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The common aldehyde dehydrogenase 2 ( ALDH2 ) alcohol flushing variant known as ALDH2*2 affects ∼8% of the world’s population. Even in heterozygous carriers, this missense variant leads to a severe loss of ALDH2 enzymatic activity and has been linked to an increased risk of coronary artery disease (CAD). Endothelial cell (EC) dysfunction plays a determining role in all stages of CAD pathogenesis, including early-onset CAD. However, the contribution of ALDH2*2 to EC dysfunction and its relation to CAD are not fully understood. In a large genome-wide association study (GWAS) from Biobank Japan, ALDH2*2 was found to be one of the strongest single-nucleotide polymorphisms associated with CAD. Clinical assessment of endothelial function showed that human participants carrying ALDH2*2 exhibited impaired vasodilation after light alcohol drinking. Using human induced pluripotent stem cell–derived ECs (iPSC-ECs) and CRISPR-Cas9–corrected ALDH2*2 iPSC-ECs, we modeled ALDH2*2 -induced EC dysfunction in vitro, demonstrating an increase in oxidative stress and inflammatory markers and a decrease in nitric oxide (NO) production and tube formation capacity, which was further exacerbated by ethanol exposure. We subsequently found that sodium-glucose cotransporter 2 inhibitors (SGLT2i) such as empagliflozin mitigated ALDH2*2 -associated EC dysfunction. Studies in ALDH2*2 knock-in mice further demonstrated that empagliflozin attenuated ALDH2*2 -mediated vascular dysfunction in vivo. Mechanistically, empagliflozin inhibited Na + /H + -exchanger 1 (NHE-1) and activated AKT kinase and endothelial NO synthase (eNOS) pathways to ameliorate ALDH2*2 -induced EC dysfunction. Together, our results suggest that ALDH2*2 induces EC dysfunction and that SGLT2i may potentially be used as a preventative measure against CAD for ALDH2*2 carriers.

Rare diseases affect millions of people worldwide, and discovering their genetic causes is challenging. More than half of the individuals analyzed by the Undiagnosed Diseases Network (UDN) remain undiagnosed. The central hypothesis of this work is that many of these rare genetic disorders are caused by multiple variants in more than one gene. However, given the large number of variants in each individual genome, experimentally evaluating combinations of variants for potential to cause disease is currently infeasible. To address this challenge, we developed the digenic predictor (DiGePred), a random forest classifier for identifying candidate digenic disease gene pairs by features derived from biological networks, genomics, evolutionary history, and functional annotations. We trained the DiGePred classifier by using DIDA, the largest available database of known digenic-disease-causing gene pairs, and several sets of non-digenic gene pairs, including variant pairs derived from unaffected relatives of UDN individuals. DiGePred achieved high precision and recall in cross-validation and on a held-out test set (PR area under the curve > 77%), and we further demonstrate its utility by using digenic pairs from the recent literature. In contrast to other approaches, DiGePred also appropriately controls the number of false positives when applied in realistic clinical settings. Finally, to enable the rapid screening of variant gene pairs for digenic disease potential, we freely provide the predictions of DiGePred on all human gene pairs. Our work enables the discovery of genetic causes for rare non-monogenic diseases by providing a means to rapidly evaluate variant gene pairs for the potential to cause digenic disease.

PURPOSE: Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome consists of congenital absence of the uterus and vagina and is often associated with renal, skeletal, cardiac, and auditory defects. The genetic basis is largely unknown except for rare variants in several genes. Many candidate genes have been suggested by mouse models and human studies. The purpose of this study was to narrow down the number of candidate genes. METHODS: Whole exome sequencing was performed on 111 unrelated individuals with MRKH; variant analysis focused on 72 genes suggested by mouse models, human studies of physiological candidates, or located near translocation breakpoints in t(3;16). Candidate variants (CV) predicted to be deleterious were confirmed by Sanger sequencing. RESULTS: Sanger sequencing verified 54 heterozygous CV from genes identified through mouse (13 CV in 6 genes), human (22 CV in seven genes), and translocation breakpoint (19 CV in 11 genes) studies. Twelve patients had ≥ 2 CVs, including four patients with two variants in the same gene. One likely digenic combination of LAMC1 and MMP14 was identified. CONCLUSION: We narrowed 72 candidate genes to 10 genes that appear more likely implicated. These candidate genes will require further investigation to elucidate their role in the development of MRKH.

