authors@Fred Hutch

Frost, Sophia

February 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
Settling the (Risk) Score for CGVHD Patients 
February 16, 2015 
    SHL Frost 
Chronic graft-versus-host disease (CGVHD) is a complication that may arise in patients who have 
received bone marrow or stem cell transplants from another person (allogeneic transplants), in which 
the transplanted immune cells attack the host's own cells. Compared with acute GVHD, the chronic 
form usually occurs later, normally after 100 days post-transplant, and lasts longer, and it is the 
leading cause of late morbidity and mortality among recipients of allogeneic hematopoietic cell 
transplantation (HCT). 
In 2011, a risk score was developed for patients with CGVHD in a study from the Center for 
International Bone Marrow Transplant Research (CIBMTR) that included a number of Fred Hutch 
investigators. CIBMTR is a voluntary research organization that involves more than 500 
transplantation centers that have agreed to share patient data and collaborate in scientific studies. 
Using multivariate analysis of a cohort of 5343 CGVHD patients, ten significant variables were 
successfully identified for predicting the overall survival and non-relapse mortality; the latter defined 
as death in continuous remission (Arora et al., 2011). The variables were age, prior acute GVHD, 
time from HCT to CGVHD, donor type, disease status at transplantation, GVHD prophylaxis, gender 
mismatch, serum total bilirubin, Karnofsky performance status (the ability to perform ordinary, daily 
activities on a scale from 0 to 100) and platelet count at the time of CGVHD diagnosis. The study 
concluded that patients could be categorized into six well-defined risk groups that adequately 
reflected the assessed outcomes, but to advance from "promising" to clinically useful the score 
needed to be validated in an independent dataset. 
To determine the reproducibility of the risk scores developed in the earlier study, the researchers 
followed up by identifying a new set of 1128 CGVHD patients that received HCT between 2005 and 
2007, and applied the same inclusion criteria and risk-score calculations. In both studies, all patients 
were registered with CIBMTR. Some discrepancies were seen between the validation cohort and the 
training cohort; most importantly, the proportion of patients assigned to the various risk groups 
differed considerably. The proportion of patients in risk groups 1 and 2 (lowest risk) were significantly 
higher in the validation study, and no patients qualified for groups 5 or 6 (highest risk). Only 12% of 
all patients in the validation cohort were assigned risk group 3 or 4, and the two groups were 
therefore combined; the final comparison was consequently carried out in three separate risk groups  
 
February 16, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
(RG1 = lowest risk, RG2 = intermediate, RG3 = highest). The disparity between the two patient 
cohorts was likely due to uniquely different baseline characteristics; nevertheless, good 
discriminatory ability was demonstrated for the CIBMTR CGVHD risk score in predicting overall 
survival and non-relapse mortality in patients with CGVHD. Simply put, better survival was seen in 
patients with a lower risk score (< RG3) than in those with a higher risk score. 
The results of this validation study were recently published in Biology of Blood and Marrow 
Transplantation, with Dr. Mary Flowers as the senior and corresponding author. Dr. Flowers is the 
director of the Adult Clinical Service Long-Term Follow-Up Program (LTFU) at Fred Hutch and 
Seattle Cancer Care Alliance. Although the lower rate of patients assigned to the highest risk groups 
(RG4 to RG6) has yet to be fully explained, another study by Dr. Flowers and colleagues described 
comparable findings when evaluating the score in patients diagnosed using the National Institutes of 
Health (NIH) consensus criteria for GVHD (Inamoto et al., 2014). 
"The CIBMTR risk score performed well in predicting differences in overall survival also when 
applied in other cohorts treated for NIH chronic GVHD in two individual centers," Dr. Flowers said, 
adding that, "Of interest, lower non-relapse mortality was observed in patients with more risk factors 
(> group 3) at the Fred Hutch cohort compared to the Princess Margaret Hospital (Toronto, ON, 
Canada) and the CIBMTR cohort. The dedicated long-term follow up program at Fred Hutch may 
have contributed to the superior outcome noted in this high-risk group." 
And there is more good news for the transplantation community; according to Dr. Flowers, the 
validated risk score can be readily introduced in the clinic: "The CIBMTR CGVHD score is easy to 
apply and can be used for prediction of major outcomes, treatment planning and enrollment in 
clinical trials." Another handy tool for improving the care of cancer patients. 
 
Arora M, Hemmer MT, Ahn KW, Klein JP, Cutler CS, Urbano-Ispizua A, Couriel DR, Alousi AM, Gale 
RP, Inamoto Y, Weisdorf DJ, Li P, Antin JH, Bolwell BJ, Boyiadzis M, Cahn J-Y, Cairo MS, Isola LM, 
Jacobsohn DA, Jagasia M, Klumpp TR, Petersdorf EW, Santarone S, Schouten HC, Wingard JR, 
Spellman SR, Pavletic SZ, Lee SJ, Horowitz MM, Flowers MED. 2014. CIBMTR Chronic GVHD Risk 
Score Predicts Mortality in an Independent Validation Cohort. Biol Blood Marrow Transplant. [Epub 
ahead of print] 
See also: Arora M, Klein JP, Weisdorf DJ, Hassebroek A, Flowers ME, Cutler CS, Urbano-Ispizua A, 
Antin JH, Bolwell BJ, Boyiadzis M, Cahn JY, Cairo MS, Isola L, Jacobsohn DA, Jagasia M, Klumpp 
TR, Lee SJ, Petersdorf EW, Santarone S, Gale RP, Schouten HC, Spellman S, Wingard JR, 
February 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 2 | Fred Hutchinson Cancer Research Center 
 
Horowitz MM, Pavletic SZ. 2011. Chronic GVHD risk score: a Center for International Blood and 
Marrow Transplant Research analysis. Blood. 117(24):6714-20 
Inamoto Y, Kim DD, Storer BE, Moon JH, Lipton JH, Kuruvilla J, Martin PJ, Flowers ME. 
2014. Application of CIBMTR risk score to NIH chronic GVHD at individual centers. Blood. 
123(3):453-5 
 
 
Image provided by Dr. Mary Flowers 
Kaplan-Meier curves showing overall survival and non-
relapse mortality (NRM) among patients with chronic graft-
versus-host disease (CGVHD) according to CIBMTR risk 
score (n = 1128). 
 


