutation of NPC1, which encodes a lysosomal transmembrane protein essential for normal transport and trafficking of cholesterol and sphingolipids. NPC1 is highly heterogeneous in both clinical phenotypes and age of onset. Previous studies have reported sub-Mendelian survival rates for mice homozygous for various Npc1 mutant alleles but have not studied the potential mechanisms underlying this phenotype. We performed the first developmental analysis of a Npc1 mouse model, Npc1em1Pav, and discovered significant fetal growth restriction in homozygous mutants beginning at E16.5. Npc1em1Pav/em1Pav mice also exhibited cyanosis, increased respiratory effort, and over 50% lethality at birth. Analysis of neonatal lung tissues revealed lipid accumulation, notable abnormalities in surfactant, and enlarged alveolar macrophages, suggesting that lung abnormalities may be associated with neonatal lethality in Npc1em1Pav/em1Pav mice. The phenotypic severity of the Npc1em1Pav model facilitated this first analysis of perinatal lethality and lung pathology in an NPC1 model organism, and this model may serve as a useful resource for developing treatments for respiratory complications seen in NPC1 patients.