Phylogenetic analysis has been widely used to test the a priori hypothesis of epidemiological clustering in suspected transmission chains of HIV-1. Among studies showing strong support for relatedness between HIV samples obtained from infected individuals, evidence for the direction of transmission between epidemiologically related pairs has been lacking. During transmission of HIV, a genetic bottleneck occurs, resulting in the paraphyly of source viruses with respect to those of the recipient. This paraphyly establishes the direction of transmission, from which the source can then be inferred. Here, we present methods and results from two criminal cases, State of Washington v Anthony Eugene Whitfield , case number 04-1-0617-5 (Superior Court of the State of Washington, Thurston County, 2004) and State of Texas v Philippe Padieu , case numbers 219-82276-07, 219-82277-07, 219-82278-07, 219-82279-07, 219-82280-07, and 219-82705-07 (219th Judicial District Court, Collin County, TX, 2009), which provided evidence that direction can be established from blinded case samples. The observed paraphyly from each case study led to the identification of an inferred source (i.e., index case), whose identity was revealed at trial to be that of the defendant.

OBJECTIVE -Ketosis-prone diabetes (KPD) is an emerging syndrome that encompasses several distinct phenotypic subgroups that share a predisposition to diabetic ketoacidosis. We investigated whether the AϪ␤Ϫ subgroup of KPD, characterized by complete insulin dependence, absent ␤-cell functional reserve, lack of islet cell autoantibodies, and strong family history of type 2 diabetes, represents a monogenic form of diabetes.RESEARCH DESIGN AND METHODS -Over 8 years, 37 patients with an AϪ␤Ϫ phenotype were identified in our longitudinally followed cohort of KPD patients. Seven genes, including hepatocyte nuclear factor 4A (HNF4A), glucokinase (GCK), HNF1A, pancreas duodenal homeobox 1 (PDX1), HNF1B, neurogenic differentiation 1 (NEUROD1), and PAX4, were directly sequenced in all patients. Selected gene regions were also sequenced in healthy, unrelated ethnically matched control subjects, consisting of 84 African American, 96 Caucasian, and 95 Hispanic subjects.RESULTS -The majority (70%) of the AϪ␤Ϫ KPD patients had no significant causal polymorphisms in either the proximal promoter or coding regions of the seven genes. The combination of six potentially significant low-frequency, heterozygous sequence variants in HNF-1␣ (A174V or G574S), PDX1 (putative 5Ј-untranslated region CCAAT box, P33T, or P239Q), or PAX4 (R133W) were found in 27% (10/37) of patients, with one additional patient revealing two variants, PDX1 P33T and PAX4 R133W. The A174V variant has not been previously reported.CONCLUSIONS -Despite its well-circumscribed, robust, and distinctive phenotype of severe, nonautoimmune-mediated ␤-cell dysfunction, AϪ␤Ϫ KPD is most likely not a predominantly monogenic diabetic syndrome. Several AϪ␤Ϫ KPD patients have low-frequency variants in HNF1A, PDX1, or PAX4 genes, which may be of functional significance in their pathophysiology.

Genetic analysis of an adult patient with an unusual course of Ketosis-Prone Diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the<i> </i>5’-UTR of <i>PDX1</i>, a beta-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm stage. Metabolomic analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the definitive endoderm to the pancreatic progenitor stage, and RNA-sequencing showed defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR-Cas9 manipulation of the <i>PDX1</i> variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the <i>PDX1</i> 5’-UTR, leading to decreased PDX1 expression at the early pancreatic progenitor stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical beta-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset beta-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multi-omics analysis of patient-specific stem cells for clinical discovery.

In the article cited above, due to a coding error, the RNA-Seq heat map in Fig. 3C displayed the first 100 genes in chromosomal order rather than the top 100 most significant genes expressed. The correct RNA-Seq heat map, shown below, accurately represents the 100 most significant genes in the RNA-Seq analysis, clustered according to gene expression value. This correction does not change any of the reported data, other figures, or conclusions. The authors apologize for the error. The online version of the article (https://doi.org/10.2337/db20-1293) has been updated with the correct heat map.

Genetic analysis of an adult patient with an unusual course of Ketosis-Prone Diabetes (KPD) and lacking islet autoantibodies demonstrated a nucleotide variant in the<i> </i>5’-UTR of <i>PDX1</i>, a beta-cell development gene. When differentiated to the pancreatic lineage, his induced pluripotent stem cells stalled at the definitive endoderm stage. Metabolomic analysis of the cells revealed that this was associated with leucine hypersensitivity during transition from the definitive endoderm to the pancreatic progenitor stage, and RNA-sequencing showed defects in leucine-sensitive mTOR pathways contribute to the differentiation deficiency. CRISPR-Cas9 manipulation of the <i>PDX1</i> variant demonstrated that it is necessary and sufficient to confer leucine sensitivity and the differentiation block, likely due to disruption of binding of the transcriptional regulator NFY to the <i>PDX1</i> 5’-UTR, leading to decreased PDX1 expression at the early pancreatic progenitor stage. Thus, the combination of an underlying defect in leucine catabolism characteristic of KPD with a functionally relevant heterozygous variant in a critical beta-cell gene that confers increased leucine sensitivity and inhibits endocrine cell differentiation resulted in the phenotype of late-onset beta-cell failure in this patient. We define the molecular pathogenesis of a diabetes syndrome and demonstrate the power of multi-omics analysis of patient-specific stem cells for clinical discovery.