Mutation screening of mismatch repair gene Mlh3 in familial esophageal cancer

AIM： To shed light on the possible role of mismatch repair gene MIh3 in familial esophageal cancer （FEC）. METHODS： A total of 66 members from 10 families suggestive of a genetic predisposition to hereditary esophageal cancer were screened for germline mutations in MIh3 with denaturing high performance liquid chromatography （DHPLC）, a newly developed method of comparative sequencing based on heteroduplex detection. For all samples exhibiting abnormal DHPLC profiles, sequence changes were evaluated by cycle sequencing. For any mutation in family members, we conducted a segregation study to compare its prevalence in sporadic esophageal cancer patients and normal controls. RESULTS： Exons of MIh3 in all samples were successfully examined. Overall, 4 missense mutations and 3 polymorphisms were identified in 4 families. MIh3 missense mutations in families 9 and 10 might be pathogenic, but had a reduced penetrance. While in families 1 and 7, there was no sufficient evidence supporting the monogenic explanations of esophageal cancers in families. The mutations were found in 33% of high-risk families and 50% of low-risk families.CONCLUSION： MIh3 is a high risk gene with a reduced penetrance in some families. However, it acts as a low risk gene for esophageal cancer in most families. Mutations of MIh3 may work together with other genes in an accumulated manner and result in an increased risk of esophageal tumor. DHPLC is a robust and sensitive technique for screening gene mutations.

Screening for Melanocortin 4 Receptor Mutations in Chinese Extremely Obese Individuals

Accumulating evidence indicates that overweight and obesity are the major international public health concern. Obesity is a major independent risk factor for chronic diseases, such as hypertension, type 2 diabetes, cardiovascular disease, stroke, and certain cancer. Disease burden due to obesity has been dramatically increasing in many countries including China in the past years. According to the Nationwide Health and Nutrition Survey （CHNS）, the prevalence of overweight and obesity among men and women in China increased by 27.6% and 8.8%, respectively, from 1993 to 2009.

Screening of Genes with Unique Mutations of Microcus

Yersinia pestis is the causative agent of bubonic and pneumonic plagues. Strains of Y. pestis are classified into four biovars： antiqua, mediaevalis, orientalis, and microtus[11. There are two microtus-related plague loci in China： the Microtus brandti plague focus in the Xilin Gol Grassland （focus L） and the Microtus fuscus plague focus in the Ojnghai-Tibet Plateau （focus M）.

Mutation Breeding of β-carotene Producing Strain B. trispora by Low Energy Ion Implantation

Ion beam bioengineering technology as a new mutation approach has been widely used in the biological breeding field. In this paper the application of low energy nitrogen ion implantation in the β-carotene producing strain, Blakeslea trispora（-） was investigated. The effects of different fermentation conditions on β-carotene production by a high yield strain were examined. Results showed that two β-carotene high yielding strains B.trispora（-） BH3-701 and BH3-728 were screened out and the averaged production of β-carotene was raised by 178.7% and 164.6% respectively after five passages in the shaking flasks. Compared with the original strain, the highest yield strain BH3-701 was potent in accumulating β-carotene, especially in the later stage, and greatly increased production efficiency.

TGFBI and CHST6 gene analysis in Chinese stromal corneal dystrophies

AIM:To investigate whether mutations in TGFBI gene or CHST6 gene correlated with stromal corneal dystrophies(CD) in 8 Chinese probands.· METHODS:Eight unrelated patients with stromal corneal dystrophies were recruited in this study;all affected members were assessed by completely ophthalmologic examinations.Genomic DNA was extracted from peripheral leukocytes,17 exons of TGFBI gene and the exon of CHST6 gene were amplified by polymerase chain reaction(PCR),sequenced directly and compared with the reference database.· RESULTS:Three heterozygous mutations in TGFBI gene were identified in six patients:c.370C>T(p.Arg124Cys) was found in exon 4 of TGFBI gene in three members,c.371G>A(p.Arg124His) was found in one patient;c.1663C>T(p.Arg555Trp) was found in exon 12 in other two members.In addition,four polymorphisms with the nucleotide changes rs1442,rs1054124,rs4669,and rs35151677 were found in TGFBI gene.Mutations were not identified in the rest of 2 affected individuals in TGFBI gene or CHST6 gene.· CONCLUSION:Within these patients,R124C,R124H and R555W mutations were co-segregated with the disease phenotypes and were specific mutations for lattice corneal dystrophy type I(LCD I),Avellino corneal dystrophy(ACD,GCDⅡ),granular corneal dystrophy type I(GCD I),respectively.Our study highlights the prevalence of codon 124 and codon 555 mutations in the TGFBI gene among the Chinese stromal corneal dystrophies patients.·