English

Settling the (Risk) Score for CGVHD Patients

Kocarnik, Jonathan

March 16, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
Multiethnic Genetic Associations with Lycopene 
Concentrations 
March 16, 2015 
     JM Kocarnik 
Lycopene is a carotenoid found in many red and pink fruits and vegetables, such as tomatoes and 
grapefruits.  Consumption of lycopene-containing foods and higher serum lycopene concentrations 
have been associated with lower risk of several chronic diseases, including cardiovascular disease 
and cancer.  Inconsistencies in quantifying these associations may be explained by genetic variation 
that influences blood lycopene levels.  While these associations have been evaluated in individuals 
of European decent, genetic effects in other populations have not been fully explored.  To address 
this issue, Drs. Niha Zubair, Marian Neuhouser, and colleagues in the Public Health Sciences 
Division conducted a study to evaluate whether genetic variation is associated with serum lycopene 
levels across ethnic groups.  As recently reported in The Journal of Nutrition, the researchers found 
three novel loci associated with serum lycopene concentrations, two of which were unique to African 
Americans. 
Lycopene is one of the most widely consumed carotenoids in the United States.  Previous studies 
suggesting that increased consumption of lycopene-rich foods may decrease cancer risk have been 
widely covered by the media, particularly for prostate cancer.  "Recently, however, the presumed 
role for lycopene in prostate cancer prevention has been disputed," said lead author Dr. Niha 
Zubair.  "These inconsistencies may stem from a lack of knowledge about the genetic variation in the 
synthesis, metabolism, and deposition of transport and binding proteins, which potentially influence 
blood lycopene concentrations."  
To evaluate this hypothesis, the authors performed a genome-wide association study of serum 
lycopene levels in the Women’s Health Initiative (WHI).  This project included women from two 
genetic studies in the WHI: the Single Nucleotide Polymorphism Health Association Resource 
(SHARe), and the Genomics and Randomized Trials Network (GARNET).  Importantly, this allowed 
the researchers to perform multiethnic analyses that included roughly 1,200 European American, 
900 African American, and 460 Hispanic American women.  "This is the first time that such a study 
has been conducted in a multiethnic population," said Dr. Zubair.  
Some 7 million common genetic variants were screened for an association with serum lycopene 
levels in each group, which were then combined for multiethnic meta-analyses.  In so doing, the 
March 16, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
authors found a significant association with three variants near the SCARB1 gene, which encodes a 
cholesterol membrane transporter.  The strongest signal was for rs1672879, where each G allele 
was associated with a 9-20% decrease in serum lycopene concentrations.  While the amount of 
decrease varied slightly by ethnicity group (see figure), the direction of effect was consistent and the 
association remained statistically significant after further adjustment for total serum cholesterol 
levels. 
In the ethnicity-specific analyses, the authors additionally found several variants in two loci that were 
associated with serum lycopene concentrations in African Americans, but not in the other ethnic 
groups.  This included four variants in theSLIT3 gene and four variants in the DHRS2 gene.  These 
variants were monomorphic in the Hispanic and European Americans, demonstrating the importance 
of performing these studies in multiple ethnic groups.  The authors note that previous studies have 
observed discrepancies between dietary intake and serum lycopene levels, and variants specific to 
African Americans may help explain some of this variance.  
Overall, this study identified three novel loci associated with serum lycopene levels, which may help 
to clarify the potential impact of this common dietary component.  Said Dr. Zubair, "Future studies 
looking at the biological function of these specific genes may provide insight into the metabolism and 
underlying function of lycopene in humans, which may further elucidate lycopene’s influence on 
disease risk and health." 
Other PHS researchers contributing to this project were Ms. Jingmin Liu and Drs. Charles 
Kooperberg, Chongzhi Di, and Ulrike Peters. 
 
Citation:  
Zubair N, Kooperberg C, Liu J, Di C, Peters U, Neuhouser ML. 2015. Genetic variation predicts 
serum lycopene concentrations in a multiethnic population of postmenopausal women. J Nutr. 
145(2):187-92. doi: 10.3945/jn.114.202150. 
 
 
 
 
 
March 16, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 3 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provided by Dr. Jonathan Kocarnik 
Percent reduction in serum lycopene concentrations per G allele 
of rs1672879, a single nucleotide polymorphism in the SCARB1 
gene. 
 


