Dose-dependent effects of Dnmt3a in an inducible murine model of KrasG12D-driven leukemia.

DNMT3A mutations are frequently found in clonal hematopoiesis and a variety of hematologic malignancies including acute myeloid leukemia (AML). An assortment of mouse models have been engineered to explore the tumorigenic potential and malignant lineage bias due to loss of function of DNMT3A in consort with commonly co-mutated genes in myeloid malignancies such as Flt3, Nras, Kras, and c-Kit. We employed several tamoxifen-inducible Cre-ERT2 murine model systems to study the effects of constitutively active KrasG12D-driven myeloid leukemia (Kras) development together with heterozygous (3aHet) or homozygous Dnmt3a deletion (3aKO). Due to the rapid generation of diverse nonhematologic tumors appearing after tamoxifen induction, we employed a transplantation model. With pretransplant tamoxifen induction, most Kras mice died quickly of T-cell malignancies regardless of Dnmt3a status. Using post-transplant induction, we observed a dose-dependent effect of DNMT3A depletion that skewed the leukemic phenotype toward a myeloid lineage- 64% of 3aKO/Kras mice had exclusively myeloid disease compared with 36% of 3aHet/Kras, and only 13% of Kras mice. 3aKO combined with Kras led to increased disease burden, multi-organ infiltration, and faster disease progression. DOT1L inhibition exerted profound anti-leukemic effects in malignant 3aKO/Kras cells, but not malignant cells with Kras mutation alone, consistent with the known sensitivity of DNMT3A-mutant leukemia to DOT1L inhibition. RNAseq from malignant myeloid cells revealed that biallelic Dnmt3a deletion was associated with loss of cell cycle regulation, MYC activation, and TNF⍺ signaling. Overall, we have developed a robust model system for mechanistic and preclinical investigations of AML with DNMT3A and Ras pathway lesions.

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.

Novel syndromic neurodevelopmental disorder caused by de novo deletion of CHASERR, a long noncoding RNA.

Genes encoding long non-coding RNAs (lncRNAs) comprise a large fraction of the human genome, yet haploinsufficiency of a lncRNA has not been shown to cause a Mendelian disease. CHASERR is a highly conserved human lncRNA adjacent to CHD2-a coding gene in which de novo loss-of-function variants cause developmental and epileptic encephalopathy. Here we report three unrelated individuals each harboring an ultra-rare heterozygous de novo deletion in the CHASERR locus. We report similarities in severe developmental delay, facial dysmorphisms, and cerebral dysmyelination in these individuals, distinguishing them from the phenotypic spectrum of CHD2 haploinsufficiency. We demonstrate reduced CHASERR mRNA expression and corresponding increased CHD2 mRNA and protein in whole blood and patient-derived cell lines-specifically increased expression of the CHD2 allele in cis with the CHASERR deletion, as predicted from a prior mouse model of Chaserr haploinsufficiency. We show for the first time that de novo structural variants facilitated by Alu-mediated non-allelic homologous recombination led to deletion of a non-coding element (the lncRNA CHASERR) to cause a rare syndromic neurodevelopmental disorder. We also demonstrate that CHD2 has bidirectional dosage sensitivity in human disease. This work highlights the need to carefully evaluate other lncRNAs, particularly those upstream of genes associated with Mendelian disorders.

Diagnostic Utility of Genome-wide DNA Methylation Analysis in Genetically Unsolved Developmental and Epileptic Encephalopathies and Refinement of a CHD2 Episignature.

Sequence-based genetic testing currently identifies causative genetic variants in ∼50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. Rare epigenetic variations ("epivariants") can drive disease by modulating gene expression at single loci, whereas genome-wide DNA methylation changes can result in distinct "episignature" biomarkers for monogenic disorders in a growing number of rare diseases. Here, we interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 516 individuals with genetically unsolved DEEs who had previously undergone extensive genetic testing. We identified rare differentially methylated regions (DMRs) and explanatory episignatures to discover causative and candidate genetic etiologies in 10 individuals. We then used long-read sequencing to identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and two copy number variants. We also identify pathogenic sequence variants associated with episignatures; some had been missed by previous exome sequencing. Although most DEE genes lack known episignatures, the increase in diagnostic yield for DNA methylation analysis in DEEs is comparable to the added yield of genome sequencing. Finally, we refine an episignature for CHD2 using an 850K methylation array which was further refined at higher CpG resolution using bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate genetic causes as ∼2% (10/516) for unsolved DEE cases.

