Genetic homogeneity, high-resolution mapping, and mutation analysis of the urofacial (Ochoa) syndrome and exclusion of the glutamate oxaloacetate transaminase gene (GOT1) in the critical region as the disease gene.

The urofacial (Ochoa) syndrome (UFS) is a rare autosomal recessive disorder characterized by abnormal facial expression and urinary abnormalities. Previously, we mapped the gene to a genomic interval of approximately 1 cM on chromosome region 10q23-24, using families from Columbia. Here we demonstrate genetic homogeneity of the syndrome through homozygosity mapping in American patients with Irish heritage. We established a physical map and identified novel polymorphic markers in the UFS critical region. Haplotype analysis using the new markers mapped the UFS gene within one YAC clone of 1,410 kb. We also determined the precise location of the gene encoding for glutamate oxaloacetate transaminase (GOT1) within the new UFS critical region and determined its genomic structure. However, mutation analysis excluded GOT1 as a candidate for the UFS gene.     

Physical mapping of the chromosome 7 breakpoint region in an SLOS patient with t(7;20) (q32.1;q13.2)

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder characterized by multiple congenital anomalies and mental retardation. SLOS has an associated defect in cholesterol biosynthesis, but the molecular genetic basis of this condition has not yet been elucidated. Previously our group reported a patient with a de novo balanced translocation [t(7;20)(q32.1;q13.2)] fitting the clinical and biochemical profile of SLOS. Employing fluorescence in situ hybridization (FISH), a 1.8 Mb chromosome 7-specific yeast artificial chromosome (YAC) was identified which spanned the translocation breakpoint in the reported patient. The following is an update of the on-going pursuit to physically and genetically map the region further, as well as the establishment of candidate genes in the 7q32.1 breakpoint region. Am. J. Med Genet. 68:279–281, 1997. © 1997 Wiley-Liss, Inc.     

Homozygosity mapping identifies the Crumbs homologue 1 (Crb1) gene as responsible for a recessive syndrome of retinitis pigmentosa and nanophthalmos

The association of retinitis pigmentosa (RP) and microphthalmia has been reported in a number of familial and isolated cases. Here, the results of genetic analysis in a familial case of early RP associated with nanophthalmos are described. Two affected sibs were ascertained from an endogamous population in Mexico. A genome-wide linkage analysis was performed by means of an Affymetrix 250K microarray. Five large regions of homozygosity were demonstrated. The largest interval comprised 15.08 Mb at chromosome 1q31-32.1 and contained the Crumbs homologue-1, CRB1, a gene responsible for a number of recessive retinal dystrophies. Nucleotide sequence analysis demonstrated a c.1125C>G transversion in CRB1 exon 5, predicting a novel p.Tyr375X variant. To our knowledge this is the first instance in which a CRB1 mutation has been associated with early RP and nanophthalmos. Our results suggest a role for CRB1 in promoting axial growth of the eye. Clinical analysis of additional subjects with retinal dystrophies due to CRB1 mutations will help to identify if the high hyperopia, a frequently observed trait in these subjects, could be related to decreased eye axial length (nanophthalmos).     

Genetic defects underlying Peutz-Jeghers syndrome (PJS) and exclusion of the polarity-associated MARK/Par1 gene family as potential PJS candidates

LKB1/STK11 germline inactivations are identified in the majority (66-94%) of Peutz-Jeghers syndrome (PJS) patients. Therefore, defects in other genes or so far unidentified ways of LKB1 inactivation may cause PJS. The genes encoding the MARK proteins, homologues of the Par1 polarity protein that associates with Par4/Lkb1, were analyzed in this study because of their link to LKB1 and cell polarity. The genetic defect underlying PJS was determined through analysis of both LKB1 and all four MARK genes. LKB1 point mutations and small deletions were identified in 18 of 23 PJS families using direct sequencing and multiplex ligation-dependent probe amplification analysis identified exon deletions in 3 of 23 families. In total, 91% of the studied families showed LKB1 inactivation. Furthermore, a MARK1, MARK2, MARK3 and MARK4 mutation analysis and an MARK4 quantitative multiplex polymerase chain reaction analysis to identify exon deletions on another eight PJS families without identified LKB1 germline mutation did not identify mutations in the MARK genes. LKB1 defects are the major cause of PJS and genes of the MARK family do not represent alternative PJS genes. Other mechanisms of inactivation of LKB1 may cause PJS in the remaining families.     

