Recurrent De Novo Mutations Affecting Residue Arg138 of Pyrroline-5-Carboxylate Synthase Cause a Progeroid Form of Autosomal-Dominant Cutis Laxa

Progeroid disorders overlapping with De Barsy syndrome (DBS) are collectively denoted as autosomal-recessive cutis laxa type 3 (ARCL3). They are caused by biallelic mutations in PYCR1 or ALDH18A1, encoding pyrroline-5-carboxylate reductase 1 and pyrroline-5-carboxylate synthase (P5CS), respectively, which both operate in the mitochondrial proline cycle. We report here on eight unrelated individuals born to non-consanguineous families clinically diagnosed with DBS or wrinkly skin syndrome. We found three heterozygous mutations in ALDH18A1 leading to amino acid substitutions of the same highly conserved residue, Arg138 in P5CS. A de novo origin was confirmed in all six probands for whom parental DNA was available. Using fibroblasts from affected individuals and heterologous overexpression, we found that the P5CS-p.Arg138Trp protein was stable and able to interact with wild-type P5CS but showed an altered sub-mitochondrial distribution. A reduced size upon native gel electrophoresis indicated an alteration of the structure or composition of P5CS mutant complex. Furthermore, we found that the mutant cells had a reduced P5CS enzymatic activity leading to a delayed proline accumulation. In summary, recurrent de novo mutations, affecting the highly conserved residue Arg138 of P5CS, cause an autosomal-dominant form of cutis laxa with progeroid features. Our data provide insights into the etiology of cutis laxa diseases and will have immediate impact on diagnostics and genetic counseling.     

Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination

Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes have yet to be identified. Here, we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white-matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in PYCR2 in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in the amount of PYCR2. When mutant cDNAs were transfected into HEK293FT cells, both variant proteins retained normal mitochondrial localization but had lower amounts than the wild-type protein, suggesting that the variant proteins were less stable. A PYCR2-deficient HEK293FT cell line generated by genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of a zebrafish PYCR2 ortholog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2’s isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human CNS during development.     

Pyrroline-5-Carboxylate Reductase in Chlorella autotrophica and Chlorella saccharophila in Relation to Osmoregulation

Pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2), which catalyzes the reduction of P5C to proline, was partially purified from two Chlorella species; Chlorella autotrophica, a euryhaline marine alga that responds to increases in salinity by accumulating proline and ions, and Chlorella saccharophila, which does not accumulate proline for osmoregulation. From the elution profile of this enzyme from an anion exchange column in Tris-HCl buffer (pH 7.6), containing sorbitol and glycine betaine, it was shown that P5C reductase from C. autotrophica was a neutral protein whereas the enzyme from C. saccharophila was negatively charged. The kinetic mechanisms of the reductase was characteristic of a ping-pong mechanism with double competitive substrate inhibition. Both enzymes showed high specificity for NADH as cofactor. The affinities of the reductases for their substrates did not change when the cells were grown at different salinities. In both algae, the apparent K_m values of the reductase for P5C and NADH were 0.17 and 0.10 millimolar, respectively. A fourfold increase in maximal velocity of the reductase was observed when C. autotrophica was transferred from 50 to 150% artificial sea water. Even though the reductase was inhibited by NaCl, KCl, and proline, it still showed appreciable activity in the presence of these compounds at molar concentrations. A possible role for the regulation of proline synthesis at the step catalyzed by P5C reductase is discussed in relation to the specificity of P5C reductase for NADH and its responses to salt treatments.     

Pyrroline-5-Carboxylate Reductase Is in Pea (Pisum sativum L.) Leaf Chloroplasts

Proline accumulation is a well-known response to water deficits in leaves. The primary cause of accumulation is proline synthesis. Δ¹-Pyrroline-5-carboxylate reductase (PCR) catalyzes the final reaction of proline synthesis. To determine the subcellular location of PCR, protoplasts were made from leaves of Pisum sativum L., lysed, and fractionated by differential and Percoll density gradient centrifugation. PCR activity comigrated on the gradient with the activity of the chloroplast stromal marker NADPH-dependent triose phosphate dehydrogenase. We conclude that PCR is located in chloroplasts, and therefore that chloroplasts can synthesize proline. PCR activities from chloroplasts and etiolated shoots were compared. PCR activity from both extracts is stimulated at least twofold by 100 millimolar KCl or 10 millimolar MgCl₂. The pH profiles of PCR activity from both extracts reveal two separate optima at pH 6.5 and 7.5. Native isoelectric focusing gels of sampies from etiolated tissue reveal a single band of PCR activity with a pl of 7.8.     

