Many encoded gene products responsible for neurodevelopmental disorders (NDs) like autism spectrum disorders (ASD), schizophrenia (SCZ), intellectual disability (ID), and idiopathic generalized epilepsy (IGE) converge on networks controlling synaptic function. An increase in KCC2 (SLC12A5) Cl− transporter activity drives the developmental GABA excitatory-inhibitory sequence, but the role of KCC2 in human NDs is essentially unknown. Here, we report two rare, non-synonymous (NS), functionally-impairing variants in the KCC2 C-terminal regulatory domain (CTRD) in human ASD (R952H and R1049C) and SCZ (R952H) previously linked with IGE and familial febrile seizures, and another novel NS KCC2 variant in ASD (R1048W) with highly-predicted pathogenicity. Exome data from 2517 simplex families in the ASD Simon Simplex Collection (SSC) revealed significantly more KCC2 CTRD variants in ASD cases than controls, and interestingly, these were more often synonymous and predicted to disrupt or introduce a CpG site. Furthermore, full gene analysis showed ASD cases are more likely to contain rare KCC2 variants affecting CpG sites than controls. These data suggest genetically-encoded dysregulation of KCC2-dependent GABA signaling may contribute to multiple human NDs.

Inherited genetic risk factors contribute toward breast cancer (BC) onset. BC risk variants can be divided into three categories of penetrance (high, moderate, and low) that reflect the probability of developing the disease. Traditional BC susceptibility gene discovery approaches that searched for high- and moderate-risk variants in familial BC cases have had limited success; to date, these risk variants explain only ∼30% of familial BC cases. Next-generation sequencing technologies can be used to search for novel high and moderate BC risk variants, and this manuscript reviews 12 familial BC whole-exome sequencing efforts. Study design, filtering strategies, and segregation and validation analyses are discussed. Overall, only a modest number of novel BC risk genes were identified, and 90% and 97% of the exome-sequenced families and cases, respectively, had no BC risk variants reported. It is important to learn from these studies and consider alternate strategies in order to make further advances. The discovery of new BC susceptibility genes is critical for improved risk assessment and to provide insight toward disease mechanisms for the development of more effective therapies.

IntroductionToll-like receptor 9 (TLR9) is an innate immune system DNA-receptor that regulates tumor invasion and immunity in vitro. Low tumor TLR9 expression has been associated with poor survival in Caucasian patients with triple negative breast cancer (TNBC). African American (AA) patients with TNBC have worse prognosis than Caucasians but whether this is due to differences in tumor biology remains controversial. We studied the prognostic significance of tumor Toll like receptor-9 (TLR9) protein expression among African American (AA) triple negative breast cancer (TNBC) patients. Germline TLR9 variants in European Americans (EAs) and AAs were investigated, to determine their contribution to AA breast cancer risk.MethodsTLR9 expression was studied with immunohistochemistry in archival tumors. Exome Variant Server and The Cancer Genome Atlas were used to determine the genetic variation in the general EA and AA populations, and AA breast cancer cases. Minor allele frequencies (MAFs) were compared between EAs (n = 4300), AAs (n = 2203), and/or AA breast cancer cases (n = 131).ResultsThirty-two TLR9 variants had a statistically significant MAF difference between general EAs and AAs. Twenty-one of them affect a CpG site. Rs352140, a variant previously associated with protection from breast cancer, is more common in EAs than AAs (p = 2.20E-16). EAs had more synonymous alleles, while AAs had more rare coding alleles. Similar analyses comparing AA breast cancer cases with AA controls did not reveal any variant class differences; however, three previously unreported TLR9 variants were associated with late onset breast cancer. Although not statistically significant, rs352140 was observed less frequently in AA cases compared to controls. Tumor TLR9 protein expression was not associated with prognosis.ConclusionsTumor TLR9 expression is not associated with prognosis in AA TNBC. Significant differences were detected in TLR9 variant MAFs between EAs and AAs. They may affect TLR9 expression and function. Rs352140, which may protect from breast cancer, is 1.6 X more common among EAs. These findings call for a detailed analysis of the contribution of TLR9 to breast cancer pathophysiology and health disparities.

