Assessment of housekeeping genes for use in normalization of real time PCR in skeletal muscle with chronic degenerative changes.

The real time PCR technique requires the normalization of the gene of interest to reference genes that are accepted to be ubiquitously expressed. The choice of the reference gene(s) needs to be determined by researchers according to the particular tissue or model of interest. The best normalization gene is not easy to decide, particularly if the investigated tissue displays architectural changes and structural reorganization. We have investigated the expression of four housekeeping genes that are widely used for the normalization purposes (TATA binding protein, beta actin, hypoxanthine-guanine phosphorybosyl transferase and glyceraldehyde-3-phosphate dehydrogenase) in a skeletal muscle degeneration model applied by the release of the Achilles tendon which leads to a time-course degeneration of the soleus and gastrocnemius muscles. This study indicates that the TATA binding protein and the beta actin gene to be the least effected in the course of degeneration induced by tenotomy in rat soleus and gastrocnemius muscle.

Serial analysis of gene expression in the hippocampus of patients with mesial temporal lobe epilepsy.

Hippocampal sclerosis constitutes the most frequent neuropathological finding in patients with medically intractable mesial temporal lobe epilepsy. Serial analysis of gene expression was used to get a global view of the gene profile in human hippocampus in control condition and in epileptic condition associated with hippocampal sclerosis. Libraries were generated from control hippocampus, obtained by rapid autopsy, and from hippocampal surgical specimens of patients with mesial temporal lobe epilepsy and the classical pattern of hippocampal sclerosis. More than 50,000 tags were analyzed (28,282, control hippocampus; 25,953, hippocampal sclerosis) resulting in 9206 (control hippocampus) and 9599 (hippocampal sclerosis) unique tags (genes), each representing a specific mRNA transcript. Comparison of the two libraries resulted in the identification of 143 transcripts that were differentially expressed. These genes belong to a variety of functional classes, including basic metabolism, transcription regulation, protein synthesis and degradation, signal transduction, structural proteins, regeneration and synaptic plasticity and genes of unknown identity of function. The database generated by this study provides an extensive inventory of genes expressed in human control hippocampus, identifies new high-abundant genes associated with altered hippocampal morphology in patients with mesial temporal lobe epilepsy and serves as a reference for future studies aimed at detecting hippocampal transcriptional responses under various pathological conditions.

Myogenic program induction in mature fat tissue (with MyoD expression).

MyoD exerts a master transcriptional control over the myogenic differentiation cascade. Here, we study different approaches to induce myogenic transdifferentiation in mature adipocytes utilizing MyoD gene transfer. Organotypic cultures of fat tissue and a long-term culture of in vitro differentiated adipocytes deduced that MyoD provoked morphological changes in mature adipocytes that can be summarized as loss of fat content, acquisition of a fusiform shape and eventual fusion with committed neighbor cells. In vivo, MyoD gene transfer into rat interscapular and inguinal fat pads demonstrated that while structural proteins of muscle lineage were expressed, they co-existed with specific adipocyte proteins. Expression of these proteins diminished over time likewise the fat content. The transdifferentiation process initiated by MyoD did not require cell cycle progression and was well tolerated by the fully differentiated and mature adipocytes.

Neurotrophin receptor immunoreactivity in the hippocampus of patients with mesial temporal lobe epilepsy.