Rare diseases affect hundreds of millions of people worldwide, and diagnosing their genetic causes is challenging. The Undiagnosed Diseases Network (UDN) was formed in 2014 to identify and treat novel rare genetic diseases, and despite many successes, more than half of UDN patients remain undiagnosed. The central hypothesis of this work is that many unsolved rare genetic disorders are caused by multiple variants in more than one gene. However, given the large number of variants in each individual genome, experimentally evaluating even just pairs of variants for potential to cause disease is currently infeasible. To address this challenge, we developed DiGePred, a random forest classifier for identifying candidate digenic disease gene pairs using features derived from biological networks, genomics, evolutionary history, and functional annotations. We trained the DiGePred classifier using DIDA, the largest available database of known digenic disease causing gene pairs, and several sets of non-digenic gene pairs, including variant pairs derived from unaffected relatives of UDN patients. DiGePred achieved high precision and recall in cross-validation and on a held out test set (PR area under the curve >77%), and we further demonstrate its utility using novel digenic pairs from the recent literature. In contrast to other approaches, DiGePred also appropriately controls the number of false positives when applied in realistic clinical settings like the UDN. Finally, to facilitate the rapid screening of variant gene pairs for digenic disease potential, we freely provide the predictions of DiGePred on all human gene pairs. Our work facilitates the discovery of genetic causes for rare non-monogenic diseases by providing a means to rapidly evaluate variant gene pairs for the potential to cause digenic disease.

Point mutations in genes encoding sarcomeric proteins are the leading cause of inherited primary cardiomyopathies. Among them are mutations in the TNNT2 gene that encodes cardiac troponin T (TnT). These mutations are clustered in the tropomyosin (Tm) binding region of TnT, TNT1 (residues 80-180). To understand the mechanistic changes caused by pathogenic mutations in the TNT1 region, six hypertrophic cardiomyopathy (HCM) and two dilated cardiomyopathy (DCM) mutants were studied by biochemical approaches. Binding assays in the absence and presence of actin revealed changes in the affinity of some, but not all, TnT mutants for Tm relative to WT TnT. HCM mutants were hypersensitive and DCM mutants were hyposensitive to Ca2+ in regulated actomyosin ATPase activities. To gain better insight into the disease mechanism, we modeled the structure of TNT1 and its interactions with Tm. The stability predictions made by the model correlated well with the affinity changes observed in vitro of TnT mutants for Tm. The changes in Ca2+ sensitivity showed a strong correlation with the changes in binding affinity. We suggest the primary reason by which these TNNT2 mutations between residues 92 and 144 cause cardiomyopathy is by changing the affinity of TnT for Tm within the TNT1 region.

Point mutations in genes encoding sarcomeric proteins often cause primary cardiomyopathies via changes in protein-protein interactions and/or alterations in calcium sensitivity. Cardiac troponin T (TnT) interacts with troponin I and troponin C (TnI, Tnc) and binds to tropomyosin (Tm) to provide control of contractility by calcium. A helical region of TnT (residues 80-150) is known to interact with Tm. There is a direct interaction of Tm residues at position d, g, a and e of the heptad repeat with residues e, a, d and g of TnT respectively. We hypothesize that the TnT mutations seen at the above mentioned positions will severe biochemical and biophysical changes than the residues at most other positions because of their direct interaction with Tm. To test the hypothesis, we have studied multiple biochemical parameters of 5 hypertrophic (HCM) and 2 dilated (DCM) cardiomyopathy-causing mutants at representative positions of the heptad repeat. Microscale thermophoresis revealed changes in binding patterns of mutants with Tm relative to wild-type. Alterations in calcium sensitivity were observed by ATPase assays. Significant changes were observed in velocities with in vitro motility analysis. We have also used the data from these mutants to better model the interaction between TnT and Tm. Our data illustrate multiple mechanistic routes by which single-residue changes in TnT alter contractility.

The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are 30 single-residue mutations in TPM1, the gene encoding α-tropomyosin. We examined seven mutant tropomyosins, E62Q, D84N, I172T, L185R, S215L, D230N, and M281T, that were chosen based on their clinical severity and locations along the molecule. The goal of our study was to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn could provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca(2+) sensitivity of human β-cardiac myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca(2+) activation, and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca(2+) concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that different substitutions perturb the structure of the regulatory complex in different ways. Moreover, we observed changes in protein stability and protein-protein interactions in these mutants. Our results suggest multiple mechanistic pathways to hypertrophic and dilated cardiomyopathies. Finally, we examined a computationally designed mutant, E181K, that is hypersensitive, confirming predictions derived from in silico structural analysis.