Multiethnic Genetic Associations with Lycopene Concentrations

Neves, Alexandre

April 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
Being a Stickler about Going to School 
April 20, 2015 
    A Neves 
Scientists and lay people alike have long been fascinated by the wide diversity of animal behaviors, 
yet the genetic basis for behavioral differences across and within species remains poorly 
understood, particularly in vertebrates. One prominent animal behavior is formation of social groups, 
which can provide a number of advantages, including predator avoidance, but can also come with 
costs, like competition for resources. Genetic mapping of social behaviors has been challenging 
because it requires robust assays that can buffer environmental influences and, yet at the same 
time, model interactions among individuals. Previous studies in the laboratory of Dr. Catherine 
Peichel (Human Biology and Basic Sciences Divisions) sought to overcome these challenges by 
developing a model school assay that used an artificial school of model fish to show that two 
different populations of threespine sticklebacks displayed distinct schooling behaviors (Wark et al., 
2011). Specifically, they showed that marine sticklebacks (Hokkaido, Japan) have a stronger 
tendency to school than benthic sticklebacks from a freshwater lake (Paxton Lake, British Columbia), 
and that these differences were heritable. A separate study by the same lab used quantitative trait 
locus (QTL) mapping in a marine-benthic F2 intercross (when F1 siblings are mated), to identify 
regions of the genome that underlie schooling ability, but this study did not identify significant QTL 
for schooling tendency. 
In a new Fred Hutch study led by staff scientist Anna Greenwood in the Peichel lab, published 
in G3 (Genes, Genomes, Genetics), the investigators attempted to increase the experimental power 
of the model school assay by employing several innovations. First, they backcrossed (when a hybrid 
is crossed with one of its parents) benthic/marine F1 hybrids to benthic fish to increase the number 
of genes that were homozygous for benthic alleles, which the authors hypothesized might result in 
reduced schooling behavior. Second, they performed a new benthic/marine F2 intercross. Third, 
each hybrid fish was tested three times to normalize for environmental effects. Finally, the authors 
developed the choice assay, where the fish could choose between schooling and taking cover under 
an artificial plant. 
The authors first compared the phenotype distribution in hybrid and parental individuals in both the 
school and the choice assays. Marine individuals spent more time with the school and exhibited a 
shorter latency, while the choice assay revealed that benthic fish spent more time with the model 
plant when compared to marine fish. Backcross or intercross individuals also showed wide 
April 20, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
phenotypic distribution, and the average behavior was more similar to benthic values for all but one 
of the traits, suggesting that benthic alleles are more likely to be dominant or semi-dominant. 
Next, the authors performed QTL analysis by genotyping fish with a genome-wide panel of single 
nucleotide polymorphism markers. Two significant QTL were identified in the backcross, one on 
chromosome 6 and the other on chromosome 21. Benthic alleles on chromosome 21 acted to 
reduce the time with the school. In contrast, an increased number of benthic alleles at chromosome 
6 correlated with more time spent with the school and a shorter latency to school. This could be 
interpreted either as the assay not being able to accurately distinguish schooling from shelter 
seeking, or alternatively, this "negative QTL" could also have a biological basis. For the choice 
assay, the investigators identified one significant QTL on chromosome 10, and benthic alleles at this 
locus correlated with decreased approaches to the school. 
In conclusion, this study showed that an enhanced experimental design was able to detect novel 
QTL for schooling tendency in sticklebacks that paves the way for identifying specific genes that 
regulate a fish's motivation to school. "Our study identifies key considerations for experimental 
design of studies that seek to identify genomic contributions to behavior, including using multiple 
genetic crosses and robust behavioral assays. Because all vertebrates share the fundamental brain 
regions that control social behavior, genes that affect social motivation in fish may also play a role in 
regulating human sociality," summarized Dr. Greenwood. 
Greenwood AK, Ardekani R, McCann SR, Dubin ME, Sullivan A, Bensussen S, Tavare S, Peichel 
CL. 2015. Genetic Mapping of Natural Variation in Schooling Tendency in the Threespine 
Stickleback. G3. Epub ahead of print. 
See also: 
Greenwood AK, Wark AR, Yoshida K, Peichel CL. 2013. Genetic and neural modularity underlie the 
evolution of schooling behavior in threespine sticklebacks. Curr Biol. 23(19):1884-8. 
Wark AR, Greenwood AK, Taylor EM, Yoshida K, Peichel CL. 2011. Heritable differences in 
schooling behavior among threespine stickleback populations revealed by a novel assay. PLoS one. 
Mar 25;6(3):e18316 
 
 
 
April 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 4 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provide by Dr. Anna Greenwood 
The model school assay: A live stickleback fish (center) schools 
with eight plastic sticklebacks. 
 


Being a Stickler about Going to School

Pattacini, Laura

May 18, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
Clues on Limiting Herpes Virus Reactivation 
May 18, 2015 
     L Pattacini 
Herpes zoster (shingles) is caused by reactivation of varicella zoster virus. Live attenuated zoster 
strain vOka vaccine is currently approved to prevent varicella zoster reactivation in persons over 50 
years of age.  The vaccine reduces the risk of shingles by 70% in persons aged 50–59 and 51%–
55% in persons ≥60 years, so is only partially effective in the elderly, the most susceptible group. 
The mechanisms by which the vaccine reduces shingles risk are largely unknown, although it is 
clear that both arms of adaptive immunity (B-cells and T-cells) are boosted. A more extensive 
characterization of the immune response generated by the boost and a classification of the peptides 
that induce an immune response are needed to design a more effective preventive strategy. "We 
believe more efficacious vaccines could be developed if the mechanisms of protection were better 
understood", explains Dr. Kerry Laing, first author of a paper published in the April issue of Journal 
of Infectious Diseases. 
In a collaborative research effort among researchers from Fred Hutch, University of Washington and 
the Center for Disease Control and Prevention, the immune responses of 12 subjects receiving the 
zoster boost were characterized. An initial increase in CD4 T-cell response that peaked at day 14 
and decayed at month 6, mainly targeted two proteins of the virus, the opening reading frames 
(ORF) 40 and 67, followed by other 9 different ORFs. Interestingly, boosting not only increased the 
magnitude but also the breadth of the response: the boost in T-cell magnitude mirrored an increase 
in the number of unique viral proteins against which T-cells reacted. Therefore, the increased 
breadth of the response following vaccination might be part of its mechanism of action and thus 
important for blocking viral reactivation. 
While this study focused on CD4 T-cell responses, future work will also focus on the effect of the 
boost on CD8 T-cells: "Of course, we are poised to extend our investigation CD8+ T-cell immunity 
with our complete antigen set to see what clues that cell-type will show us," said Dr Laing. 
The study analyzes the response to zoster vaccination and opens the way to the design of more 
effective vaccines. However, the reported results also raise questions about what immune response 
is needed for protection. In fact, increasing the level or breadth of T-cells alone may not be enough 
to protect from viral reactivation: protection may need, instead, a response to the "right" set of 
peptides. "Using our complete set of viral proteins to compare responses in people protected by 
May 18, 2015 SCIENCE SPOTLIGHT 
 
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zoster vaccination versus people not protected would be a great benefit to understanding this 
further," proposed Dr. Laing. 
This knowledge could conceivably facilitate the development of similar vaccines for related 
alphaherpesviruses (HSV1 and HSV2), for which there is no current cure or effective vaccine. In 
fact, as the zoster vaccine is the only FDA-approved therapeutic vaccine currently on the market, it 
offers a wealth of opportunity to understand how the immune system may work to control recurrent 
infections. 
 
