HLA Typing Status of Hospitalized Pediatric Patients with Sickle Cell Disease: Impact of Socioeconomics and an Initiative to Offer Typing to All Patients with Siblings

Background: Hematopoietic stem cell transplant (HSCT) using an HLA-identical sibling donor is a well-established cure for sickle cell disease (SCD). Historically, HSCT was only offered to patients with SCD who had suffered severe complications; however, given improved HSCT outcomes, it is now reasonable to consider HSCT for most patients with SCD who have an HLA-identical sibling. Thus, HLA typing of all full siblings of patients with SCD should be a clinical priority to ensure patients are aware of and have access to this therapeutic option. The primary objectives of this study are to describe the baseline prevalence of HLA typing among a cohort of hospitalized pediatric patients with SCD and to evaluate whether having had HLA typing is associated with certain characteristics. Secondarily, the study describes the acceptability of HLA typing among patients with a healthy (non-SCD) full sibling who had not already had HLA typing at baseline after dedicated outreach to these families. Methods: Between January 1, 2020 and December 31, 2020 a REDCap database of all hospitalized patients with SCD was prospectively maintained. Patient demographic and clinical information was abstracted via retrospective chart review. To evaluate socioeconomic status, a neighborhood area deprivation index (ADI) was determined for each patient using their home address and the Neighborhood Atlas website (https://www.neighborhoodatlas.medicine.wisc.edu/). ADI is a validated ranking (0-100) of Census Block Groups, considering income, education, employment, and housing quality. A higher ADI represents greater socioeconomic disadvantage. As part of a clinical outreach initiative, patients' families who had not already had typing at the time of their hospitalization were contacted to determine if the patient has a healthy full sibling and, if applicable, offer sibling HLA typing. This outreach was originally planned to occur in person at the time of hospitalization or clinic follow-up, but due the COVID-19 pandemic it was paused and when resumed conducted mostly via telephone. Results: During the 52-week study period, 291 patients with SCD were hospitalized at the study pediatric institution. Seventy-one patients (24%) had already completed HLA typing at the time of their first hospitalization during the study period. These patients with HLA typing at baseline were significantly more likely to have a diagnosis of sickle cell anemia (HbSS/HbSβ 0 genotype) and be on disease-modifying therapy (hydroxyurea or chronic transfusion) compared to patients without typing at baseline (Table). Age and sex were not significantly different between patients with and without typing (Table). The group of patients who did not have HLA typing at baseline had a significantly greater ADI (mean 29.7 vs. 24.0, p=.008) and proportion of patients with a high disadvantage ADI score ≥40 (23% vs. 10%, p=.02), Figure. Of the 220 patients with no history of HLA typing, the sibling status of 187 patients was determined via outreach to these families as of July 2021. Among these 187 patients, 81 (43%) reported having a healthy full sibling. Among these 81 patients with siblings, after being offered family HLA typing, 42 (52%) were interested and referred for HLA typing, 29 (36%) were undecided, and 10 (12%) declined typing. Conclusion: Hospitalized pediatric patients with SCD who had already been HLA typed were more likely to have a severe SCD genotype and be on disease-modifying therapy as expected. Patients who had not had HLA typing were more likely to live in a socioeconomically disadvantaged neighborhood. This finding suggests that dedicated outreach to all families regarding HLA typing is needed. Our clinical initiative to offer typing to all hospitalized patients with healthy full siblings was feasible, with a majority of families interested in pursuing HLA typing. Continued work is needed to ensure patients with SCD have equal access to curative therapy regardless of socioeconomic status. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Evaluating the accuracy of variant calling methods using the frequency of parent-offspring genotype mismatch

