Deep exome sequencing identifies enrichment of deleterious mosaic variants in neurodevelopmental disorder genes and mitochondrial tRNA regions in bipolar disorder.

Bipolar disorder (BD) is a global medical issue, afflicting around 1% of the population with manic and depressive episodes. Despite various genetic studies, the genetic architecture and pathogenesis of BD have not been fully resolved. Besides germline variants, postzygotic mosaic variants are proposed as new candidate mechanisms contributing to BD. Here, we performed extensive deep exome sequencing (DES, ~300×) and validation experiments to investigate the roles of mosaic variants in BD with 235 BD cases (194 probands of trios and 41 single cases) and 39 controls. We found an enrichment of developmental disorder (DD) genes in the genes hit by deleterious mosaic variants in BD (P = 0.000552), including a ClinVar-registered pathogenic variant in ARID2. An enrichment of deleterious mosaic variants was also observed for autism spectrum disorder (ASD) genes (P = 0.000428). The proteins coded by the DD/ASD genes with non-synonymous mosaic variants in BD form more protein-protein interaction than expected, suggesting molecular mechanisms shared with DD/ASD but restricted to a subset of cells in BD. We also found significant enrichment of mitochondrial heteroplasmic variants, another class of mosaic variants, in mitochondrial tRNA genes in BD (P = 0.0102). Among them, recurrent m.3243 A > G variants known as causal for mitochondrial diseases were found in two unrelated BD probands with allele fractions of 5-12%, lower than in mitochondrial diseases. Despite the limitation of using peripheral tissues, our DES investigation supports the possible contribution of deleterious mosaic variants in the nuclear genome responsible for severer phenotypes, such as DD/ASD, to the risk of BD and further demonstrates that the same paradigm can be applied to the mitochondrial genome. These results, as well as the enrichment of heteroplasmic mitochondrial tRNA variants in BD, add a new piece to the understanding of the genetic architecture of BD and provide general insights into the pathological roles of mosaic variants in human diseases.

Systematic analysis of exonic germline and postzygotic de novo mutations in bipolar disorder.

Bipolar disorder is a severe mental illness characterized by recurrent manic and depressive episodes. To better understand its genetic architecture, we analyze ultra-rare de novo mutations in 354 trios with bipolar disorder. For germline de novo mutations, we find significant enrichment of loss-of-function mutations in constrained genes (corrected-P = 0.0410) and deleterious mutations in presynaptic active zone genes (FDR = 0.0415). An analysis integrating single-cell RNA-sequencing data identifies a subset of excitatory neurons preferentially expressing the genes hit by deleterious mutations, which are also characterized by high expression of developmental disorder genes. In the analysis of postzygotic mutations, we observe significant enrichment of deleterious ones in developmental disorder genes (P = 0.00135), including the SRCAP gene mutated in two unrelated probands. These data collectively indicate the contributions of both germline and postzygotic mutations to the risk of bipolar disorder, supporting the hypothesis that postzygotic mutations of developmental disorder genes may contribute to bipolar disorder.

Potent and orally active ET(A) selective antagonists with 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acid structures.

The synthesis and structure-activity relationships of a series of 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids are described. Our efforts have been focused on modification of the aryl ring at the 5-position and the alkyl substituent at the 2-position of the bottom 4-methoxyphenyl ring in an effort to develop orally available ET(A) selective antagonists with safer profiles in terms of the P-450 enzyme inhibitory activity. Incorporation of a hydroxymethyl group as an alkyl substituent in methylenedioxyphenyl and 6-dihydrobenzofuran derivatives led to the identification of orally bioavailable ET(A) selective antagonists 1f and 7f. These compounds also showed not only excellent binding affinity (IC(50) < 0.10nM, more than 800-fold selectivity for the ET(A) receptor over the ET(B) receptor) but also sufficient oral bioavailability, 48% and 56%, respectively, in rats. Furthermore, these compounds did not exhibit either competitive or mechanism-based inhibition of human cytochrome P450 enzymes.

Structure-activity relationships of a novel class of endothelin receptor selective antagonists; 6-carboxy-2-isopropylamino-5,7-diarylcyclopenteno[1,2-b]pyridines.