Laing KJ, Russell RM, Dong L, Schmid DS, Stern M, Magaret A, Haas JG, Johnston C, Wald A, 
Koelle DM. 2015. Zoster vaccination increases the breadth of CD4+ T cells responsive to varicella 
zoster virus. Journal of Infectious Diseases. Epub ahead of print. 
 
 
Image provided by Dr. Kerry Laing 
T-cell responses before and after zoster vaccination in 12 study 
subjects (A). The median number of ORF responses in the cohort 
increased approximately 4-fold following vaccination (P = .0495), 
returning to approximately 2-fold above baseline after 6 months (P 
> .999) (B). 
 


Clues on Limiting Herpes Virus Reactivation

Zentner, Gabriel

May 18, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
Mechanism of Promoter Opening by TFIIH 
May 18, 2015 
     GE Zentner 
A critical step in the initiation of gene transcription is the formation of the open complex (OC).  OC 
formation occurs when an RNA polymerase and its associated transcription factors bind to promoter 
DNA, resulting in the unwinding of ~11 base pairs (bp) of DNA upstream of the transcription start 
site.  Opening of promoter DNA depends on the general transcription factor TFIIH, which contains 
two subunits (XPD/Rad3 and XPB/Ssl2, human/yeast protein names) that hydrolyze ATP in the 
presence of DNA, but only XPB/Ssl2 is required for DNA unwinding in the OC.  Both XPB and XPD 
also play crucial roles in nucleotide excision repair.  Moreover, mutations in several TFIIH subunits 
cause various xeroderma pigmentosum (XP) complementation groups (XPB and XPD), XP 
associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTF) by disrupting 
transcription and/or DNA repair (Singh et al., 2015).  Thus, understanding the mechanisms of TFIIH 
action is of great interest.  However, how TFIIH functions in DNA unwinding during OC formation has 
remained controversial.  While the XPD/Rad3 subunit acts as a helicase, it is dispensable for 
transcription, and several properties of XPB/Ssl2 indicate that it cannot function as a traditional 
helicase.  However, it may function as a DNA translocase, tracking along DNA and leaving unwound 
DNA in its wake. 
"Our motivation for this work was to understand the mechanism of why transcription initiation by 
RNA polymerase II is uniquely dependent on ATP hydrolysis – an observation first made in 
1982.  Why is Pol II different from all other Pols in this ATP requirement?  How does Ssl2 work and, 
does this tell us something about the mechanism of DNA unwinding for all Pols?" said Dr. Steven 
Hahn (Basic Sciences Division).  "[Hahn lab postdoc] Sebastian Grünberg first proposed that 
Ssl2/XPB might be a DNA translocase, rotating and inserting DNA into the Pol II active site cleft, 
leading to tortional strain and DNA unwinding.  Previously, the only biochemical activity observed 
with this protein was DNA helicase activity.  In this work, [Hahn lab research assistant] James 
Fishburn, working with our biophysicist collaborators in the Galburt lab [at Washington University in 
St. Louis], showed that Ssl2 is a DNA translocase with properties that can explain many previous 
observations such as the instability of Pol II open complexes, the weak ATPase function of Ssl2, and 
its ability to stimulate Pol II escape from the promoter after transcription initiation." 
The authors first tested potential helicase and translocase functions of TFIIH components using in 
vitro assays.  They assayed TFIIH helicase activity by testing the ability of purified TFIIH containing  
May 18, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
wild-type (WT) or a predicted helicase-deficient Rad3 mutant.  TFIIH containing mutant Rad3 was 
indeed defective in helicase activity but, interestingly, was able to support wild-type levels of 
transcription by Pol II, suggesting that the helicase activity of TFIIH is dispensable for Pol II OC 
formation.  The ability of TFIIH to function as a translocase was next assayed, and revealed a 
robust, ATP-dependent translocase activity. 
DNA translocases are thought to track in a particular direction along the strand of DNA, leading to 
DNA unwinding behind them.  Ssl2 binds DNA downstream of where DNA unwinding occurs in the 
OC, and so the directionality of its translocase activity has important implications for the mechanism 
of DNA unwinding.  To investigate the directionality of Ssl2’s translocase activity, the authors tested 
the ability of Ssl2 to displace a short triplex-forming oligonucleotide (TFO) bound to a longer stretch 
of double-stranded DNA (dsDNA). Ssl2 displaced the TFO when it was bound to the top but not the 
bottom strand of the dsDNA, suggesting that Ssl2 tracks in the 5’ → 3’ direction.  The authors further 
tested this by introducing gaps in the top and bottom strands.  This showed that a gap in the top 
strand strongly inhibited TFO displacement, while a bottom strand gap had little effect, a result 
consistent with 5’ → 3’ translocase activity of Ssl2.  Furthermore, blocking Ssl2 translocation using 
dye-modified nucleotides on the top strand resulted in inhibition of in vitro transcription. 
These results strongly support the a idea model that in which TFIIH functions as a translocase in 
transcriptional initiation.  "Our results have important implications for understanding how Pol II can 
scan for downstream transcription start sites, the molecular basis for mutations that cause human 
disease (xeroderma pigmentosum) and for its role in nucleotide excision repair where it acts in the 
first step of DNA unwinding during removal of DNA lesions," said Dr. Hahn. 
 
Fishburn J, Tomko E, Galburt E, Hahn S. 2015. Double-stranded DNA translocase activity of 
transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation. Proc 
Natl Acad Sci USA 112(13):3961-3966. 
Also see: Singh A, Compe E, Le May N, Egly J-M. 2015. TFIIH Subunit Alterations Causing 
Xeroderma Pigmentosium and Trichothiodystrophy Specifically Disturb Several Steps During 
Transcription. Am J Hum Genet 96(2):194-207. 
 