Mutational screening of BASP1 and transcribed processed pseudogene TPΨg-BASP1 in patients with Mbius syndrome

Moebius syndrome is a rare disorder primarily characterized by congenital facial palsy, frequently accompanied by ocular abduction anomalies and occasionally associated with orofacial, limb and musculoskeletal malformations. Abnormal development of cranial nerves Ⅴ through Ⅻ underlines the disease pathogenesis. Although a genetic etiology for Moebius syndrome was proposed, molecular genetic studies to identify the causative gene（s） are scarce. In this study, we selected two candidate genes. One is BASP1 residing in a human chromosome 5p15.1-p15.2, syntenic to mouse chromosome 15qA2-qB2, to which a mouse model with facial nerve anomalies was mapped. The other is transcribed processed pseudogene TPψg-BASP1, which is located on chromosome 13q flanking the putative locus for Moebius syndrome and might be involved in the regulation of the transcripts encoded by BASP1. Mutation analyses in nineteen patients excluded these genes as being candidates for Moebius syndrome.

Genetic Analysis of the PKHD1 Gene with Long-rang PCR Sequencing

PKHD1 gene mutations are found responsible for autosomal recessive polycystic kidney disease（ARPKD）. However, it is inconvenient to detect the mutations by common polymerase chain reaction（PCR） because the open reading frame of PKHD1 is very long. Recently, long-range（LR） PCR is demonstrated to be a more sensitive mutation screening method for PKHD1 by directly sequencing. In this study, the entire PKHD1 coding region was amplified by 29 reactions to avoid the specific PCR amplification of individual exons, which generated the size of 1 to 7 kb products by LR PCR. This method was compared to the screening method with standard direct sequencing of each individual exon of the gene by a reference laboratory in 15 patients with ARPKD. The results showed that a total of 37 genetic changes were detected with LR PCR sequencing, which included 33 variations identified by the reference laboratory with standard direct sequencing. LR PCR sequencing had 100% sensitivity, 96% specificity, and 97.0% accuracy, which were higher than those with standard direct sequencing method. In conclusion, LR PCR sequencing is a reliable method with high sensitivity, specificity and accuracy for detecting genetic variations. It also has more intronic coverage and lower cost, and is an applicable clinical method for complex genetic analyses.

High-resolution melting-based TILLING of γ ray-induced mutations in rice

Targeting Induced Local Lesions IN Genomes （TILLING） is a reverse genetics strategy for the high-throughput screening of induced mutations.γ, radiation, which often induces both insertion/deletion （Indel） and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting （HRM） analysis. Pooled rice （Oryza sativa） samples mixed at a 1：7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ, ray-induced mutants in rice. For demonstration, a γ, ray-mutagenized M2 rice population （n=4560） was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution （G→A） and one single nucleotide insertion （A） were identified in OsLCT1, and one tdnucleotide （TTC） deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ, ray mutagenesis to the breeding of rice and other seed crops.