TMEM161B regulates cerebral cortical gyration, Sonic Hedgehog signaling, and ciliary structure in the developing central nervous system.

Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.

RNA Sequencing as a Diagnostic Tool.

This JAMA Insights discusses RNA sequencing, which allows detection of qualitative and quantitative changes in RNA expression across the genome in clinical samples, and how it may improve molecular diagnostic rates achieved by diagnostic exome sequencing and whole-genome sequencing alone.

miRNA-34c Suppresses Osteosarcoma Progression In Vivo by Targeting Notch and E2F.

The expression of microRNAs (miRNAs) is dysregulated in many types of cancers including osteosarcoma (OS) due to genetic and epigenetic alterations. Among these, miR-34c, an effector of tumor suppressor P53 and an upstream negative regulator of Notch signaling in osteoblast differentiation, is dysregulated in OS. Here, we demonstrated a tumor suppressive role of miR-34c in OS progression using in vitro assays and in vivo genetic mouse models. We found that miR-34c inhibits the proliferation and the invasion of metastatic OS cells, which resulted in reduction of the tumor burden and increased overall survival in an orthotopic xenograft model. Moreover, the osteoblast-specific overexpression of miR-34c increased survival in the osteoblast specific p53 mutant OS mouse model. We found that miR-34c regulates the transcription of several genes in Notch signaling (NOTCH1, JAG1, and HEY2) and in p53-mediated cell cycle and apoptosis (CCNE2, E2F5, E2F2, and HDAC1). More interestingly, we found that the metastatic-free survival probability was increased among a patient cohort from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) OS, which has lower expression of direct targets of miR-34c that was identified in our transcriptome analysis, such as E2F5 and NOTCH1. In conclusion, we demonstrate that miR-34c is a tumor suppressive miRNA in OS progression in vivo. In addition, we highlight the therapeutic potential of targeting miR-34c in OS. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A novel, de novo intronic variant in POGZ causes White-Sutton syndrome.

White-Sutton syndrome (WHSUS), which is caused by heterozygous pathogenic variants in POGZ, is characterized by a spectrum of intellectual disabilities and global developmental delay with or without features of autism spectrum disorder. Additional features may include hypotonia, behavioral abnormalities, ophthalmic abnormalities, hearing loss, sleep apnea, microcephaly, dysmorphic facial features, and rarely, congenital diaphragmatic hernia (CDH). We present a 6-year-old female with features of WHSUS, including CDH, but with nondiagnostic clinical trio exome sequencing. Exome sequencing reanalysis revealed a heterozygous, de novo, intronic variant in POGZ (NM_015100.3:c.2546-20T>A). RNA sequencing revealed that this intronic variant leads to skipping of exon 18. This exon skipping event results in a frameshift with a predicted premature stop codon in the last exon and escape from nonsense-mediated mRNA decay (NMD). To our knowledge, this case is the first case of WHSUS caused by a de novo, intronic variant that is not near a canonical splice site within POGZ. These findings emphasize the limitations of standard clinical exome filtering algorithms and the importance of research reanalysis of exome data together with RNA sequencing to confirm a suspected diagnosis of WHSUS. As the sixth reported case of CDH with heterozygous pathogenic variants in POGZ and features consistent with WHSUS, this report supports the conclusion that WHSUS should be considered in the differential diagnosis for patients with syndromic CDH.

Somatic Dnmt3a inactivation leads to slow, canonical DNA methylation loss in murine hematopoietic cells.

Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) are the most common cause of clonal hematopoiesis and are among the most common initiating events of acute myeloid leukemia (AML). Studies in germline and somatic Dnmt3a knockout mice have identified focal, canonical hypomethylation phenotypes in hematopoietic cells; however, the kinetics of methylation loss following acquired DNMT3A inactivation in hematopoietic cells is essentially unknown. Therefore, we evaluated a somatic, inducible model of hematopoietic Dnmt3a loss, and show that inactivation of Dnmt3a in murine hematopoietic cells results in a relatively slow loss of methylation at canonical sites throughout the genome; in contrast, remethylation of Dnmt3a deficient genomes in hematopoietic cells occurs much more quickly. This data suggests that slow methylation loss may contribute, at least in part, to the long latent period that characterizes clonal expansion and leukemia development in individuals with acquired DNMT3A mutations in hematopoietic stem cells.

Transcriptome-directed analysis for Mendelian disease diagnosis overcomes limitations of conventional genomic testing.

BACKGROUNDTranscriptome sequencing (RNA-seq) improves diagnostic rates in individuals with suspected Mendelian conditions to varying degrees, primarily by directing the prioritization of candidate DNA variants identified on exome or genome sequencing (ES/GS). Here we implemented an RNA-seq-guided method to diagnose individuals across a wide range of ages and clinical phenotypes.METHODSOne hundred fifteen undiagnosed adult and pediatric patients with diverse phenotypes and 67 family members (182 total individuals) underwent RNA-seq from whole blood and skin fibroblasts at the Baylor College of Medicine (BCM) Undiagnosed Diseases Network clinical site from 2014 to 2020. We implemented a workflow to detect outliers in gene expression and splicing for cases that remained undiagnosed despite standard genomic and transcriptomic analysis.RESULTSThe transcriptome-directed approach resulted in a diagnostic rate of 12% across the entire cohort, or 17% after excluding cases solved on ES/GS alone. Newly diagnosed conditions included Koolen-de Vries syndrome (KANSL1), Renpenning syndrome (PQBP1), TBCK-associated encephalopathy, NSD2- and CLTC-related intellectual disability, and others, all with negative conventional genomic testing, including ES and chromosomal microarray (CMA). Skin fibroblasts exhibited higher and more consistent expression of clinically relevant genes than whole blood. In solved cases with RNA-seq from both tissues, the causative defect was missed in blood in half the cases but none from fibroblasts.CONCLUSIONSFor our cohort of undiagnosed individuals with suspected Mendelian conditions, transcriptome-directed genomic analysis facilitated diagnoses, primarily through the identification of variants missed on ES and CMA.TRIAL REGISTRATIONNot applicable.FUNDINGNIH Common Fund, BCM Intellectual and Developmental Disabilities Research Center, Eunice Kennedy Shriver National Institute of Child Health & Human Development.

The safety and clinical effects of administering a multiantigen-targeted T cell therapy to patients with multiple myeloma.

Multiple myeloma (MM) is an almost always incurable malignancy of plasma cells. Despite the advent of new therapies, most patients eventually relapse or become treatment-refractory. Consequently, therapies with nonoverlapping mechanisms of action that are nontoxic and provide long-term benefit to patients with MM are greatly needed. To this end, we clinically tested an autologous multitumor-associated antigen (mTAA)-specific T cell product for the treatment of patients with high-risk, relapsed or refractory MM. In this study, we expanded polyclonal T cells from 23 patients with MM. T cells whose native T cell receptors were reactive toward five myeloma-expressed target TAAs (PRAME, SSX2, MAGEA4, Survivin, and NY-ESO-1) were enriched ex vivo. To date, we have administered escalating doses of these nonengineered mTAA-specific T cells (0.5 × 107 to 2 × 107 cells/m2) to 21 patients with MM, 9 of whom were at high risk of relapse after a median of 3 lines of prior therapy and 12 with active, relapsed or refractory disease after a median of 3.5 prior lines. The cells were well tolerated, with only two transient, grade III infusion-related adverse events. Furthermore, patients with active relapsed or refractory myeloma enjoyed a longer than expected progression-free survival and responders included three patients who achieved objective responses concomitant with detection of functional TAA-reactive T cell clonotypes derived from the infused mTAA product.

Remethylation of Dnmt3a-/- hematopoietic cells is associated with partial correction of gene dysregulation and reduced myeloid skewing.

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a-/- bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a-/- hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a-/- mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic "state" created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.