盐胁迫下小麦苗叶片吡咯-5-羧酸还原酶活性和游离脯氨酸积累

受NaCl、KCl或MsCl_2胁迫的小麦幼苗,当外部溶液的渗透势由—160 kPa下降到—900 kPa时,叶片吡咯—5—羧酸还原酶(PSC)活性增高;渗透势由—900 kPa降低到—1500kPa,酶活性下降;胁迫 1d和 2d的幼苗,还原酶活性显著增加;3～6d酶活性无大变化。游离脯氨酸含量随溶液渗透势下降和培养时间的延长而提高。胁迫解除后酶活性和脯氨酸含量均降低。受NcCl胁迫义在ABA影响下的幼苗,P5C还原酶活性和脯氨酸含量高于仅受NaCl胁迫的幼苗。     

Pyrroline-5-Carboxylate Reductase in Soybean Nodules : Comparison of the Enzymes in Host Cytosol, Bradyrhizobium japonicum Bacteroids, and Cultures

Characteristics of pyrroline-5-carboxylate reductase (P5CR) from Bradyrhizobium japonicum bacteroids and cultured rhizobia were compared with those of the enzyme in soybean nodule host cytosol. Reductase from host cytosol differed from that in bacteroids in: (a) the effect of pH on enzymic activity, (b) the capacity to catalyze both reduction of pyrroline-5-carboxylic acid and NAD⁺-dependent proline oxidation, (c) apparent affinities for pyrroline-5-carboxylic acid, and (d) sensitivities to inhibition by NADP⁺ and proline. The K₁ for proline inhibition of P5CR in bacteroid cytosol was 1.8 millimolar. The properties of P5CR in B. japonicum and bacteroid cytosol were similar. The specific activities of P5CR in the cytosolic fractions of the nodule host and the bacteroid compartment were also comparable.     

2型糖尿病患者家系DGAT1基因K378N多态性研究

目的:探讨西安地区汉族人DGAT1基因K378N多态性及其与2型糖尿病(T2DM)的相关性.方法:应用荧光偏振-模板依赖的染料掺入反应法(TOI-FP)对T2DM患者(T2DM组)76例、T2DM患者家系中非DM一级亲属(NDR组)59例的DGAT1基因K378N多态性进行检测,同时测定相关临床和生化指标,并与45名正常人(NC组)相比较.结果:DGAT1 K378等位基因频率在T2DM组和NDR组依次为87.5%和83.9%,而NC组为54.4%,差异均具有统计学意义(P＜0.05).T2DM组、NDR组和NC组不同基因型DGAT1受栓者血浆甘油三酯(TG)水平差异无显著性(P＞0.05).结论:DGAT1基因K378N多态性与西安地区人群T2DM的发病相关.该多态性与患者血浆TG水平无明显相关.     

Complementation Cloning and Characterization of the Pyrroline 5-Carboxylate Reductase Gene from Drosophila melanogaster

The first insect cDNA and genomic sequences encoding pyrroline 5-carboxylate reductase (EC 1.5.1.2) have been isolated from Drosophila melanogaster. The cDNA sequence was identified by interspecies complementation of an E. coli proline auxotroph and encodes a protein 280 amino acids in length with 25–41% identity to pyrroline 5-carboxylate reductases isolated from other organisms. The corresponding gene is single copy and is located at cytological position 91E-F, and in one of the P1 clones in that region. With a single 61-bp intron, and an impressively small 135- to 200-bp region that presumably acts as a bidirectional promoter, the gene itself shows remarkable economy. The calculated molecular weight of 29,700 predicts that the native enzyme is likely an octomer. Sequencing of the promoter region and expression studies, as well as the known function of the enzyme in redox regulation and the high levels of free proline in insects, suggest that this housekeeping gene encodes an enzyme with a crucial role in intermediary metabolism.     

2型糖尿病家系患者LEPR基因Gln223Arg多态性的研究

目的：研究黑龙江地区汉族人2型糖尿病家系的LEPR基因Gln223Arg多态性,探讨其与2型糖尿病发病的关系。方法：应用聚合酶链式反应-限制性内切酶长度多态性（PCR-RFLP）技术,对来自于黑龙江地区120个2型糖尿病家系中的210例2型糖尿病患者及319例正常对照的LEPR基因Gln223Arg（668 A→G）位点进行基因分型。结果：LEPR基因Gln223Arg三种基因型在病例组和对照组间整体分布有统计学意义（P=0.034,df=2）;除AG基因型（x2=4.550,P〈0.01）外,其余各基因型及等位基因在病例组和对照组间分布未见显著性差异（P〉0.05）。结论：LEPR基因Gln223Arg多态性与黑龙江地区汉族人2型糖尿病有关,LEPR基因可能为中国人2型糖尿病发病的相关易感基因。     

