The clinical utility and diagnostic implementation of human subject cell transdifferentiation followed by RNA sequencing.

RNA sequencing (RNA-seq) has recently been used in translational research settings to facilitate diagnoses of Mendelian disorders. A significant obstacle for clinical laboratories in adopting RNA-seq is the low or absent expression of a significant number of disease-associated genes/transcripts in clinically accessible samples. As this is especially problematic in neurological diseases, we developed a clinical diagnostic approach that enhanced the detection and evaluation of tissue-specific genes/transcripts through fibroblast-to-neuron cell transdifferentiation. The approach is designed specifically to suit clinical implementation, emphasizing simplicity, cost effectiveness, turnaround time, and reproducibility. For clinical validation, we generated induced neurons (iNeurons) from 71 individuals with primary neurological phenotypes recruited to the Undiagnosed Diseases Network. The overall diagnostic yield was 25.4%. Over a quarter of the diagnostic findings benefited from transdifferentiation and could not be achieved by fibroblast RNA-seq alone. This iNeuron transcriptomic approach can be effectively integrated into diagnostic whole-transcriptome evaluation of individuals with genetic disorders.

Generation of five induced pluripotent stem cell lines from patients with MECP2 Duplication Syndrome.

MECP2 Duplication Syndrome (MDS) is a rare, severe neurodevelopmental disorder arising from duplications in the Xq28 region containing the MECP2 gene that predominantly affects males. We generated five human induced pluripotent stem cell (iPSC) lines from the fibroblasts of individuals carrying between 0.355 and 11.2 Mb size duplications in the chromosomal locus containing MECP2. All lines underwent extensive testing to confirm MECP2 duplication and iPSC-related features such as morphology, pluripotency markers, and trilineage differentiation potential. These lines are a valuable resource for molecular and functional studies of MDS as well as screening for a variety of therapeutic approaches.

Preinfusion factors impacting relapse immunophenotype following CD19 CAR T cells.

Relapse following chimeric antigen receptor (CAR) T-cell therapy directed against CD19 for relapsed/refractory B-acute lymphoblastic leukemia (r/r B-ALL) remains a significant challenge. Three main patterns of relapse predominate: CD19 positive (CD19pos) relapse, CD19 negative (CD19neg) relapse, and lineage switch (LS). Development and validation of risk factors that predict relapse phenotype could help define potential pre- or post-CAR T-cell infusion interventions aimed at decreasing relapse. Our group sought to extensively characterize preinfusion risk factors associated with the development of each relapse pattern via a multicenter, retrospective review of children and young adults with r/r B-ALL treated with a murine-based CD19-CAR construct. Of 420 patients treated with CAR, 166 (39.5%) relapsed, including 83 (50%) CD19pos, 68 (41%) CD19neg, and 12 (7.2%) LS relapses. A greater cumulative number of prior complete remissions was associated with CD19pos relapses, whereas high preinfusion disease burden, prior blinatumomab nonresponse, older age, and 4-1BB CAR construct were associated with CD19neg relapses. The presence of a KMT2A rearrangement was the only preinfusion risk factor associated with LS. The median overall survival following a post-CAR relapse was 11.9 months (95% CI, 9-17) and was particularly dismal in patients experiencing an LS, with no long-term survivors following this pattern of relapse. Given the poor outcomes for those with post-CAR relapse, study of relapse prevention strategies, such as consolidative hematopoietic stem cell transplantation, is critical and warrants further investigation on prospective clinical trials.

Blinatumomab Nonresponse and High-Disease Burden Are Associated With Inferior Outcomes After CD19-CAR for B-ALL.