 
May 18, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 5 | Fred Hutchinson Cancer Research Center 
 
 
 
 
Image provided by Dr. Steven Hahn 
(Left) Ssl2 binds DNA downstream of the site of DNA opening and 
translocates 5’ ? 3’ in an ATP-dependent manner, leading to (right) 
unwinding of promoter DNA. 
 


Mechanism of Promoter Opening by TFIIH

June 15, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
A New Infection Model Incorporates Genetic 
Diversity 
June 15, 2015 
    L Pattacini 
West Nile virus (WNV) is a member of the Flaviviridae family and is transmitted to humans by 
mosquito bites. So far, no vaccine or treatment is available and the only prophylactic strategies 
consist of preventing mosquito bites by using insect repellents or wearing protective clothing. The 
outcome of the infection is variable: while most people do not manifest any symptoms, about 1 in 5 
develop a fever and of these, 1% has a severe, sometimes fatal, neurologic illness characterized by 
encephalitis, meningitis and acute flaccid paralysis. 
Limited knowledge is available on the immune response to WNV in humans due to the high 
prevalence of non-symptomatic cases. Although several studies have been performed on inbred B6 
mice, the infection in this model is less informative because it lacks the genetic diversity observed in 
humans. 
A collaborative effort between Fred Hutch, University of Washington, University of North Carolina at 
Chapel Hill and Emory University led to the generation of a new model to study the immune 
response to WNV infection, the Collaborative Cross (CC) mouse. The CC model was obtained by 
crossing eight different mouse strains, including 5 classical inbred strains and 3 wild type derived 
strains. Recombinant inbred strains were generated by three generation of funnel breeding followed 
by at least 20 generation of inbred mating to develop each line. 
Researchers Jessica Graham and Jessica Swarts from Jennifer Lund's Laboratory (Vaccine and 
Infectious Disease Division), together with their collaborators, investigated the effects of WNV 
infection on different CC strains and reported their results last month on the journal mBio. 
Their results showed that the model was closer to ideal based on its ability to recapitulate the 
variability of the human disease outcome. First, three different disease phenotypes were observed: 
asymptomatic, symptomatic and asymptomatic with central nervous system (CNS) involvement. 
Asymptomatic mice did not show clinical signs, the ones with CNS involvement had inflammatory 
infiltrates in the brain, and symptomatic mice showed severe clinical scores and weight loss starting 
at day 7 after infection. Early innate immune responses, estimated by the expression of interferon 
beta and IFIT1, a molecule modulated by type I interferons, were associated with a better outcome.  
June 15, 2015 SCIENCE SPOTLIGHT 
 
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In fact, while in asymptomatic mice the two genes were up-regulated at early time points and then 
decreased, in symptomatic mice they remained elevated at later time points as a possible 
consequence of the lack of viral control. A more complex situation was observed in the adaptive 
immune responses. While WNV-specific IgM and T cells increased at early time points to then 
decrease after the infection was cleared from the periphery in asymptomatic mice, the symptomatic 
strains showed diverse responses. 
Finally, the researchers addressed the genetic variability of the CC mice by evaluating the 
relationship between two different allele classes and disease outcome for the gene Oas1b, encoding 
a protein with known antiviral function. The researchers showed that strains homozygous for the 
classical inbred allele had an enhanced propensity for a symptomatic disease outcome, while the 
wild-type allele was correlated with a symptomatic disease outcome. 
"The CC model allows us to link rare outcomes with host genotype to identify genes of susceptibility 
and protection from WNV infection and disease," explains Dr. Graham. "This can assist us in 
identifying unique correlates of protection from neuroinvasive viral infections to inform therapeutic 
and vaccine strategies for controlling WNV infection in humans." In sum, the new CC mouse model 
better recapitulate the variety of WNV infection outcomes observed in humans: do we have a new, 
better model for infectious disease research? 
 
Graham JB, Thomas S, Swarts J, McMillan AA, Ferris MT, Suthar MS, Treuting PM, Ireton R, Gale 
MJ, Lund JM. 2015. Genetic diversity in the collaborative cross model recapitulates human west nile 
virus disease outcomes. MBio 6(3):e00493-15. 
 
 
 
 
 
 
 
June 15, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 6 | Fred Hutchinson Cancer Research Center 
 
 
 
Image provided by Ms. Jessica Swarts 
Schematics representing the generation of the genetic diversity in the CC 
mouse model. 
 


A New Infection Model Incorporates Genetic Diversity

Zentner, Gabriel E.

July 20, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
Chromatin Remodelers Check in on DNA 
Replication 
July 20, 2015 
     GE Zentner 
 
 
 
The Isw2 and Ino80 chromatin 
remodeling complexes associate with 
origins to promote chromatin 
accessibility, allowing deactivation of 
the S-phase checkpoint.  
 
Image provided by Laura Lee 
 
 
 