桃拉综合征病毒VP1高变区蛋白抗体制备及初步特性分析

目的:制备高效价的桃拉综合征病毒主要结构蛋白VP1高变区蛋白抗体,分析其免疫特性,为研究免疫诊断试剂做参考。方法:将p ET-VP1重组载体转化入大肠杆菌BL21,在IPTG诱导下进行表达。重组VP1蛋白纯化后免疫新西兰家兔,用琼脂扩散试验及ELISA检测抗体效价,用与VP1纯化蛋白特异性结合的单克隆噬菌体做竞争抑制试验。结果:p ETVP1重组蛋白在大肠杆菌BL21中呈可溶性高效表达。抗血清经琼扩试验鉴定效价达1∶26,ELISA效价达1∶217。与VP1蛋白特异结合的单克隆噬菌体能阻断部分抗血清与VP1蛋白的结合活性,但不影响最终检测阳性的判断。结论:制备的VP1高变区蛋白抗体效价高,VP1蛋白少数区域的位点变异并不影响多克隆抗体的结合活性,可用作免疫检测诊断试剂。     

Enchondromatosis (Ollier disease, Maffucci syndrome) is not caused by the PTHR1 mutation p.R150C

Enchondromatosis (Ollier disease, Maffucci syndrome) is a rare developmental disorder characterized by multiple enchondromas. Not much is known about its molecular genetic background. Recently, an activating mutation in the parathyroid hormone receptor type 1 (PTHR1) gene, c.448C>T (p.R150C), was reported in two of six patients with enchondromatosis. The mutation is thought to result in upregulation of the IHH/PTHrP pathway. This is in contrast to previous studies, showing downregulation of this pathway in other cartilaginous tumors. Therefore, we investigated PTHR1 in enchondromas and chondrosarcomas from 31 enchondromatosis patients from three different European countries, thereby excluding a population bias. PTHR1 protein expression was studied using immunohistochemistry, revealing normal expression. The presence of the described PTHR1 mutation was analyzed, using allele-specific oligonucleotide hybridization confirmed by sequence analysis, in tumors from 26 patients. In addition, 11 patients were screened for other mutations in the PTHR1 gene by sequence analysis. Using both allele-specific oligonucleotide hybridization and sequencing, we could neither confirm the previously found mutation nor find any other mutations in the PTHR1 gene. These results indicate that the PTHR1 gene is not, in contrast to previous suggestions, the culprit for enchondromatosis.     

Comparison of the sequence of the secretory glycoprotein A (gA) gene in Md5 and BC-1 strains of Marek's disease virus type 1

DNA fragments containing the secretory glycoprotein A (gA) gene of Marek's disease virus type 1 (MDV 1) were cloned from the DNA libraries of very virulent Md5 and virulent BC-1 strains and sequenced. Two open reading frames (ORF1 and ORF2) were identified for both strains. The ORF1 has the potential to code for a protein of 501 amino acids with a molecular weight of 56 kD that contains strong hydrophobic regions in both the amino and carboxyl termini, and nine potential N-linked glycosylation sites, while the ORF2 is capable of coding for a 24-kD protein. These results indicate that the ORF1 codes for the unprocessed form of gA. Between the Md5 and BC-1 strains, only two sequence mismatches exist in the DNA fragment. More differences appear to exist in the gA sequence of the MDV 1 GA strain (12), which lacks a strong hydrophobic anchor sequence. Similarities between the predicted amino acid sequences of the MDV 1 gA and the proteins of the other herpesviruses such as herpes simplex type I gC, pseudorabies virus gIII, and varicella zoster virus gpV were noted.     

Partial monosomy of chromosome 1p36.3: Characterization of the critical region and delineation of a syndrome

We describe 5 patients ranging in age from 3 to 47 years, with karyotypic abnormalities resulting in monosomy for portion of 1p36.3, microcephaly, mental retardation, prominent forehead, deep-set eyes, depressed nasal bridge, flat midface, relative prognathism, and abnormal ears. Four patients have small hands and feet. All exhibited selfabusive behavior. Additional findings in some of the patients include brain anomalies, optic atrophy, hearing loss and skeletal deformities. The breakpoints within chromosome 1 were designated at 1p36.31 (3 cases), 1p36.32 (1 case) and 1p36.33 (1 case), Thus, the smallest region of deletion overlap is 1p36.33→1pter. Detection of the abnormal 1 relied on high resolution G-band analysis. Fluorescence in situ hybridization (FISH) utilizing a DNA probe (Oncor D1Z2) containing the repetitive sequences in distal 1p36, confirmed a deletion of one 1 homologue in all 5 cases. The abnormal 1 resulted from a de novo deletion in only one patient. The remaining patients were either confirmed (3 cases) or suspected (1 case) to have unbalanced translocations. Despite the additional genetic imbalance present in these four cases, monosomy of 1p36.33 appears to be responsible for a specific clinical phenotype. Characterization of this phenotype should assist in the clinical diagnosis of this chromosome abnormality. © 1995 Wiley-Liss, Inc.     

非同位素PCR及Southern印迹杂交分析检测脆性X综合征FMR-1基因突变

目的建立一种非同位素的检测脆性X综合征智力缺陷基因(FMR-1)突变的方法.方法采用PCR技术结合Southern 印迹杂交技术对FMR-1基因进行分析,进而确定其突变类型.结果共对190例样本进行了检测,其中2例为女性前突变携带者,1例为女性全突变患者.结论非同位素PCR方法可以简便、安全、可靠地检测FMR-1基因中(CGG)n 重复拷贝数,可作为临床上对脆性X综合征的筛查的首选方法;而非同位素Southern印迹杂交可对结果不确定者进一步进行检测.     