Recent evidence supports a critical role of neurotrophins in the regulation of both neuronal survival and synaptic transmission during epileptogenesis. We have examined the immunohistochemical expression of high- (tyrosine kinase receptors, trk) and low-affinity (p75) neurotrophin receptors (NTRs) in the hippocampal specimens from 18 patients with chronic temporal lobe epilepsy [TLE; 14 patients with hippocampal sclerosis (HS) and four with focal lesions (tumours) not involving the hippocampus proper]. Nonepileptic autopsy brains (n = 6) and surgical specimens from tumour patients without epilepsy (n = 3) were used as controls. Immunoreactivity (IR) for the trk receptors (trkA, trkB, trkC) was detected in normal human brain within the pyramidal neurones of hippocampal cornus ammoni (CA) regions and in the dentate gyrus. There were no detectable differences in the neuronal trk IR patterns in the hippocampus between control and TLE cases with HS, except for a decrease in neuronal density in regions where cell death had occurred (CA1, CA3 and CA4). In contrast, a consistent increase in trkA IR was observed in reactive astrocytes in CA1 and dentate gyrus. The low-affinity p75 neurotrophin receptor (p75(NTR)) was expressed in low levels in postnatal normal hippocampus. In contrast, neuronal p75(NTR) IR was detected in 10/14 cases of HS in spared neurones within the CA and hilar regions of the hippocampus. Double labelling revealed that p75(NTR)-positive neurones also contain trk receptor IR. In six cases with prominent glial activation strong p75(NTR) IR was observed in microglial cells within the sclerotic hippocampus. The present results indicate that changes in NTR expression are still detectable in the hippocampus of patients with chronic TLE and involve both glial and neuronal cells. Reactive astrocytes were immunoreactive for trkA, whereas activated microglia cells were reactive for p75(NTR), suggesting different functions for specific NTRs in the development of reactive gliosis. Moreover, the increased expression of p75(NTR) in hippocampal neurones of TLE patients may critically influence the neuronal survival during the epileptogenic process.

Frequencies of TAP1 and TAP2 gene polymorphisms in the Anatolian population.

Anatolia has a long and complex record of immigration from various regions. Here, we have used TAP1 and TAP2 gene polymorphisms as genetic markers to study the relationship between the Anatolian population and other populations. A neighbour-joining tree was constructed indicating the relatedness of European populations and the Anatolian population with respect to TAP1 and TAP2 allele frequencies.

Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome.

Chanarin-Dorfman syndrome (CDS) is a rare autosomal recessive form of nonbullous congenital ichthyosiform erythroderma (NCIE) that is characterized by the presence of intracellular lipid droplets in most tissues. We previously localized a gene for a subset of NCIE to chromosome 3 (designated "the NCIE2 locus"), in six families. Lipid droplets were found in five of these six families, suggesting a diagnosis of CDS. Four additional families selected on the basis of a confirmed diagnosis of CDS also showed linkage to the NCIE2 locus. Linkage-disequilibrium analysis of these families, all from the Mediterranean basin, allowed us to refine the NCIE2 locus to an approximately 1.3-Mb region. Candidate genes from the interval were screened, and eight distinct mutations in the recently identified CGI-58 gene were found in 13 patients from these nine families. The spectrum of gene variants included insertion, deletion, splice-site, and point mutations. The CGI-58 protein belongs to a large family of proteins characterized by an alpha/beta hydrolase fold. CGI-58 contains three sequence motifs that correspond to a catalytic triad found in the esterase/lipase/thioesterase subfamily. Interestingly, CGI-58 differs from other members of the esterase/lipase/thioesterase subfamily in that its putative catalytic triad contains an asparagine in place of the usual serine residue.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay in two unrelated Turkish families.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is an early onset form of hereditary spastic paraplegia with a peculiar clinical presentation. In addition to cerebellar findings which manifest first with ataxic gait in early life and spasticity, on an evolutionary basis, there is axonal neuropathy, prominent myelinated fibers in the optic fundus, and evidence of cerebellar atrophy that can be detected by cranial MRI. Intelligence is usually normal, however lower IQs have also been documented. This disorder mainly originates from the Charlevoix-Saguenay region of Quebec. Here, we report two Turkish families linked to the disease locus on chromosome 13 q12. There was homozygosity and segregation of disease haplotypes in both families. This form of spastic ataxia may be more common than originally presumed.

Newborn PKU screening in Turkey: at present and organization for future.