PURPOSE: CD19-targeted chimeric antigen receptor T cells (CD19-CAR) and blinatumomab effectively induce remission in relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) but are also associated with CD19 antigen modulation. There are limited data regarding the impact of prior blinatumomab exposure on subsequent CD19-CAR outcomes. PATIENTS AND METHODS: We conducted a multicenter, retrospective review of children and young adults with relapsed or refractory ALL who received CD19-CAR between 2012 and 2019. Primary objectives addressed 6-month relapse-free survival (RFS) and event-free survival (EFS), stratified by blinatumomab use. Secondary objectives included comparison of longer-term survival outcomes, complete remission rates, CD19 modulation, and identification of factors associated with EFS. RESULTS: Of 420 patients (median age, 12.7 years; interquartile range, 7.1-17.5) treated with commercial tisagenlecleucel or one of three investigational CD19-CAR constructs, 77 (18.3%) received prior blinatumomab. Blinatumomab-exposed patients more frequently harbored KMT2A rearrangements and underwent a prior stem-cell transplant than blinatumomab-naïve patients. Among patients evaluable for CD19-CAR response (n = 412), blinatumomab nonresponders had lower complete remission rates to CD19-CAR (20 of 31, 64.5%) than blinatumomab responders (39 of 42, 92.9%) or blinatumomab-naive patients (317 of 339, 93.5%), P < .0001. Following CD19-CAR, blinatumomab nonresponders had worse 6-month EFS (27.3%; 95% CI, 13.6 to 43.0) compared with blinatumomab responders (66.9%; 95% CI, 50.6 to 78.9; P < .0001) or blinatumomab-naïve patients (72.6%; 95% CI, 67.5 to 77; P < .0001) and worse RFS. High-disease burden independently associated with inferior EFS. CD19-dim or partial expression (preinfusion) was more frequently seen in blinatumomab-exposed patients (13.3% v 6.5%; P = .06) and associated with lower EFS and RFS. CONCLUSION: With the largest series to date in pediatric CD19-CAR, and, to our knowledge, the first to study the impact of sequential CD19 targeting, we demonstrate that blinatumomab nonresponse and high-disease burden were independently associated with worse RFS and EFS, identifying important indicators of long-term outcomes following CD19-CAR.

Endozoochory by mallard in New Zealand: what seeds are dispersed and how far?

In Europe and North America waterfowl are major dispersers of aquatic and terrestrial plants, but in New Zealand their role has yet to be investigated. Mallards were introduced to New Zealand in the late 1800s, and today they are the most abundant and widespread waterfowl in the country. To assess seed dispersal, we radiomarked 284 female mallards from two study sites during the pre-breeding (June-August) and breeding (August-December) periods in 2014-2015, and examined movements that occurred within 24, 48 or 72 h when seed dispersal by endozoochory is considered likely. During June and July 2015, we collected 29 faecal samples from individual female mallards during radiomarking and 24 samples from mallard flocks. We recovered 69 intact seeds from the faecal samples and identified 12 plant taxa. Of the plant seeds identified and dispersed by mallards in this study, 40% were members of the Asteraceae family, nine plant species were alien to New Zealand, and the indigenous-status of three unidentified taxa could not be determined. Two taxa (and 9% of seeds) were germinated following gut passage: an unidentified Asteraceae and Solanum nigrum. During the pre-breeding and breeding periods, movement of females within 24 h averaged 394 m (SD = 706 m) and 222 m (SD = 605 m) respectively, with maximum distances of 3,970 m and 8,028 m. Maxima extended to 19,230 m within 48 h. Most plant species recorded are generally assumed to be self-dispersed or dispersed by water; mechanisms that provide a much lower maximum dispersal distance than mallards. The ability of mallards to disperse viable seeds up to 19 km within 48 h suggests they have an important and previously overlooked role as vectors for a variety of wetland or grassland plant species in New Zealand.

Reproductive effort and success of wild female mallards: does male quality matter?

In accordance with the differential allocation hypothesis, females are expected to increase their reproductive investment when mated to high-quality males. In waterfowl, reproductive, investment increased when captive female mallards (Anas platyrhynchos) were mated to more attractive males, but information for wild ducks is lacking. Studies of waterfowl mating systems have focused primarily on the importance of plumage coloration of males and female mate choice, whereas investigations of reproductive ecology examine female attributes and virtually ignore the role of males in investment decisions. Here, we used unique data for 253 pairs of wild mallards to test whether females mated to high-quality males would increase reproductive effort and reproduce more successfully. We derived measurements of female and male body size and condition, and indices of male plumage quality, and related these traits to patterns of reproductive effort and performance of females. Consistent with predictions, yearling females nested earlier and had higher nest survival when mated to males with better plumage scores. Furthermore, when paired with larger bodied males, yearling females renested more often, and nest and brood survival increased among older females. Although the strength of male effects varied with breeding stage and female age or experience, this is one of a few studies to demonstrate an additive effect of male quality on investment and success of females, in free-ranging birds.