 
To ensure faithful transmission of genetic information, all dividing cells must replicate their DNA 
during S-phase of the cell cycle.  However, replicating cells are particularly susceptible to DNA 
damage and nucleotide depletion, leading to replication stress and, ultimately, stalling of replication 
forks.  Replication stress triggers a mechanism known as the S-phase checkpoint, which allows cells 
to properly complete DNA replication before proceeding with the remainder of the cell cycle.  The 
mechanisms by which stalled replication forks activate the S-phase checkpoint in budding yeast 
have been studied extensively, but how the checkpoint is deactivated to allow cells to resume cell 
cycle progression is much less well understood.  Previous work from the laboratory of Dr. Toshio 
Tsukiyama (Basic Sciences Division) suggested that Isw2 and Ino80 chromatin remodeling 
complexes had roles in attenuating the S-phase checkpoint (Au et al, 2011).  To follow up this 
July 20, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
observation, Tsukiyama lab graduate student Laura Lee and postdoctoral fellow Dr. Jairo Rodriguez 
performed a systematic analysis of the effect of the Isw2 and Ino80 complexes on S-phase 
checkpoint activity. 
The authors began their analysis by assessing the levels of Rad53, a protein kinase involved in 
activation of the S-phase checkpoint, in wild-type (WT) cells and cells lacking Isw2, the catalytic 
subunit of the Isw2 complex, and Nhp10, a component of the Ino80 complex, in the presence of the 
DNA damaging agents hydroxyurea (HU), which results in nucleotide depletion, and 
methylmethanesulfonate (MMS), which causes DNA damage by base alkylation.  They found that 
Rad53 was much more stable in the isw2 nhp10 mutant, and that autophosphorylation of Rad53 was 
moderately increased by MMS treatment.  The isw2 nhp10 cells also displayed a delay in S-phase 
progression that was not due to incomplete DNA replication.  These results suggest that Isw2 and 
Ino80 function to prevent overactivation of the S-phase checkpoint in the presence of MMS. 
Using genetic crosses, the authors next tested potential pathways through which Isw2 and Ino80 
might attenuate the S-phase checkpoint.  They crossed the isw2 nhp10 strain to strains with 
mutations in factors involved in replication fork protection, DNA replication, and DNA damage 
response pathways, reasoning that if Isw2 and Ino80 were functioning in any of these pathways, a 
single mutant and triple mutant would show similar MMS sensitivity, as the pathway would already 
be compromised in the single mutant.  However, they found that the isw2 nhp10 deletion enhanced 
MMS sensitivity of all strains tested, indicating that Isw2 and Ino80 function outside of these 
pathways. 
To identify factors required for Isw1 and Ino80 checkpoint function, the authors used Rad53 
autophosphorylation as a read out, reasoning as before that if a particular protein was required for 
Isw2 and Ino80 checkpoint attenuation, there would be no difference in Rad53 autophosphorylation 
between a single mutant and triple mutant.  Strikingly, increased Rad53 autophosphorylation was 
observed in all experiments, indicating that Isw2 and Ino80 function either through unknown 
checkpoint proteins or through replication protein A (RPA), which could not be tested due to the 
severe growth phenotype of the rpa isw2 nhp10 strain. 
Based on the data thus far, the authors proposed two mechanisms by which Isw2 and Ino80 could 
downregulate checkpoint activity: 1) by facilitating removal of a checkpoint protein from chromatin 
and 2) by downregulating checkpoint protein activity.  To address the first possibility, the authors 
analyzed the levels of chromatin-bound checkpoint factors in WT and isw2 nhp10 cells, finding that 
bulk levels of these factors on chromatin was essentially unchanged. 
July 20, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 7 | Fred Hutchinson Cancer Research Center 
 
As it was previously found that Isw2 and Ino80 are enriched at stalled replication forks, the authors 
wondered if Isw2 and Ino80 might affect chromatin structure at replication forks to influence 
checkpoint activity.  Using an assay called normalized chromatin accessibility to MNase (NCAM), the 
authors tested chromatin accessibility across the genome in WT and isw2 nhp10 cells.  This 
revealed decreased chromatin accessibility at replicating regions in the double mutant. 
"In our studies we found that chromatin remodeling factors are important for regulating both the 
checkpoint and chromatin accessibility during DNA replication," said Ms. Lee.  "These results 
highlight the significance of chromatin remodeling factors in establishing proper DNA replication, 
which is essential for genomic integrity."  Together, these data suggest a model in which Isw2 and 
Ino80 promote chromatin accessibility at replication forks to facilitate progression of 
replication.  Further studies will undoubtedly reveal the mechanistic underpinnings of this intriguing 
new function for these chromatin remodelers. 
Lee L, Rodriguez J, Tsukiyama T. 2015. Chromatin remodeling factors Isw2 and Ino80 regulate 
checkpoint activity and chromatin structure in S phase. Genetics 199(4):1077-1091. 
See also: Au TJ, Rodriguez J, Vincent JA, Tsukiyama T. 2011. ATP-dependent chromatin 
remodeling factors tune S phase checkpoint activity. Mol Cell Biol 31(22):4454-4463. 
 


Chromatin Remodelers Check in on DNA Replication

August 17, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
A more meaningful, less stressful, distress 
measure 
Aug. 17, 2015  
     SHL Frost 
 
 
Receiver operator characteristic (ROC) 
analysis illustrates that a CTXD score 
cut point of 1.1 results in a sensitivity of 
0.91 and a specificity of 0.58, using the 
Center for Epidemiological Studies 
Depression (CESD) standard cut point 
of 16. This indicates that the CTXD 
captures nearly all the cases of 
depression on the CESD (sensitivity) 
while also identifying elevated distress 
that is distinct from depression 
(specificity). 
 