2型糖尿病家系患者LEPR基因Gln223Arg多态性的研究

目的:研究黑龙江地区汉族人2型糖尿病家系的LEPR基因Gln223Arg多态性,探讨其与2型糖尿病发病的关系。方法:应用聚合酶链式反应-限制性内切酶长度多态性(PCR-RFLP)技术,对来自于黑龙江地区120个2型糖尿病家系中的210例2型糖尿病患者及319例正常对照的LEPR基因Gln223Arg(668 A→G)位点进行基因分型。结果:LEPR基因Gln223Arg三种基因型在病例组和对照组间整体分布有统计学意义(P=0.034,df=2);除AG基因型(x2=4.550,P<0.01)外,其余各基因型及等位基因在病例组和对照组间分布未见显著性差异(P>0.05)。结论:LEPR基因Gln223Arg多态性与黑龙江地区汉族人2型糖尿病有关,LEPR基因可能为中国人2型糖尿病发病的相关易感基因。     

RIN2 Deficiency Results in Macrocephaly, Alopecia, Cutis Laxa, and Scoliosis: MACS Syndrome

Inherited disorders of elastic tissue represent a complex and heterogeneous group of diseases, characterized often by sagging skin and occasionally by life-threatening visceral complications. In the present study, we report on an autosomal-recessive disorder that we have termed MACS syndrome (macrocephaly, alopecia, cutis laxa, and scoliosis). The disorder was mapped to chromosome 20p11.21-p11.23, and a homozygous frameshift mutation in RIN2 was found to segregate with the disease phenotype in a large consanguineous kindred. The mutation identified results in decreased expression of RIN2, a ubiquitously expressed protein that interacts with Rab5 and is involved in the regulation of endocytic trafficking. RIN2 deficiency was found to be associated with paucity of dermal microfibrils and deficiency of fibulin-5, which may underlie the abnormal skin phenotype displayed by the patients.     

The Role of a Novel TRMT1 Gene Mutation and Rare GRM1 Gene Defect in Intellectual Disability in Two Azeri Families

Cognitive impairment or intellectual disability (ID) is a widespread neurodevelopmental disorder characterized by low IQ (below 70). ID is genetically heterogeneous and is estimated to affect 1–3% of the world’s population. In affected children from consanguineous families, autosomal recessive inheritance is common, and identifying the underlying genetic cause is an important issue in clinical genetics. In the framework of a larger project, aimed at identifying candidate genes for autosomal recessive intellectual disorder (ARID), we recently carried out single nucleotide polymorphism-based genome-wide linkage analysis in several families from Ardabil province in Iran. The identification of homozygosity-by-descent loci in these families, in combination with whole exome sequencing, led us to identify possible causative homozygous changes in two families. In the first family, a missense variant was found in GRM1 gene, while in the second family, a frameshift alteration was identified in TRMT1, both of which were found to co-segregate with the disease. GRM1, a known causal gene for autosomal recessive spinocerebellar ataxia (SCAR13, MIM#614831), encodes the metabotropic glutamate receptor1 (mGluR1). This gene plays an important role in synaptic plasticity and cerebellar development. Conversely, the TRMT1 gene encodes a tRNA methyltransferase that dimethylates a single guanine residue at position 26 of most tRNAs using S-adenosyl methionine as the methyl group donor. We recently presented TRMT1 as a candidate gene for ARID in a consanguineous Iranian family (Najmabadi et al., 2011). We believe that this second Iranian family with a biallelic loss-of-function mutation in TRMT1 gene supports the idea that this gene likely has function in development of the disorder.     

Elevated Accumulation of Proline in NaCl-Adapted Tobacco Cells Is Not Due to Altered Delta-Pyrroline-5-Carboxylate Reductase

Tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that are adapted to 428 millimolar NaCl accumulate proline mainly due to increased synthesis from glutamate. These cells were used to evaluate the possible role of Δ¹-pyrroline-5-carboxylate reductase in the regulation of proline biosynthesis. No increase in the specific activity of Δ¹-pyrroline-5-carboxylate reductase in crude extracts throughout the growth cycle was observed in NaCl-adapted cells compared to unadapted cells. The enzyme from both cell types was purified extensively. On the basis of affinity for the substrates NADPH, NADH, and Δ¹-pyrroline-5-carboxylate, pH profiles, chromatographic behavior during purification, and electrophoretic mobility of the native enzyme, the activities of the enzyme from the two sources were similar. These data suggest that the NaCl-dependent regulation of proline synthesis in tobacco cells does not involve induction of pyrroline-5-carboxylate isozymes or changes in its kinetic properties.     