The use of NGS datasets has increased dramatically over the last decade, however, there have been few systematic analyses quantifying the accuracy of the commonly used variant caller programs. Here we used a familial design consisting of diploid tissue from a single Pinus contorta parent and the maternally derived haploid tissue from 106 full-sibling offspring, where mismatches could only arise due to mutation or bioinformatic error. Given the rarity of mutation, we used the rate of mismatches between parent and offspring genotype calls to infer the SNP genotyping error rates of FreeBayes, HaplotypeCaller, SAMtools, UnifiedGenotyper, and VarScan. With baseline filtering HaplotypeCaller and UnifiedGenotyper yielded one to two orders of magnitude larger numbers of SNPs and error rates, whereas FreeBayes, SAMtools and VarScan yielded lower numbers of SNPs and more modest error rates. To facilitate comparison between variant callers we standardized each SNP set to the same number of SNPs using additional filtering, where UnifiedGenotyper consistently produced the smallest proportion of genotype errors, followed by HaplotypeCaller, VarScan, SAMtools, and FreeBayes. Additionally, we found that error rates were minimized for SNPs called by more than one variant caller. Finally, we evaluated the performance of various commonly used filtering metrics on SNP calling. Our analysis provides a quantitative assessment of the accuracy of five widely used variant calling programs and offers valuable insights into both the choice of variant caller program and the choice of filtering metrics, especially for researchers using non-model study systems.

Evaluating the accuracy of variant calling methods using the frequency of parent-offspring genotype mismatch

The use of NGS datasets has increased dramatically over the last decade, however, there have been few systematic analyses quantifying the accuracy of the commonly used variant caller programs. Here we used a familial design consisting of diploid tissue from a single Pinus contorta parent and the maternally derived haploid tissue from 106 full-sibling offspring, where mismatches could only arise due to mutation or bioinformatic error. Given the rarity of mutation, we used the rate of mismatches between parent and offspring genotype calls to infer the SNP genotyping error rates of FreeBayes, HaplotypeCaller, SAMtools, UnifiedGenotyper, and VarScan. With baseline filtering HaplotypeCaller and UnifiedGenotyper yielded one to two orders of magnitude larger numbers of SNPs and error rates, whereas FreeBayes, SAMtools and VarScan yielded lower numbers of SNPs and more modest error rates. To facilitate comparison between variant callers we standardized each SNP set to the same number of SNPs using additional filtering, where UnifiedGenotyper consistently produced the smallest proportion of genotype errors, followed by HaplotypeCaller, VarScan, SAMtools, and FreeBayes. Additionally, we found that error rates were minimized for SNPs called by more than one variant caller. Finally, we evaluated the performance of various commonly used filtering metrics on SNP calling. Our analysis provides a quantitative assessment of the accuracy of five widely used variant calling programs and offers valuable insights into both the choice of variant caller program and the choice of filtering metrics, especially for researchers using non-model study systems.

Kin-mediated plasticity in alternative reproductive tactics

Conditional strategies occur when the relative fitness pay-off from expressing a given phenotype is contingent upon environmental circumstances. This conditional strategy model underlies cases of alternative reproductive tactics, in which individuals of one sex employ different means to obtain reproduction. How kin structure affects the expression of alternative reproductive tactics remains unexplored. We address this using the mite Rhizoglyphus echinopus , in which large males develop into aggressive ‘fighters’ and small males develop into non-aggressive ‘scramblers.’ Because only fighters kill their rivals, they should incur a greater indirect fitness cost when competing with their relatives, and thus fighter expression could be reduced in the presence of relatives. We raised mites in full-sibling or mixed-sibship groups and found that fighters were more common at higher body weights in full-sibling groups, not less common as we predicted (small individuals were almost exclusively scramblers in both treatments). This result could be explained if relatedness and cue variability are interpreted signals of population density, since fighters are more common at low densities in this species. Alternatively, our results may indicate that males compete more intensely with relatives in this species. We provide the first evidence of kin-mediated plasticity in the expression of alternative reproductive tactics.

Evaluation of Long-Term Shortleaf Pine Progeny Tests in the Ouachita and Ozark National Forests, USA

Between the late 1970s and the early 1990s, the USDA Forest Service installed 155 shortleaf pine (Pinus echinata Mill.) progeny tests in national forests across the Southern Region of the United States. Using control-pollinated crosses from the Mount Ida Seed Orchard, 84 of these progeny tests were established in the Ouachita and Ozark-St. Francis National Forests in Arkansas and Oklahoma. Each of these 84 test locations had, on average, 33 full-sibling families representing three local geographic seed sources (East Ouachita, West Ouachita, and Ozark). Though largely abandoned years ago, the progeny tests that remain provided an opportunity to determine if significant genetic and genetic × environment variance exists for performance traits (d.b.h., tree height, and survival) decades after installation. In 2018 and 2019, we remeasured d.b.h. and height and determined survival in 15 fully stocked progeny tests. Family variances were significant (p < 0.01) for both d.b.h. and height but not for survival (p > 0.05). Seed sources differed significantly (p < 0.05) for d.b.h., with more pronounced latitudinal differences. Additionally, we determined that individual tree and full-sibling family mean heritabilities were moderate (0.15 and 0.72, respectively, for d.b.h and 0.09 and 0.41, for height), suggesting relatively high genetic to environmental variation and good potential for genetic improvement. We also found that shortleaf pine families were broadly adapted in this region since family-by-test variances were non-significant (p > 0.05).