The major isoform of cardiac myosin heavy chain (MHC) expressed in mice is the faster alpha isoform (encoded by the Myh6 gene) whereas in humans it is the slower beta isoform (MYH7). Existing mouse models of inherited cardiomyopathies either have the human mutations introduced into the endogenous Myh6 or express mutant MYH7 as a transgene. Studies which have compared the same cardiomyopathy mutation in Myh6 versus MYH7 show contrasting effects in the two cases (Lowey et al., JBC, 2008 & 2013). To overcome these problems we have replaced the coding region of the mouse Myh6 locus with that of human MYH7 locus, including introns and microRNA genes that control MHC expression. Homozygous humanized mice (Myh6humMYH7/Myh6humMYH7) die in utero, however, heterozygotes (Myh6humMYH7/Myh6+) survive. Heterozygotes express human beta isoform at ∼50% of total MHC, with incorporation into the sarcomere, as seen by immunofluorescence with a β-MHC-specific antibody. Functionally, the heterozygous mice show no significant differences relative to wild-type controls, appearing at the correct Mendelian ratios with no difference in mortality. The heterozygotes exhibited slightly less pathological hypertrophic response to isoproterenol administration relative to wild-type controls, as measured by echocardiography, left ventricle mass and expression of relevant genes by qPCR. These observations, when considered in the context of an earlier study (Jones et al., J.Clin.Invest 1996) showing cardiac contractility defects in heterozygous Myh6 knockout mice, suggest that human MYH7 is rescuing cardiac function in our heterozygous mice. Our mouse should provide a more accurate model for studying MYH7 mutations, the largest single cause of inherited cardiomyopathy. The future direction is to study the contractility of cardiac fibers and myofibrils isolated from mutant mice to understand the primary contractile effects and disease progression prior to remodelling of the heart.

A rapid method for the determination of unsaturation of oils and fats containing non-conjugated ethylenic linkages has been developed, using hypochlorous acid as the reagent. The method has several advantages,viz., that the estimation is conducted with an aqueous solution of sodium hypochlorite reagent, and moreover no oil solvent is required for the estimation. In all the methods currently employed, the reagents used must be perfectly anhydrous otherwise incomplete addition will take place, giving low results. Since a reaction time of 4 to 5 min. is recommended, the estimations are much more rapid than the customary Wijs or Hanus methods. No discrimination as to time factor need be made for oils possessing different degree of saturation. All oils, whether belonging to non-drying or drying groups, give accurate results within the specified time. There is no secondary reaction except in the case of castor oil, but even in that case the secondary reactions are not appreciable within the first hour of reaction. The present method compares favorably with the standard procedures currently employed for determination of unsaturation.

1. The unsaturation produced during the biological oxidation of fatty acidsin vitro can be determined by use of a hypochlorous acid reagent, which readily adds at the double bond of the system as follows: $$\begin{gathered} - CH - CH - OH \hfill \\ || + HOCl \to \hfill \\ - CH - CH - Cl \hfill \\ \end{gathered} $$ 2. The addition is quantitative and complete within 10 minutes. The use of 15-minute reaction time at 37°C. is recommended. 3. The method can be employed for the semi-micro estimation of unsaturated acids. 4. There is no chance of substitution and hence prolonged reaction times are without any influence. 5. The results are affected by the presence of ketone bodies, but beta-hydroxy acids cause no interference with the estimation.

Summary The experiments carried out on the oxidation of butyric acid by A. niger indicate that the butyric acid oxidation in vitro by molds can be believed to take place according to the beta -oxidation theory as well as according to the dehydrogenation mechanism, as the presence of acetoacetic acid in the oxidation products of butyric acid might be regarded as positive proof in support of the former hypothesis, and the development of slight unsaturation being indicative of the latter. The pH optimum for the oxidation of butyric, β -hydroxybutyric, and crotonic acids has also been determined. With these preliminary results in hand it has been possible to carry out a more detailed and quantitative study on the mechanism of butyric acid decomposition by the mold and this will form the subject of our next communication.