Image provided by Dr. Karen Syrjala 
 
 
A cancer diagnosis is never an easy challenge, and the distress that follows is often fluctuating but 
enduring. It adversely affects the well-being of cancer patients from the initial finding, through 
treatment and follow-up to survivorship or end of life. This cancer-related stress impacts not only 
overall quality of life, but also satisfaction with medical care and adherence to treatment 
recommendations. Some studies have even demonstrated decreased survival rates with increased 
stress levels. The evidence for distress monitoring is compelling, but current standardized measures 
of anxiety and clinical depression account poorly for the wide-ranging complexity of cancer-related 
emotions. 
Because of the high risk of long-term complications and death after hematopoietic cell 
transplantation (HCT), efforts have been made to evaluate the associated distress using a number of 
existing measures. However, one single, non-duplicative, non-overlapping measure that captures all 
important aspects for understanding cancer-related distress within one test would more likely provide 
a useful tool for routine screening. With this in mind, Drs. Karen Syrjala, Jean Yi, and Shelby Langer 
August 17, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
from the Clinical Research Division set out to define a reliable and valid distress measure for HCT-
treated cancer patients. Importantly, the measure needed to be minimally burdensome to patients. 
"To be useful in research or clinical care, it is important to be brief but relevant to people's 
experiences," Dr. Syrjala explained. The development and psychometric properties of the resulting 
Cancer and Treatment (CTXD) measure was recently published in Psycho-Oncology. 
Potentially stressful aspects were revealed through qualitative interviews with 48 transplant 
recipients regarding feelings about the disease and related treatment, starting at the point before 
HCT when most transplant recipients experience peak distress. The first CTXD version comprised 
46 items that were iteratively selected based on feedback from patients and additional input from 
physicians, nurses, and other healthcare providers. A cohort of 176 patients from 8 transplant 
centers then completed the baseline CTXD, along with other measures, and after statistical analysis 
the initial 46 items were reduced to 22. In this optimized version, 6 factors were distinguished that 
explained 69% of the variance in distress between patients: uncertainty, health burden, identity, 
medical demands, finances, and family strain. The tradeoff between sensitivity (true positive) and 
specificity (true negative) for identifying elevated distress was described through receiver operating 
characteristic (ROC) analysis. A large majority of cases of elevated depression, defined by the 
Center for Epidemiologic Studies Depression (CESD) scale, were captured by a CTXD score > 1.1 
(sensitivity rate 91%). High-distress cases lacking parallel elevated depression were also identified 
(specificity 58%), indicating that the measured qualities of distress extended beyond depression. 
The investigators hope that the new measure will improve the screening of distress among cancer 
patients for both research and clinical care, thereby expediting decisions about psychosocial 
interventions. "The measure can replace the screening of depression and anxiety separately, since it 
evaluates those aspects of clinical need, but also help to identify those patients needing 
psychosocial care for issues that might not be about clinical depression or general anxiety. Fear of 
recurrence, living with uncertainty about the future, and worry about finances and family are ever-
present concerns for lots of people after a cancer diagnosis," Dr. Syrjala explained. Ideally, clinicians 
could use the information to personalize patient referral needs. "The bottom line goal is to use the 
routine assessment of distress to improve the care of our patients so they not only get a referral for 
psychosocial care when needed, but a referral to the right resources at the right time for them," she 
concluded. 
The CTXD is not specific for HCT and may therefore also be useful in other settings. After having 
studied the measure over time after HCT, the team has proceeded with CTXD data collection from 
other cancer patients as well. The correlations found so far among different patient groups have 
August 17, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
been quite surprising, according to Dr. Syrjala. "None of the items we needed were specific to the 
HCT experience, but were shared by patients with other cancers - like thoughts about dying or how 
the family would get through treatment and recovery." 
Still, much remains to be investigated. Can the CTXD help predict who is at greater risk of poor 
outcomes during or after treatment? Will treatment improve the distress indicated by the CTXD? The 
researchers have many hypotheses to test before the full potential of the new distress measure is 
revealed. Hopefully, they are not too stressed out about it. 
Syrjala KL, Yi JC, Langer SL. 2015. Psychometric properties of the Cancer and Treatment Distress 
(CTXD) measure in hematopoietic cell transplantation patients. Psychooncology. doi: 
10.1002/pon.3861.  [Epub ahead of print] 
This study was supported by funding from the National Cancer Institute. 
 


A more meaningful, less stressful, distress measure

August 17, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
The around-the-world journey of flu 
Aug. 17, 2015  
 
     L Pattacini 
 
 
Graphic illustrating estimated 
path and rate of virus migration 
for four strains of influenza: 
H3N2, H1N1, Vic and Yam. 
 
Image provided by Dr. Trevor 
Bedford. 
 
 
The flu season can seem so far away during hot and sunny summer days, and most of us do not 
think about next year’s flu. For scientists in charge of producing influenza vaccines, however, there 
is no time to rest. In fact, summer in the northern hemisphere means flu season in the southern. 
Furthermore, every year the World Health Organization recommends which strains should be 
included in next season’s vaccine. As several different viruses circulate at the same time, the 
seasonal vaccine includes two influenza A viruses, H3N2 and H1N1 (where H indicates the subtype 
of haemagglutinin and N the subtype of neuraminidase, two proteins expressed on the viral surface) 
and two influenza B viruses. While the global circulation pattern of the H3N2 virus has been partially 
characterized previously, the migration patterns of the other viruses are not fully known.  
All this complexity, together with the need of timely production of effective vaccines, calls for year-
long action. "Influenza circulation patterns suggest that H3N2 viruses, in particular, emerge from 
India, China and Southeast Asia. Focusing surveillance on these areas will help to catch new 
antigenic variants early. Also, when confronted with a choice in circulating lineages, our work 
suggests that, other factors being equal, an E-SE Asian lineage will more likely spread to the rest of 
the world. Both these perspectives can inform vaccine strain selection", explained Dr. Trevor 
Bedford from the Vaccine and Infectious Disease Division at Fred Hutch. To understand the 'travel 
itinerary' of influenza, Dr. Bedford and his collaborators compared the global circulation patterns of 
haemagglutinin genes for two influenza A viruses (H2N3 and H1N1) and two influenza B viruses, 
August 17, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 8 | Fred Hutchinson Cancer Research Center 
 
(B/Victoria/2/1987-like [Vic] and B/Yamagata/16/1988-like [Yam]). Findings from these efforts were 
reported in a paper published in the July issue of Nature. 
Similarly to H3N2, viruses H1N1, Vic, and Yam originate in China, Southeast Asia and India. Their 
global circulation patterns, however, differ substantially from H3N2 viruses. In the case of H1N1, 
three co-circulating genetic lineages started segregating in 2004-2005 from one common strain, and 
circulated in Southeast Asia, China and India, until this last lineage eventually dominated and spread 
worldwide. Vic and Yam also revealed a different pattern, with lineages frequently seeding outside 
East and Southeast Asia. Furthermore, H3N2 viruses move between regions more frequently than 
the other analyzed viruses.  
Other differences between these strains can lead to differential immune response.  Slower antigenic 
evolution of H1N1 and the B viruses allows a greater fraction of adults to develop an immune 
response against them, while children are more sensitive and their infection rate is higher. 
Conversely, the fast paced H3N2 influenza is more likely to infect adults. The reason H3N2 is 
hypothesized to maintain its fast pace of transmission is that adults are more likely to travel around 
the world by airplane and to carry around with them the flu that they contracted in another country. 
Has that ever happened to you? I did bring back to Seattle a flu that I got in Italy when I was there 
for my Christmas vacations. From then on, I have never forgotten again to vaccinate. Will other 
adults make the same choice? Will influenza patterns change again as more adults decide to 
vaccinate?  
To know, we will have to wait for Dr. Bedford to solve this enigma! 
Bedford T, Riley S, Barr IG, Broor S, Chadha M, Cox NJ, Daniels RS, Gunasekaran CP, Hurt AC, 
Kelso A, Klimov A, Lewis NS, Li X, McCauley JW, Odagiri T, Potdar V, Rambaut A, Shu Y, Skepner 
E, Smith DJ, Suchard MA, Tashiro M, Wang D, Xu X, Lemey P, Russell CA. 2015. Global circulation 
patterns of seasonal influenza viruses vary with antigenic drift. Nature. 523: 217-220. 
The research was supported by grants from the NIH and other US and international funding 
agencies. 
Supplementary information: Video. 
 