The synthesis and structure-activity relationships of 6-carboxy-2-isopropylamino-5,7-diarylcyclopenteno[1,2-b]pyridine class of ET(A) receptor selective antagonists were described. These derivatives were prepared from the optically active key intermediates (3, 4, 10, and 13). Optimization of the substituent at the 2-position of the bottom 4-methoxyphenyl ring of the lead compound 1 led to identification of 2-hydroxy-1-methylethoxy (2g and h), hydroxyalkyl (2i, m, and p), 3-methoxy-2-methylpropyl (2t and u), N-acetyl-N-methylaminomethyl (2v), and 2-(dimethylcarbamoyl)propyl (2w) derivatives that showed greater than 1000-fold selectivity for the ET(A) receptor over the ET(B) receptor with excellent binding affinity (IC(50)<0.10 nM). Further screening of these compounds by assessing the plasma exposures at 1 h, 4 h, and 8 h after oral administration (3 or 10 mg/kg) in rats led to identification of the hydroxymethyl (2i) and 3-methoxy-2-methylpropyl (2u) derivatives exhibiting good oral bioavailability in rats.

Approach to obstructive sleep apnea syndrome at Tokyo Dental College, Ichikawa General Hospital.

In this communication, we report the current status of OSAS (Obstructive Sleep Apnea Syndrome) in the southern region of Higashikatsushika around Ichikawa City, our effort to improve patient QOL as well as to establish diagnostic and therapeutic methods, and the results of a comparison of therapeutic options with the focus on improvement of compliance by using nCPAP (nasal continuous positive airway pressure). We examined 112 patients who visited the Otolaryngology Department at Tokyo Dental College, Ichikawa General Hospital, with the chief complaint of nocturnal snoring or sleep apnea from January 2001 to April 2003 and underwent all-night PSG (polysomnography). Based upon the results of these all-night PSGs, 89 and 23 patients were diagnosed as having OSAS and simple snoring, respectively. Using the AHI classification of severity, 58 and 31 patients were assessed as having severe OSAS and mild OSAS, respectively. (1) nCPAP was tried in 61 patients, and 39 patients (63%) were able to continue it. After the introduction of nCPAP, surgery was performed in 18 patients (30%). As a result, weaning from nCPAP was successfully achieved in 10 cases, compliance with nCPAP was improved in six cases, alleviation of symptoms (decreased pressure) was seen in one case, and aggravation was noted in one case. In addition, four patients (7%) unilaterally discontinued nCPAP. (2) Surgery was performed in 34 patients, and 18 of them had surgery after nCPAP was tried. (3) We asked the dental department to make OAs (oral appliances) for 31 patients but seven of them did not attend the department, so a total of 24 patients used OAs. Fourteen patients (58%) were able to tolerate an OA for 3 months or more. Based on these results, we are hoping to achieve a better control of OSAS by combining nCPAP and other modalities.

A convenient synthetic method of a 5,7-diarylcyclopenteno-[1,2-b]pyridine-6-carboxylate: a key intermediate for potent endothelin receptor antagonists.

A convenient method for the synthesis of the title intermediate 4 was described. The key steps of this synthesis involved: (1) regioselective addition reaction of arylzinc reagent to quinolic anhydride in 42% isolated yield, (2) conversion of a ketoacid to an enone, which was achieved in 65% yield by intramolecular Knoevenagel reaction of beta-ketoester generated by condensation of an acid imidazolide with an ester enolate, followed by dehydration assisted with silica gel, and (3) stereoselective reduction of an allyl alcohol in 75% yield with zinc under acidic conditions. This synthesis enabled us to provide hundreds of grams of without chromatographic purification.

6-Carboxy-5,7-diarylcyclopenteno[1,2-b]pyridine derivatives: a novel class of endothelin receptor antagonists.

Compounds (2-5) with a 6-carboxy-5,7-diarylcyclopentenopyridine skeleton were designed, synthesized, and identified as a new class of potent non-peptide endothelin receptor antagonists. The regio-isomer 2 was found to show potent inhibitory activity with an IC(50) value of 2.4 nM against (125)I-labeled ET-1 binding to human ET(A) receptors and a 170-fold selectivity for ET(A) over ET(B) receptors. Furthermore, 2 displayed more potent in vivo activity than did the indan-type compound 1 in a mouse ET-1 induced lethality model, suggesting the potential of 2 as a new lead structure. Derivatization on substituted phenyl groups at the 5- and 7-positions of 2 revealed that a 3,4-methylenedioxyphenyl group at the 5-position and a 4-methoxyphenyl group at the 7-position were optimal for binding affinity. Further derivatization of 2 by incorporating a substituent into the 2-position of the 4-methoxyphenyl group led to the identification of a more potent ET(A) selective antagonist 2p with an IC(50) value of 0.87 nM for ET(A) receptors and a 470-fold selectivity. In addition, 2p showed highly potent in vivo efficacy (AD(50): 0.04 mg/kg) in the lethality model.