Haploinsufficiency for DNA methyltransferase 3A predisposes hematopoietic cells to myeloid malignancies.

The gene that encodes de novo DNA methyltransferase 3A (DNMT3A) is frequently mutated in acute myeloid leukemia genomes. Point mutations at position R882 have been shown to cause a dominant negative loss of DNMT3A methylation activity, but 15% of DNMT3A mutations are predicted to produce truncated proteins that could either have dominant negative activities or cause loss of function and haploinsufficiency. Here, we demonstrate that 3 of these mutants produce truncated, inactive proteins that do not dimerize with WT DNMT3A, strongly supporting the haploinsufficiency hypothesis. We therefore evaluated hematopoiesis in mice heterozygous for a constitutive null Dnmt3a mutation. With no other manipulations, Dnmt3a+/- mice developed myeloid skewing over time, and their hematopoietic stem/progenitor cells exhibited a long-term competitive transplantation advantage. Dnmt3a+/- mice also spontaneously developed transplantable myeloid malignancies after a long latent period, and 3 of 12 tumors tested had cooperating mutations in the Ras/MAPK pathway. The residual Dnmt3a allele was neither mutated nor downregulated in these tumors. The bone marrow cells of Dnmt3a+/- mice had a subtle but statistically significant DNA hypomethylation phenotype that was not associated with gene dysregulation. These data demonstrate that haploinsufficiency for Dnmt3a alters hematopoiesis and predisposes mice (and probably humans) to myeloid malignancies by a mechanism that is not yet clear.

CpG Island Hypermethylation Mediated by DNMT3A Is a Consequence of AML Progression.

DNMT3A mutations occur in ∼25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3AR882H, has dominant negative activity that reduces DNA methylation activity by ∼80% in vitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patients with or without DNMT3AR882 mutations. Non-leukemic hematopoietic cells with DNMT3AR882H displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing and was essentially absent in AMLs with DNMT3AR882 mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Our findings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3AR882, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression.

PML-RARA requires DNA methyltransferase 3A to initiate acute promyelocytic leukemia.

The DNA methyltransferases DNMT3A and DNMT3B are primarily responsible for de novo methylation of specific cytosine residues in CpG dinucleotides during mammalian development. While loss-of-function mutations in DNMT3A are highly recurrent in acute myeloid leukemia (AML), DNMT3A mutations are almost never found in AML patients with translocations that create oncogenic fusion genes such as PML-RARA, RUNX1-RUNX1T1, and MLL-AF9. Here, we explored how DNMT3A is involved in the function of these fusion genes. We used retroviral vectors to express PML-RARA, RUNX1-RUNX1T1, or MLL-AF9 in bone marrow cells derived from WT or DNMT3A-deficient mice. Additionally, we examined the phenotypes of hematopoietic cells from Ctsg-PML-RARA mice, which express PML-RARA in early hematopoietic progenitors and myeloid precursors, with or without DNMT3A. We determined that the methyltransferase activity of DNMT3A, but not DNMT3B, is required for aberrant PML-RARA-driven self-renewal ex vivo and that DNMT3A is dispensable for RUNX1-RUNX1T1- and MLL-AF9-driven self-renewal. Furthermore, both the PML-RARA-driven competitive transplantation advantage and development of acute promyelocytic leukemia (APL) required DNMT3A. Together, these findings suggest that PML-RARA requires DNMT3A to initiate APL in mice.

Association of gene variants with lipid levels in response to fenofibrate is influenced by metabolic syndrome status.