Cell-mediated immunity to cytomegalovirus (CMV) and herpes simplex virus (HSV) antigens in the acquired immune deficiency syndrome: Interleukin-1 and interleukin-2 modifyin vitro responses

Peripheral blood lymphocytes (PBL) were obtained from five patients with the acquired immune deficiency syndrome (AIDS), six homosexual males with lymphadenopathy, and five normal heterosexual controls. Modulation of virus-specific immunity was assayedin vitro by measuring the lymphocyte blastogenic response and the production of lymphokine (leukocyte inhibition factor; LIF) by PBL stimulated with herpes simplex virus (HSV) or cytomegalovirus (CMV) antigens in the presence or absence of interleukin-1 (IL-1) and interleukin-2 (IL-2). PBL from the control and lymphadenopathy subjects responded to both antigens in the lymphocyte transformation assay (LT) measured on day 7, and the responses were significantly enhanced in cultures grown in the presence of antigen and IL-2 (1 U/ml). PBL from the AIDS patients were unresponsive, but responsiveness was restored by the addition of IL-2. The addition of IL-1 (0.02 µg/ml) to antigen-stimulated PBL cultures failed to enhance the proliferative responses in all three study groups. LIF production was assayed in the supernatants from day 1 PBL cultures. LIF was not produced by PBL from AIDS patients grown in the presence of viral antigens, whereas three of five patients from the lymphadenopathy group, and three of five control subjects gave rise to positive responses. The addition of IL-1 to the antigenstimulated cultures enhanced LIF production in the control and lymphadenopathy groups but not in the AIDS patients. The addition of IL-2 did not modulate LIF production by antigen-stimulated PBL from the control oR AIDS patients while suppressing the LIF response of the similarly stimulated PBL from the lymphadenopathy patients.     

Catalog of 46 single-nucleotide polymorphisms (SNPs) in the microsomal glutathione S-transferase 1 (MGST1) gene

A major goal in our laboratory is to understand the role of common genetic variations among individual patients as regards susceptibility to common diseases and differences in therapeutic efficacy and/or side effects of drugs. As an addition to the high-density SNP (single-nucleotide polymorphism) maps of 12 glutathione S-transferase and related genes reported earlier, we provide here an SNP map of the microsomal glutathione S-transferase 1 (MGST1) gene. Among 48 healthy Japanese volunteers examined, we identified a total of 46 SNPs at this locus, 36 of which had not been reported before: 4 in the promoter region, 34 in introns, 3 in the 3′ untranslated region, and 5 in the 3′ flanking region. No SNP was found in 5′ untranslated or coding regions. The ratio of transition to transversion was approximately 1.2 : 1. Among the 13 insertion–deletion polymorphisms was a 2-bp deletion in the coding region of MGST1 in DNA from one of the volunteers, which resulted in a frame-shift mutation. Since the gene product encoded by this mutant allele would lack the C-terminal half including the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) domain, MGST1 activity is likely to be reduced in the carrier's cells. The SNP map presented here adds to the archive of tools for studying complex genetic diseases, population migration patterns, and a variety of pharmacogenetic possibilities. Received: June 27, 2001 / Accepted: July 16, 2001     

Myotonia congenita and myotonic dystrophy in the same family: coexistence of a CLCN1 mutation and expansion in the CNBP (ZNF9) gene

Myotonia is characterized by hyperexcitability of the muscle cell membrane. Myotonic disorders are divided into two main categories: non-dystrophic and dystrophic myotonias. The non-dystrophic myotonias involve solely the muscle system, whereas the dystrophic myotonias are characterized by multisystem involvement and additional muscle weakness. Each category is further subdivided into different groups according to additional clinical features or/and underlying genetic defects. However, the phenotypes and the pathological mechanisms of these myotonic disorders are still not entirely understood. Currently, four genes are identified to be involved in myotonia: the muscle voltage-gated sodium and chloride channel genes SCN4A and CLCN1, the myotonic dystrophy protein kinase (DMPK) gene, and the CCHC-type zinc finger, nucleic acid binding protein gene CNBP. Additional gene(s) and/or modifying factor(s) remain to be identified. In this study, we investigated a large Norwegian family with clinically different presentations of myotonic disorders. Molecular analysis revealed CCTG repeat expansions in the CNBP gene in all affected members, confirming that they have myotonic dystrophy type 2. However, a CLCN1 mutation c.1238C>G, causing p.Phe413Cys, was also identified in several affected family members. Heterozygosity for p.Phe413Cys seems to exaggerate the severity of myotonia and thereby, to some degree, contributing to the pronounced variability in the myotonic phenotype in this family.