Multi-year pair-bonding in Murray cod (Maccullochella peelii)

Mating strategies in fishes are known to include polygyny, polyandry and monogamy and provide valuable insights regarding powerful evolutionary forces such as sexual selection. Monogamy is a complex of mating systems that has been relatively neglected. Previous work on mating strategies in fishes has often been based on observation and focused on marine species rather than freshwater fishes. SNPs are increasingly being used as a molecular ecology tool in non-model organisms, and methods of probabilistic genetic analysis of such datasets are becoming available for use in the absence of parental genotypes. This approach can be used to infer mating strategies. The long-term pair bonding seen in mammals, reptiles and birds has not been recorded in freshwater fishes—in every other respect an extremely diverse group. This study shows that multi-year pair bonding occurs in an Australian Percichthyid fish that exhibits paternal care of eggs and larvae. Using SNPs, full sibling pairs of larvae were found over multiple years in a three-year study. Stable isotope signatures of the larvae support the genetic inference that full sibling pairs shared a common mother, the ultimate source of that isotopic signature during oogenesis. Spatial and temporal clustering also suggests that the full sibling larvae are unlikely to be false positive identifications of the probabilistic identification of siblings. For the first time, we show multi-year pair bonding in a wild freshwater fish. This will have important conservation and management implications for the species. This approach could provide insights into many behavioural, ecological and evolutionary questions, particularly if this is not a unique case. Our findings are likely to initiate interest in seeking more examples of monogamy and alternative mating strategies in freshwater fishes, particularly if others improve methods of analysis of SNP data for identification of siblings in the absence of parental genotypes.

Family variation in surface feeding behavior of steelhead fry predicts growth rate under hatchery conditions

Hatchery reared salmonid fish often have lower fitness than their natural-origin counterparts when spawning in the wild. Although this difference appears to result from rapid adaptation to captivity, it is not known what traits are under selection. We hypothesize that variation in traits that confer a growth rate advantage to some individuals in the novel hatchery environment are under strong selection because survival at sea is correlated with size at release. Here we show that full sibling families of steelhead trout (Oncorhynchus mykiss) show substantial variation in propensity to feed at the surface as fry, and that the more surface-oriented families grew faster under hatchery conditions. We hypothesize that surface-oriented fry gain an initial growth advantage that persists through size at release. Because surface orientation is a correlate of generalized boldness, hatcheries may inadvertently select for that phenotype, which could explain the fitness differences observed between hatchery and natural-origin fish in the wild.

Metabolite Quantitative Trait Loci for flavonoids provide new insights into the genetic architecture of strawberry (Fragaria x ananassa) fruit quality

Flavonoids are products from specialized metabolism that contribute to fruit sensorial (colour) and nutritional (antioxidant properties) quality. Here, using a pseudo full-sibling F1 progeny previously studied for fruit sensorial quality of cultivated strawberry (Fragaria x ananassa), we explored over two successive years the genetic architecture of flavonoid-related traits using LC-ESI-MS (13 compounds including anthocyanins, flavonols and flavan-3-ols) and colorimetric assays (anthocyanins, flavonoids, phenolics, FRAP and TEAC antioxidant capacity). Network correlation analysis highlighted the high connectivity of flavonoid compounds within each chemical class and low correlation with colorimetric traits except anthocyanins. Mapping onto the female and male linkage maps of 152 flavonoid metabolic QTLs (mQTLs) and of 26 colorimetric QTLs indicated co-localization on few linkage groups of major flavonoid- and taste-related QTLs previously uncovered. These results pave the way for the discovery of genetic variations underlying flavonoid mQTLs and for marker-assisted selection of strawberry varieties with improved sensorial and nutritional quality.