R555W mutation of TGF, βI related to granular corneal dystrophy in Chinese patients

Background Mutations in the transforming growth factor beta I （TGFBI） gene cause several types of autosomal-dominant corneal dystrophies. We investigated the role of this gene in a Chinese family affected by granular corneal dystrophy （GCD）.Methods Family history and phenotypic data were recorded. The diagnosis of GCD was made on the basis of clinical evaluation. The genomic DNA was extracted from peripheral blood leukocytes. All the exons and flanking intron-exon boundary sequences of TGFβ1 were amplified by polymerase chain reaction （PCR） and screened for mutation by direct DNA sequencing.Results A heterozygous C to T transition at nucleotide c.1663 （CGG to TGG R555W） of TGFβ1 gene was present in two affected members but was absent in the rest of the family members. Conclusion A recurrent pathogenic R555W of TGFβ1 gene mutation is identified, which appears to be the predominant mutations causing GCD in different populations.

Screening for tetrahydrobiopterin deficiency among hyperphenylalaninemia patients in Southern China

Abstract Objectives To assess the incidence of tetrahydrobiopterin (BH4)deficiency among patients with hyperphenylalaninemia (HPA) in southern Chinese and evaluate clinical outcome and gene mutations in tetrahydrobiopterin deficient patients.Methods Urinary neopterin (N) and biopterin (B) was analyzed in 87 patients with hyperphenylalaninemia by high-performance liquid chromatography. Further combined loading tests with phenylalanine(Phe) (100*!mg/kg) and tetrahydrobiopterin (BH4) (7.5*!mg/kg) were performed in suspected patients with abnormal urinary pterin profiles. Gene mutation analysis was performed for patients with BH4 deficiency and their parents. BH4 deficient patients were treated with BH4 and neurotransmitter precursors after diagnosis. Blood phenylalanine levels, clinical symptoms and mental development were followed up.Results Eleven patients were diagnosed as having BH4 deficiency caused by 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency. The incidence of tetrahydrobiopterin (BH4) deficiency among patients with hyperphenylalaninemia (HPA) in southern Chinese was 10%. Combined loading tests with phenylalanine and oral BH4 were done in 4 of 11 patients and their phenylalanine levels were decreased to normal 4-6h after BH4 administration. Four different mutations (P87S, N52S, D96N and G144R) in the PTPS gene were detected in 5 families. Five PTPS-deficient patients were treated with synthetic BH4, neurotransmitter precursors (L-dopa plus carbidopa, and 5-hydroxytryptophan). They had satisfactory physical and mental development after treatment. One patient with partial PTPS deficiency had normal growth and mental development without treatment. Conclusions Our results emphasize that screening for BH4 deficiency should be carried out in all patients with hyperphenylalaninemia in order to minimize the misdiagnosis. Patients with BH4 deficiency should be treated early with BH4 and a combination of neurotransmitter precursors.

Genetic analysis and serum level of cartilage oligomeric matrix protein in patients with pseudoachondroplasia

Background Pseudoachondroplasia （PSACH） is an autosomal-dominant osteochondrodysplasia due to mutations in the gene encoding cartilage oligomeric matrix protein （COMP）.Clinical diagnosis of PSACH is based primarily on family history, physical examination, and radiographic evaluation.There is evidence that decreased serum COMP concentration may serve as a diagnostic marker in PSACH.Here, we investigated the role of this gene and the serum COMP concentration in Chinese patients with PSACH.Methods A family with three patients and a sporadic case were recruited.Genomic and phenotypic data were recorded.The diagnosis of PSACH was made on the base of clinical evaluation.The genomic DNA was extracted from peripheral blood leukocytes.The 8-19 exons and flanking intron-exon boundary sequences of COMP were amplified by polymerase chain reaction （PCR） and screened for mutation by direct DNA sequencing.Serum COMP concentrations of 4 patients and age-compatible control group of 20 unrelated healthy subjects were analyzed on the basis of an ELISA Kit for human cartilage oligomeric matrix protein.Results A deletion （c.1447-1455del） was identified in exon 13 in the sporadic case.The mean serum COMP concentrations of four patients （3.12±2.28） were significantly lower than those of control group （10.86±2.21, P 〈0.05）.There was no overlap in the distribution of serum COMP concentration between PSACH patients and controls.Conclusions Mutations in COMP gene are responsible for the PSACH.Serum COMP concentration may be suggested as an additional diagnostic marker to aid clinical findings in suspected cases of PSACH.