From the Divisions of Developmental and Stem Cell Biology (H.S., A.E., M.R., E.M.M.A.M., R.W., J.M., J.O.S., E.I., K.S., J.H., K.K., G.C., D.B.S., S.L.D.), Vascular Biology (G.J.M., R.S.), and Molecular, Structural, and Computational Biology (D.T.H., J.W.K.H., E.G.), Victor Chang Cardiac Research Institute, the Faculties of Medicine and Science, University of New South Wales (H.S., A.E., J.O.S., E.I., D.T.H., G.J.M., J.W.K.H., K.K., R.S., E.G., G.C., D.B.S., S.L.D.), Liverpool Hospital, Department of Clinical Genetics (A.E., A.C.), the Department of Clinical Genetics (A.E., J.S., F.C., D.O.S.) and the Heart Centre for Children (D.S.W.), Children's Hospital at Westmead, the Discipline of Genetic Medicine (A.E., J.S., F.C., D.O.S.) and the Medical School (D.S.W.), University of Sydney, and the Faculty of Medicine and Health Sciences, Macquarie University (C.K.L., G.J.G.) - all in Sydney, the School of Biological Sciences, University of Adelaide, Adelaide, SA (J.N.H., P.Q.T.), and the Institute of Health and Biomedical Innovation, Queensland University of Technology (A.M.M.-L., P.J.L., M.A.B., E.L.D.), the Translational Research Institute (A.M.M.-L., P.J.L., M.A.B., E.L.D.), the Department of Endocrinology, Royal Brisbane and Women's Hospital (E.L.D.), and the University of Queensland School of Medicine (E.L.D.), Brisbane - all in Australia; and Spectrum Health Medical Group, Medical Genetics, Grand Rapids, MI (P.R.M.).
N Engl J Med. 2017 Aug 10;377(6):544-552. doi: 10.1056/NEJMoa1616361.
Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients.
We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system.
Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs21). Another patient carried heterozygous KYNU variants (p.Y156 and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects.
Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).
先天性畸形可表现为多种表型的组合，这些表型的同时出现频率高于随机出现的频率。在许多此类情况下，确定遗传原因都极具挑战性。我们在无亲缘关系的患者中寻找心脏、脊柱和肾脏等部位缺陷的遗传原因。
我们使用基因组测序来鉴定具有多种先天性畸形的个体的家族中潜在的致病性基因突变。我们使用体外酶活性测定和量化患者血浆代谢物的方法来测试变体的功能。我们使用 CRISPR（成簇规律间隔短回文重复）-Cas9 系统在具有相似变体的小鼠模型中进行基因工程改造。
我们在编码犬尿氨酸途径中酶的两个基因中发现了变体，即 3-羟基邻氨基苯甲酸 3,4-加双氧酶（HAAO）和犬尿氨酸酶（KYNU）。三名患者携带纯合变体，预测 HAAO 或 KYNU 蛋白发生功能丧失性变化（HAAO p.D162*、HAAO p.W186或 KYNU p.V57Efs21）。另一名患者携带杂合 KYNU 变体（p.Y156和 p.F349Kfs4）。突变酶的体外活性大大降低。烟酰胺腺嘌呤二核苷酸（NAD）通过犬尿氨酸途径从色氨酸从头合成。患者的循环 NAD 水平降低。由于 NAD 缺乏，Haao 基因敲除或 Kynu 基因敲除小鼠的胚胎中出现了与患者相似的缺陷。在基因敲除小鼠中，妊娠期间预防 NAD 缺乏可避免缺陷。
NAD 合成的中断导致 NAD 缺乏和人类及小鼠的先天性畸形。妊娠期间补充烟酰胺可预防小鼠的畸形。（由澳大利亚国家卫生和医学研究委员会等资助）。