Submergence Enhances Expression of a Gene Encoding 1-Aminocyclopropane-1-Carboxylate Oxidase in Deepwater Rice

Partial submergence greatly stimulates internodal growth indeepwater rice (Oryza sativa L.). Previous work has shown thatthe effect of submergence is, at least in part, mediated byethylene, which accumulates in the air spaces of submerged internodes.To investigate the expression of the genes encoding ethylenebiosynthetic enzymes during accelerated growth of deepwaterrice, we cloned a 1-aminocyclopropane- 1-carboxylate (ACC) oxidasecDNA (OSACO1) from internodes of submerged plants and measuredthe activity of the enzyme in tissue extracts with an improvedassay. We found an increase in ACC oxidase mRNA levels and enzymeactivity after 4 to 24 h of submergence. Thus, it is likelythat ethylene biosynthesis in internodes of deepwater rice iscontrolled, at least in part, at the level of ACC oxidase. (Received January 6, 1996; Accepted April 6, 1996)     

畸精子症病人的睾丸特异性表达基因SPEM1筛查分析

目的:探讨睾丸特异性表达基因SPEM1突变与畸精子症患者之间的关系.方法:收集从2005年4月至2007年3月临床上不明原因的畸精症患者113份外周血标本以及100份正常生育能力男子的外周血标本,抽提其DNA.然后采用PCR技术、变性高效液相色谱技术(DHPLC)以及测序等手段对全部DNA样本进行该基因的突变筛查.结果:在畸精症患者中发现1个新的未见报道的多态性位点;尚未发现有基因突变或微缺失.结论:SPEM1基因突变或缺失不是引起本组畸精子症病人的主要致病基因.该基因在对畸精子症所致不育的诊断价值尚需进一步研究.     

hMLH1和hMSH2种系突变携带者累积患癌风险研究

目的：探讨遗传性非息肉病性结直肠癌（HNPCC）家系中错配修复基因hMLH1和hMSH2种系突变携带者发生HNPCC相关恶性肿瘤的累积风险度。方法：通过随访14个HNPCC家系中222例hMLH1或hMSH2种系突变携带者与非携带者,应用SPSS14.0统计软件包分析种系突变携带者在不同年龄点发生HNPCC相关恶性肿瘤的累积风险度及两种基因种系突变累积患癌风险的差异。结果：hMLH1或hMSH2种系突变携带者肿瘤发生率为63.8%（60/94）,非突变携带者肿瘤发生率为0.8%（1/128）,种系突变携带者发生恶性肿瘤的相对危险度为非突变携带者的317.6倍;种系突变携带者发生各种HNPCC相关恶性肿瘤的累积风险度随年龄的增加逐渐增大,在60岁时发生各种HNPCC相关恶性肿瘤、结直肠癌、胃癌等的平均累积风险度分别为92.4%、81.3%、29.6%,40岁以前发生胃癌的平均风险度较低（6.1%）;hMLH1与hMSH2种系突变携带者发生各种HNPCC相关恶性肿瘤、结直肠癌、胃癌等累积风险度的差异无统计学意义（均为P＆gt;0.05）。结论：hMLH1或hMSH2种系突变携带者为HNPCC家系中患癌高危人群,发生HNPCC相关恶性肿瘤的风险度随年龄的增加而增大,最常发生恶性肿瘤的部位为胃和结直肠;hMLH1与hMSH2种系突变携带者发生各种HNPCC相关恶性肿瘤的累积风险度无明显差异。     

VAMP1 Mutation Causes Dominant Hereditary Spastic Ataxia in Newfoundland Families

Our group previously described and mapped to chromosomal region 12p13 a form of dominantly inherited hereditary spastic ataxia (HSA) in three large Newfoundland (Canada) families. This report identifies vesicle-associated membrane protein 1 (VAMP1), which encodes a critical protein for synaptic exocytosis, as the responsible gene. In total, 50 affected individuals from these families and three independent probands from Ontario (Canada) share the disease phenotype together with a disruptive VAMP1 mutation that affects a critical donor site for the splicing of VAMP1 isoforms. This mutation leads to the loss of the only VAMP1 isoform (VAMP1A) expressed in the nervous system, thus highlighting an association between the well-studied VAMP1 and a neurological disorder. Given the variable phenotype seen in the affected individuals examined here, we believe that VAMP1 should be tested for mutations in patients with either ataxia or spastic paraplegia.     

Molecular Mechanisms of Carcinogenesis Associated with MEN1 Gene Mutation

Russian Journal of Genetics - Despite recent advances in genomics and the discovery of numerous genes involved in carcinogenesis, the mechanisms of malignant transformation of different tissues...