Although an average woman in the United States can have a 12.5% lifetime risk of developing breast cancer (BC), inherited genetic risk variants can greatly influence a woman’s overall lifetime risk. Genes that harbor BC risk variants are referred to as BC susceptibility genes. Unfortunately, mutations in known BC susceptibility genes only explain approximately 35% of hereditary BC cases, leaving a large portion, approximately 65%, genetically unexplained. With the introduction of next-generation sequencing, several attempts have been carried out to identify additional hereditary BC susceptibility genes using whole-exome sequencing. The majority of these studies were relatively unsuccessful; however, Cybulski et al. (2015) successfully associated two rare truncation variants in RECQL, p.K555delinsMYKLIHYSFR and p.R215*, in a Polish and French-Canadian cohort, respectively. Subsequently, two additional studies were carried out in northern and southern regions of China, which also confirmed that overtly deleterious coding mutations in RECQL are associated with familial BC. Herein, we investigated RECQL variants in BC-affected African Americans (AAs) and European Americans (EAs). To our knowledge, this study represents the first attempt to identify an association between RECQL variants and BC cases in the United States. We initially screened all RECQL coding exons using polymerase chain reaction and Sanger sequencing in 49 BC cases (19 AAs and 30 EAs) from Alabama. Primarily synonymous variants were detected in both ethnicities; thus, in order to further investigate the role of RECQL synonymous variants in BC risk, we analyzed RECQL in blood-derived exomes of 168 and 580 BC-affected AAs and EAs, respectively, from The Cancer Genome Atlas (TCGA) project using an in-house bioinformatics pipeline. A case-control analysis revealed that all rare synonymous variants detected in EA BC cases were associated with BC, and p.S64= was significantly associated in both ethnicities. Interestingly, only one truncation variant was detected (p.Y492*) in all 748 BC cases analyzed. Unlike previous findings, this preliminary analysis suggests that rare RECQL synonymous variants may also increase an individual’s lifetime risk of developing BC; further investigation is warranted. Citation Format: Madison R. Chandler, Sydney Bergstresser, Anna LW Huskey, Elizabeth Stallworth, Amber Davis, Holly Dean, Brandon Johnson, Nancy D. Merner. Investigation of RECQL variants in European and African American breast cancer cohorts [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A29.

Over 60% of the Alabama population is medically underserved, including the entire population of many Alabama counties. These counties are generally rural and have transportation barriers, thus a lack of access to healthcare results in poor cancer outcomes. These healthcare gaps, in addition to lower education levels and mistrust towards researcher/healthcare providers, highlight the challenges but also the need to make a difference in this underserved and understudied population. In general, recruitment for human genetic research studies presents major challenges, and, despite federal mandates to include women and minorities in federally funded research, African Americans (AAs) remain underrepresented in cancer genetic research. Recruitment of participants requires extensive efforts and strategic planning in order to overcome the difficulties, such as distrust towards medical research and logistical challenges of recruiting under-served and under-represented individuals. These considerations led to the establishment of two IRB-approved protocols, hospital recruitment (#14-232) and community-based recruitment (CBR; #15-111) to recruit cancer individuals/families from Alabama and identify genetic risk factors. Hospital recruitment was established through a partnership with East Alabama Medical Center (EAMC), and CBR targets medically underserved individuals through a mechanism aimed to educate and build trust in the community. Through connections fostered by community-partners, CBR team members travel to rural communities all over Alabama to be introduced to, educate, and recruit patients/survivors and their family members. All CBR team members are phlebotomy trained. Recruitment particularly focuses on cancer associated with Hereditary Breast and Ovarian Cancer (HBOC) Syndrome since there are two disparities within the AA population that may be genetically linked. Firstly, AA women are generally diagnosed with a more aggressive and less treatable breast cancer (BC) sub-type and have a higher incidence rate of BC under the age of 40 compared to Caucasians. Secondly, AA males are generally diagnosed with prostate cancer (PC) at a younger age and with larger tumors compared to Caucasians. Considering that (1) an early age of onset is a hallmark of hereditary cancer, (2) hereditary BC is associated with an increased risk of ovarian cancer (OvC) and PC, and (3) the Black Women's Health Study has demonstrated there is a strong familial component of AA BC, inherited and potentially overlapping genetic risk factors may explain these disparities. Thus, the study enrollment criteria include individuals (1) diagnosed with BC, OvC, or PC under the age of 45 and/or (2) diagnosed with BC, OvC, or PC with have a cancer family history. Ultimately, in an effort to recruit informative families for genetic study, both cancer-affected and unaffected family members of study participants can join the study. To date, 133 DNA samples from 74 cancer families are stored in the Merner DNA Bank; 33 and 41 probands have been recruited through EAMC and CBR, respectively. CBR has been the most effective mechanism of AA BC recruitment (20/36) compared to hospital recruitment (5/32). Probands are screened for known and candidate susceptibility genes using the BRAP (BReast And Prostate) gene-screening panel. This aims to provide insight towards the true contribution of mutations in known susceptibility genes, and identify non-mutation individuals/families for novel HBOC susceptibility gene discovery efforts. Additionally, this study offers each participant the option to receive her/his confidential research-based gene screening results and aims to translate those results to the clinic by providing free UAB telegenetic counseling to underserved individuals through the use of telemedicine carts at equipped Alabama County Health Departments. Citation Format: Nancy Merner, Madison Chandler, Amber Davis, Ebony Jackson, Erin Bilgili, Cameron Turner, Kasey Shepp, Stephanie Spina, Brandon Johnson. Cancer genetics research in Alabama: Recruitment mechanisms to reach the underserved. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr A47.