The around-the-world journey of flu

September 21, 2015 SCIENCE SPOTLIGHT 
 
1 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
Rpd3 helps put yeast cells to bed 
September 21, 2015 
     GE Zentner 
 
 
 
 
In the presence of abundant nutrients, 
Rpd3 binds mainly to gene bodies, 
allowing promoter histones to be 
highly acetylated. Upon nutrient 
consumption, quiescence-specific TFs 
recruit Rpd3 to promoters to 
deacetylate histones and globally 
repress transcription. 
 
Image provided by Dr. Jeffrey 
McKnight. 
 
 
 
 
Quiescence is a state of reduced cellular activity, characterized by cell cycle arrest, increased 
longevity and stress resistance, as well as decreased transcription, protein synthesis, and metabolic 
activity. However, the molecular details of the establishment of quiescence, particularly how 
transcription is globally repressed, are still unclear. To better understand this transcriptional shutoff, 
postdoctoral fellow Dr. Jeffrey McKnight in the laboratory of Dr. Toshio Tsukiyama and colleagues in 
the laboratory of Dr. Linda Breeden (Basic Sciences Division) analyzed chromatin structure during 
the transition from growth to quiescence in budding yeast. "We were fascinated by the programmed 
ability of budding yeast to survive for so many weeks in water without any nutritional supplements. 
This was a beautiful system for trying to identify conserved regulators and mechanisms of stress 
tolerance and longevity during quiescence," said Dr. McKnight. "We found Rpd3, a conserved 
histone deacetylase with minimal known contributions in nutrient-rich conditions, was essential for 
quiescence entry and survival." 
The authors first analyzed gene expression in cells growing in rich medium (log), cells growing until 
glucose was exhausted from medium (diauxic shift, DS), and quiescent (Q) cells. Q cells showed a 
September 21, 2015 SCIENCE SPOTLIGHT 
 
2 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
30-fold decrease in overall mRNA levels compared to log cells and a 15-fold decrease in mRNA 
versus DS cells. Further analysis revealed decreased binding of RNA polymerase II and the 
transcription pre-initiation complex to the genome, suggesting that the global transcriptional shutoff 
occurs at the level of transcriptional initiation. 
Hypothesizing that chromatin might play a role in the Q transcriptional shutoff, the authors compared 
chromatin structure between log and Q cells. Q cells displayed increased nucleosome occupancy at 
promoters, indicating the establishment of a repressive chromatin architecture. Q cells also 
displayed a global decrease in histone acetylation, which is generally associated with transcriptional 
activation. These chromatin alterations were strongly correlated with transcriptional shutoff, 
suggesting that chromatin is indeed the means by which Q cells repress transcription.  
Many of the observed chromatin shifts were associated with binding sites for transcription factors 
(TFs) proposed to function through the lysine deacetylase Rpd3, and so the authors next 
investigated the potential function of this protein in Q cells. Cultures of cells lacking Rpd3 displayed 
a lower fraction of Q cells following nutrient exhaustion, likely due to reduced viability, and also had a 
shortened lifespan. Rpd3 is present in two distinct complexes, Rpd3S and Rpd3L, and genetic 
analysis suggested that the promoter-associated Rpd3L complex, but not Rpd3S, is involved for the 
establishment of the Q state. Indeed, cells lacking Rpd3 were impaired in the establishment of the 
repressive chromatin architecture in Q cells. 
The authors next profiled the genome-wide binding of Rpd3 in log and Q cells. In log cells, relatively 
few Rpd3 binding sites were identified, and most Rpd3 signal was observed in in gene bodies. 
However, in Q cells, a large-scale redistribution of Rpd3 to promoters occurred. Furthermore, 
several hundred of these peaks were dependent on the quiescence-specific Stb3 and Xbp1 TFs. 
These results convincingly demonstrate that establishment of repressive chromatin by Rpd3 is 
critical for entry into quiescence by yeast cells. Of note, understanding the role of Rpd3 in 
establishing quiescence may have implications for the treatment of disease. "These findings are 
particularly interesting, since targeted inhibition of the human Rpd3 orthologs is effective in treating 
drug-resistant leukemias and latent HIV infections. Our mechanistic investigation of transcriptional 
quiescence in yeast may therefore shed light on the mechanisms of drug resistance of quiescent 
cancers," said Dr. McKnight. 
McKnight JN, Boerma JW, Breeden LL, Tsukiyama T. 2015. Global Promoter Targeting of a 
Conserved Lysine Deacetylase for Transcriptional Shutoff during Quiescence Entri. Mol Cell 
59(5):732-743. 
September 21, 2015 SCIENCE SPOTLIGHT 
 
3 Volume 5, Issue 9 | Fred Hutchinson Cancer Research Center 
 
Funding source: Research was supported by Leukemia and Lymphoma Society Career 
Development Program grant CDP-5078-14 for J.N.M., R01GM111428 for T.T., NCI P30-CA015704-
38 for L.L.B. and T.T., and R21-AG048595 for L.L.B 
 
 


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