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PubMed2005

Molecular engineering approaches for DNA sequencing and analysis.

High-throughput DNA sequencing development for mutation screening and identification is essential to realize the goal of pharmacogenomics and personalized medicine, which will lead to a new era in clinical medicine and healthcare. Molecular engineering approaches to modify the building blocks of DNA by introducing functional groups for purification and detection has led to the development of high-throughput genetic analysis technologies. This review is focused on the following two DNA sequencing approaches. The first approach is based on the use of molecular affinity and mass spectrometry to perform quick and highly accurate mutation screening, heterozygote identification and insertion/deletion detection. The second approach is based on a sequencing-by-synthesis platform that has the potential for generating DNA sequencing data in a massive, parallel manner. The basic principles, fundamental challenges and methods of implementation of these exciting new technologies will be discussed.

Animals↗
PubMed2005

Clinical characteristics of pheochromocytoma patients with germline mutations in SDHD.

PURPOSE: We examined the value of SDHD mutation screening in patients presenting with apparently sporadic and familial pheochromocytoma for the identification of SDHD-related pheochromocytomas. PATIENTS AND METHODS: This retrospective study involved 126 patients with adrenal or extra-adrenal pheochromocytomas, including 24 patients with a family history of multiple endocrine neoplasia 2, von Hippel-Lindau disease, neurofibromatosis type 1, or paraganglioma (PGL). Conformation-dependent gel electrophoresis and sequence determination analysis of germline and tumor DNA were used to identify SDHD alterations. The clinical and molecular characteristics of sporadic and hereditary tumors were compared. We reviewed the literature and compared our results with those from previously published studies. RESULTS: Pathogenic germline SDHD mutations were identified in three patients: two (2.0%) of the 102 apparently sporadic pheochromocytoma patients and one patient with a family history of PGL. These patients presented with multifocal disease (two of three multifocal patients) or with a single adrenal tumor (one of 82 patients). In the literature, mutations are mostly found in patients </= 35 years of age or presenting with multifocal or extra-adrenal disease. All patients with an SDHD mutation developed extra-adrenal tumors (pheochromocytomas or PGLs) at presentation or during follow-up. CONCLUSION: SDHD gene mutations in patients presenting with apparently sporadic adrenal pheochromocytoma are rare. We recommend SDHD mutation screening for patients presenting with a family history of pheochromocytoma or PGL, multiple tumors, isolated adrenal or extra-adrenal pheochromocytomas, and age </= 35 years. Analysis of SDHD can also help to distinguish synchronous primary tumors from abdominal metastases.

Adolescent↗
PubMed1999

A recurrent COL7A1 mutation, R2814X, in British patients with recessive dystrophic epidermolysis bullosa.

Mutations in the type VII collagen gene, COL7A1, underlie all forms of dystrophic epidermolysis bullosa (DEB). The identification of COL7A1 mutations in DEB is complicated because the COL7A1 gene contains 118 distinct exons and most mutations are specific to individual families. In an attempt to simplify mutation screening procedures we searched for recurrent mutations in genomic DNA from 38 British patients with recessive DEB using polymerase chain reaction (PCR), heteroduplex analysis and direct nucleotide sequencing. We identified a recurrent premature termination codon, R2814X, on three out of 76 alleles. Previously we identified the COL7A1 mutations R578X and 7786delG as other frequent molecular abnormalities in British recessive DEB patients. Taken together, these three mutations account for approximately 25% of the molecular pathology of this disease in our population discovered thus far and we recommend initial screening for these mutations by PCR and restriction analysis before undertaking more exhaustive COL7A1 gene analysis. Such an approach is likely to reveal underlying COL7A1 mutations in a significant number of cases.

Collagen↗
PubMed2002

Better speech performance in cochlear implant patients with GJB2-related deafness.

OBJECTIVE: We applied mutation screening in seven cochlear implant users to identify those persons with GJB2-related deafness to determine whether etiology of deafness was predictive of speech performance after implantation. METHODS: Direct sequence of GJB2 was conducted over seven cochlear implant users with prelingual hearing impairment and their speech, language and cognitive performance was examined. RESULTS: The three persons with GJB2-related deafness had a mean vocabulary of 1243 words compared to a mean vocabulary of 195 words in the four children with GJB2-unrelated deafness, although the number of patients examined here was limited. The developmental quotient (DQ) of cognitive ability also was higher in those children with GJB2-related deafness. CONCLUSIONS: These preliminary results suggest that better speech performance after cochlear implantation may be observed in persons with GJB2-related deafness. In the future, detailed phenotypic studies and mutation screening for non-syndromic hearing loss may play an important role in the preoperative assessment of prelingually-deafened children.

Audiometry↗
PubMed2002

Screening E-cadherin in gastric cancer families reveals germline mutations only in hereditary diffuse gastric cancer kindred.