African American (AA) women are diagnosed with breast cancer (BC) at a higher rate under the age of 40 compared to Caucasian women and are more often diagnosed with triple-negative (TN) BC, a less treatable and more aggressive BC sub-type. Since an early age of onset is a key characteristic of hereditary BC, genetic risk factors are likely contributing towards these BC disparities. Genes that harbor genetic risk variants are referred to as BC susceptibility genes. Prior to searching for novel BC susceptibility genes, previously identified BC susceptibility genes should initially be excluded. Since the introduction of next-generation sequencing, gene-screening panels have been implemented; this innovative approach involves designing probes for target-capture of genes of interest, amplification, and sequencing on a massively parallel array. AA BC genetics is understudied; however, recently, Churpek et al. published the first article to screen AA hereditary/early onset/TN BC cases for a panel of known BC susceptibility genes. This study identified 68 pathogenic germline mutations in 65 of 289 (22%) AA cases; 18% had a BRCA1 or BRCA2 mutation. The authors acknowledged the paucity of their study and that more gene panel screening studies are needed. Currently, it appears that the majority and spectrum of BC susceptibility genes/risk variants that contribute towards the disproportionate number of aggressive and young onset AA BCs remain to be identified. Our group has custom-designed an Agilent Technologies (AT) HaloPlex probe kit, named BRAP (BReast And Prostate) that targets 87 known and candidate BC and prostate cancer (PC) susceptibility genes using the AT SureDesign program. PC genes were included on the panel since AA males are frequently diagnosed with PC in BC families, more susceptible to PC compared to Caucasians, and normally diagnosed at a younger age with larger tumors. Furthermore, men carrying BRCA1/2 mutations have a higher risk of PC, so further genetic overlap will likely be detected through BRAP screening. Ultimately, the probes were designed to generate 100 base pair (bp) reads on an Illumina platform. All coding exons as well as the 5' and 3' untranslated regions of the 87 genes were targeted, totaling 499.5 kbp. The designed probes covered 98.9% of the targeted region. Forty-six seemingly unrelated individuals (24 AA and 22 Caucasians) from Alabama who were diagnosed with BC under the age of 45 and/or had a BC family history were selected for the initial BRAP capture; the HaloPlex HS Target Enrichment System For Illumina Sequencing Protocol (Version C0) was followed. Each individually prepared and indexed library was quantified using a Bioanalyzer 2100. The targeted DNA from 44 of the 46 individuals were successfully captured and amplified. The successful samples were pooled for sequencing; the sample was loaded onto one flow cell lane and run on an Illumina HiSeqTM 2500 at the HudsonAlpha Genomic Services Laboratory. The sequencing data was downloaded and is being processed using an in-house, bioinformatics pipeline. Two of the screened individuals served as positive controls since upon recruitment one reported a mutation in BRCA1 and the other a mutation in BRCA2. The pipeline has been optimized using the control with BRCA1 p.M1775R, which has been successfully identified. Variant calling files are being processed for the remaining screened individuals. This data provides great insight towards the array of mutations in known BC and PC susceptibility genes that contributes towards hereditary AA BC. Furthermore, non-mutation individuals/families identified through BRAP screening will be whole-genome sequenced in order to identify novel, BC susceptibility genes that may contribute towards disease disparity. Citation Format: Madison R. Chandler, Kasey J. Shepp, Stephanie M. Spina, Nancy D. Merner. A breast cancer susceptibility gene screening panel for variant discovery and gene exclusion in an African American cohort from Alabama. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B35.