OBJECTIVE: Fenofibrate therapy reduces serum triglycerides (TG) and increases high-density lipoprotein-cholesterol (HDL-C) and thus addresses the atherogenic dyslipidemia associated with metabolic syndrome (MetS). Our hypothesis is that genetic factors contribute to the variability of lipid response to fenofibrate differently in subjects with MetS and without MetS. METHODS: We investigated the association in 25 candidate genes with lipid responses to a 3-weeks trial on fenofibrate in subjects with and without MetS. We employed growth curve mixed models to generate the response phenotypes to fenofibrate in TG, HDL-C, and low-density lipoprotein-cholesterol (LDL-C) and examined the genetic associations accounting for family dependencies. RESULTS: After correcting for multiple testing (p<0.05) and accounting for significant differences in the association effect sizes between subjects with and without MetS (p<0.05), variants of APOA5 (rs662799) and APOE (rs429358) were associated with HDL-C and LDL-C responses in MetS subjects, while APOA4 (rs675) was associated with TG response in non-MetS subjects. There was also suggestive evidence that MetS may interact with APOA4 (p=0.017), APOA5 (p=0.06), and APOE (p=0.09) to the variation to lipid responses. CONCLUSIONS: Genetic effects that contributed to the variability of lipid responses to fenofibrate may differ in subjects with and without MetS. This research may provide guidance for more personalized and effective therapies.

Hundreds of variants clustered in genomic loci and biological pathways affect human height.

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.

New loci associated with kidney function and chronic kidney disease.

Chronic kidney disease (CKD) is a significant public health problem, and recent genetic studies have identified common CKD susceptibility variants. The CKDGen consortium performed a meta-analysis of genome-wide association data in 67,093 individuals of European ancestry from 20 predominantly population-based studies in order to identify new susceptibility loci for reduced renal function as estimated by serum creatinine (eGFRcrea), serum cystatin c (eGFRcys) and CKD (eGFRcrea < 60 ml/min/1.73 m(2); n = 5,807 individuals with CKD (cases)). Follow-up of the 23 new genome-wide-significant loci (P < 5 x 10(-8)) in 22,982 replication samples identified 13 new loci affecting renal function and CKD (in or near LASS2, GCKR, ALMS1, TFDP2, DAB2, SLC34A1, VEGFA, PRKAG2, PIP5K1B, ATXN2, DACH1, UBE2Q2 and SLC7A9) and 7 loci suspected to affect creatinine production and secretion (CPS1, SLC22A2, TMEM60, WDR37, SLC6A13, WDR72 and BCAS3). These results further our understanding of the biologic mechanisms of kidney function by identifying loci that potentially influence nephrogenesis, podocyte function, angiogenesis, solute transport and metabolic functions of the kidney.

Epistatic interactions of CDKN2B-TCF7L2 for risk of type 2 diabetes and of CDKN2B-JAZF1 for triglyceride/high-density lipoprotein ratio longitudinal change: evidence from the Framingham Heart Study.

UNLABELLED: Fifteen known type 2 diabetes (T2D) gene variants were assessed for their associations with T2D status in 228 T2D families from the Framingham Heart Study (FHS) Original, Offspring, and Children Cohorts. Bayesian approach was used to test single-single-nucleotide polymorphism (SNP) association followed by logistic regression. Bayesian and logic regression approaches were used to test multiple SNP association searching for the best combinations of variants followed by logistic regression reconfirmation. The significant variants for T2D risk were also tested for their main and interacting effects on triglyceride (TG)/high-density lipoprotein (HDL) ratio change derived from four point measures across time. This slope phenotype was made available using mixed model growth curve approach from 155 T2D families in the FHS Offspring Cohort. RESULTS: CDKN2B rs10811661 (p = 0.042), TCF7L2 rs4506565 (p = 0.004), and JAZF1 rs864745 (p = 0.04) were individually associated with risk of T2D (OR = 1.0-2.0; effect size <1%). CDKN2B and TCF7L2 were found with significant main (p = 0.02, 0.01) and interacting (p = 0.05) effects for increased (OR = 3.0) risk of T2D. CDKN2B and JAZF1 were found with significant main (p = 0.0002 and 0.034) and interacting (p = 0.001) effects on increased (beta = 0.42) TG/HDL ratio longitudinal change. These interacting effects were independent of effects of age and sex with effect sizes of 0.3-0.4% for risk of T2D or TG/HDL ratio longitudinal change. CONCLUSION: These synthetic approaches allowed for successful detection of CDKN2B and TCF7L2 interacting effect for T2D risk and CDKN2B and JAZF1 interacting effect on TG/HDL ratio increase over time among T2D families in the FHS. These interacting effects were consistent in conferring risk of T2D or progressive insulin resistance with modest effect sizes.