The International Gastric Cancer Linkage Consortium (IGCLC) predicted that up to 25% of families fulfilling the criteria for hereditary diffuse gastric cancer (HDGC) would harbor CDH1 germline mutations. This was based on observations from the low number of diffuse gastric cancer families described at the time, and its validation would require analysis of larger numbers. Here we report the results of germline CDH1 mutation screening in 39 kindred with familial aggregation of gastric cancer, a subset of which fulfills the criteria defined by the IGCLC for HDGC. CDH1 germline mutations were detected in four of 11 (36.4%) HDGC families. No mutations were identified in 63.6% of HDGC families or in kindred with familial aggregation of gastric cancer not fulfilling criteria for HDGC. These results add support to the evidence that only HDGC families harbor germline mutations in CDH1 and that genes other than CDH1 remain to be identified.

Adult↗
PubMed1995

Molecular analysis and predictive testing in retinoblastoma.

Predictive testing using molecular analysis is an integral part of contemporary retinoblastoma management. We have made extensive use of segregation analysis for risk assessment in both familial and sporadic disease. Investigation of loss of heterozygosity in tumor samples proved to be invaluable for the identification of linkage phase. In many families, however, accurate carrier risk assessment depends on direct identification of the causative R B I mutation. Consequently, we have developed methods for rapid mutation screening. Using these techniques, mutation analysis can now be offered to an increasing number of individuals.

DNA Mutational Analysis↗
PubMed2004

A novel NPHS2 gene mutation in Turkish children with familial steroid-resistant nephrotic syndrome.

We report in this paper two siblings aged 8 and 17 months who were clinically diagnosed with familial steroid-resistant nephrotic syndrome (SRNS). By mutation screening of the NPHS2 gene, a homozygous missense mutation, P118L, was detected in both children. This study is the first systematic investigation of NPHS2 gene mutations in Turkish children with familial SRNS. If this mutation is a hot spot of mutation in the Turkish population, screening this novel mutation in Turkish children with SRNS may be of great clinical use to prevent unnecessary treatment modalities, provide accurate genetic counselling and predict the prognosis of the disease.

Drug Resistance↗
PubMed1998

An evaluation of common breast cancer gene mutations in a population of Ashkenazi Jews.

OBJECTIVES: In view of the recent reports of recurrent mutations in BRCA1 and BRCA2 in the Ashkenazi Jewish population, we have undertaken to assess the frequency of these mutations in this population attending for genetic counselling and risk assessment of familial breast cancer. DESIGN: Mutation screening for the 185delAG and the 5382insC mutations in BRCA1 and the 6174delT mutation in BRCA2 was performed on DNA samples from either subjects affected by breast or ovarian cancer or obligate gene carriers. The likelihood of the cancers being hereditary in each family was calculated. SUBJECTS: Blood samples were obtained from 26 affected subjects or obligate gene carriers from 23 Ashkenazi Jewish families, all with a history of either early onset breast or ovarian cancers, or multiple cases of breast or ovarian cancer. RESULTS: Twelve mutations have been identified in the 23 families (52%) of which eight (67%) were the 185delAG mutation, three (25%) were the 6174delT mutation, and one (8%) was the 5382insC mutation. While the majority of these mutations were identified in families with a greater than 50% probability of being hereditary under the CASH segregation model, three mutations were identified in families with a 35% or less probability. CONCLUSIONS: Genetic screening of the recurrent mutations in Ashkenazi Jewish families will lead to the availability of predictive testing in a reasonably large proportion, even if the family history of breast/ovarian cancer is not particularly strong. In our view it is possible to reassure high risk unaffected members of these families, if the screening is negative for these mutations, even if a sample from an affected member of the family is unavailable for previous screening.

Adult↗
PubMed2002

Clinical phenotype and molecular diagnosis of multiple epiphyseal dysplasia with relative hip sparing during childhood (EDM2).

We report on a family of 19 individuals over four generations in which 12 members are affected with a variant of multiple epiphyseal dysplasia. Beginning in childhood, the disease leads to pain and stiffness of knees, ankles, elbows and finger joints. Some adult patients repeatedly suffer from free articular bodies resulting in locking of the joint. Finally, affected individuals are prone to the development of early degenerative joint disease. Mutation screening of candidate regions revealed a novel point mutation at position -1 in the COL9A2 exon 3/intron 3 splicing region. This G --> C substitution most probably induces an alteration of the splicing process. Family screening was carried out by both automated sequencing and by digestion of amplicons with BsaWI. We confirmed the nucleotide substitution in eight clinically affected family members as well as in three presymptomatic young children. Electron microscopy showed that the diameter of collagen fibrils from arthroscopically removed free articular bodies of two patients was not obviously different from that of normal articular cartilage. Together with previous reports our results indicate that mutations leading to skipping of exon 3 within the COL3 domain of the alpha2-chain of collagen type IX may be relatively common in patients with a special subtype of multiple epiphyseal dysplasia (MED) in which the hips are not markedly affected at early age (EDM2). In these patients and their families, mutation screening of the candidate regions may help to confirm the diagnosis, lead to appropriate advice for lifestyle and well based genetic counseling.

Adult↗
PubMed1996

Systematic screening for mutations in the 5'-regulatory region of the human dopamine D1 receptor (DRD1) gene in patients with schizophrenia and bipolar affective disorder.

A possible dysregulation of dopaminergic neurotransmission has been implicated in a variety of neuropsychiatric diseases. In the present study we systematically searched for the presence of mutations in the 5'-flanking region of the dopamine D1 receptor (DRD1) gene. This region has previously been shown to contain a functional promoter [Minowa et al., 1992: Proc Natl Acad Sci 89:3045-3049; Minowa et al., 1993: J Biol Chem 268:23544-23551]. We investigated 119 unrelated individuals (including 36 schizophrenic patients, 38 bipolar affective patients, and 45 healthy controls) using single-strand conformation analysis (SSCA). Eleven overlapping PCR fragments covered 2,189 bp of DNA sequence. We identified six single base substitutions: -2218T/C, -2102C/A, -2030T/C, -1992G/A, -1251G/C, and -800T/C. None of the mutations was found to be located in regions which have important influence on the level of transcriptional activity. Allele frequencies were similar in patients and controls, indicating that genetic variation in the 5'-regulatory region of the DRD1 gene is unlikely to play a frequent, major role in the genetic predisposition to either schizophrenia or bipolar affective disorder.

Alleles↗
PubMed2000

Screening for mutations in the peripheral myelin genes PMP22, MPZ and Cx32 (GJB1) in russian charcot-marie-tooth neuropathy patients; irina V. Mersiyanova, sookhrat M. Ismailov, alexandr V. Polyakov, elena L. Dadali, valeriy P. Fedotov, eva nelis, ann Lofgren, vincent timmerman, christine van broeckhoven, and oleg V. Evgrafov (Article was originally published in human mutation 15:340-347, 2000)

The authors wish to correct a mistake which occurred in the reporting of one of the mutations. The mutation in Cx32 Met34Lys is wrongly described as 100A>G. The correct description of the mutation should be 101T>A (Met34Lys).

Journal Article↗
PubMed1995

Systematic screening for mutations in the promoter and the coding region of the 5-HT1A gene.

In the present study we sought to identify genetic variation in the 5-HT1A receptor gene which through alteration of protein function or level of expression might contribute to the genetic predisposition to neuropsychiatric diseases. Genomic DNA samples from 159 unrelated subjects (including 45 schizophrenic, 46 bipolar affective, and 43 patients with Tourette's syndrome, as well as 25 healthy controls) were investigated by single-strand conformation analysis. Overlapping PCR (polymerase chain reaction) fragments covered the whole coding sequence as well as the 5' untranslated region of the 5-HT1A gene. The region upstream to the coding sequence we investigated contains a functional promoter. We found two rare nucleotide sequence variants. Both mutations are located in the coding region of the gene: a coding mutation (A-->G) in nucleotide position 82 which leads to an amino acid exchange (Ile-->Val) in position 28 of the receptor protein and a silent mutation (C-->T) in nucleotide position 549. The occurrence of the Ile-28-Val substitution was studied in an extended sample of patients (n = 352) and controls (n = 210) but was found in similar frequencies in all groups. Thus, this mutation is unlikely to play a significant role in the genetic predisposition to the diseases investigated. In conclusion, our study does not provide evidence that the 5-HT1A gene plays either a major or a minor role in the genetic predisposition to schizophrenia, bipolar affective disorder, or Tourette's syndrome.

Base Sequence↗
PubMed1995

Complete screening of mutations in the coding sequence of the CFTR gene in a sample of CF patients from Russia: identification of three novel alleles.

To date, a large number of mutations causing the disease, cystic fibrosis, have been reported worldwide. Having analysed the coding sequence of a sample of cystic fibrosis (CF) patients from Russia, we have identified three novel CF mutations. Two of them, 175 del C in exon 1 and 624 del T in exon 5, are frameshift mutations, predicted to result in premature termination of the CFTR transcript. The third mutation is missense and occurs in exon 12 (D572N). The profile of mutations in this sample of Russian CF patients is particular, with two mutations in exon 13 (2143 del T and 2184 ins A), accounting for 12% of the non-delta F508 alleles.

Alleles↗
PubMed2002

Molecular analysis of Turkish mucopolysaccharidosis IVA (Morquio A) patients: identification of novel mutations in the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS; EC 3.1.6.4). The deficiency of N-acetylgalactosamine-6-sulfate sulfatase leads to lysosomal accumulation of undegraded glycosaminoglycans, keratan sulfate and chondroitin-6-sulfate. Mutation screening of the GALNS gene was performed by SSCP and direct sequence analyses using genomic DNA samples from 10 Morquio A patients. By nonradioactive SSCP screening, 6 different gene mutations and 2 polymorphisms were identified in 10 severely affected MPS IVA patients. Five of the mutations and one of the polymorphisms are novel. The vast majority of the gene alterations were found to be single nucleotide deletions (389delG, 929delG, and 763delT) or insertions (1232-1233insT). The other two mutations were one previously identified missense mutation (Q473X) and one novel nonsense (P179S) mutation. Together they account for 95% of the disease alleles of the patients investigated. Beside mutations, one previously identified E477 polymorphism and one novel W520 polymorphism were found among Turkish MPS IVA patients.

Adolescent↗