At present, pkenylketonuria screening is a national child health program in Turkey which is carried out collaboratively by the Ministry of Health and three University Children's Hospitals in Ankara, Istanbul and Izmir. Since 1986 the number of cities included in the screening program has gradually increased, now and it covers all the metropolises the country. A total of 383 babies were found with persistent hyperphenylalaninemia (1:4,172) among 1,605,582 babies screened by the Guthrie test at the Hacettepe Screening Center in Ankara. By taking into account pretreatment phenylalanine levels and phenlyalanine tolerances at five years of age, the numbers of classical and mild-moderate phenylketonuria and mild hyperphenylalaninemia cases were 216, 102 and 58, respectively. The major problems encountered in the screening program and in management of the detected cases were unsatisfactory sample collection, early discharge from maternity hospitals, difficulties in reaching some detected cases, and noncompliance with dietary therapy due to illiterate parents or to lack of social insurance. To screen and treat all newborns for phenylketonuria and to include at least hypothyroidism in the screening program, there is a need for a more disciplinary intersectoral approach than exists at present.

Mutation frequency of Familial Mediterranean Fever and evidence for a high carrier rate in the Turkish population.

Familial Mediterranean Fever (FMF) is a recessive disorder characterised by episodes of fever and neutrophil-mediated serozal inflammation. The FMF gene (MEFV) was recently identified and four common mutations characterised. The aim of this study was to determine the carrier rate in the Turkish population and the mutation frequency in the clinically diagnosed FMF patients. We found a high frequency of carriers in the healthy Turkish population (20%). The distribution of the five most common MEFV mutations among healthy individuals (M694V 3%, M680I 5%, V726A 2%, M694I 0% and E148Q 12%) was significantly different (P<0.005) from that found in patients (M694V 51.55%, M680I 9.22%, V726A 2.88%, M694I 0.44% and E148Q 3.55%).

Mitochondrial DNA analysis in the Turkish Leber's hereditary optic neuropathy population.

PURPOSE: To define the prevalence of a panel of mitochondrial DNA (mtDNA) mutations associated with Leber's hereditary optic neuropathy (LHON) in the Turkish LHON population. LHON-associated mtDNA mutations have been found in LHON patients from around the world, but the Turkish LHON population has not been studied. METHODS: Thirty-two Turkish patients were defined clinically as having LHON on the basis of painless, subacute, bilateral optic neuropathy and the exclusion of other causes of subacute optic neuropathy. mtDNA was extracted from blood of the 32 probands and assayed for a panel of primary and secondary LHON-associated mtDNA mutations by polymerase chain reaction (PCR)-based methods. We studied three well-known LHON-associated primary mutations (at nucleotide positions 11778, 3460 and 14484) and one common secondary mutation (at nucleotide 15257) in all 32 probands. In addition to these mutations, 18 of the 32 probands were tested for the Complex IV, COX III gene, LHON associated 9804 and 9438 mutations and secondary LHON mutations at nucleotide positions 3394, 4160, 4216, 4917, 5244, 7444, 7706, 13708, 13730 and 15812. RESULTS: Among the 32 probands tested for four common LHON mutations, 3 carried the 14484 mutation, 1 carried the 11778 mutation, 1 carried the 3460 mutation and 1 carried the 15257 mutation. Among the 18 LHON patients who tested for additional mutations, 1 proband harboured the 9804 mutation and 4 carried the secondary mutations at nucleotide positions 4216, 4917 and 13708. CONCLUSION: The results of mtDNA analysis of the Turkish LHON patients appear to be different from those of previous reports.

Vacuoliting megalencephalic leukoencephalopathy with subcortical cysts, mapped to chromosome 22qtel.

The leukodystrophies form a complex group of orphan genetic disorders that primarily affect myelin, the main constituent of the brain white matter. Among the leukodystrophies of undetermined etiology, a new clinical entity called "vacuoliting megalencephalic leukoencephalopathy" (VL) was recently recognized. VL is characterized by diffuse swelling of the white matter, large subcortical cysts, and megalencephaly with infantile onset. Family studies in several ethnic groups have suggested an autosomal recessive mode of inheritance. We mapped the VL gene to chromosome 22qtel, within a 3-cM linkage interval between markers D22S1161 and n66c4 (maximum LOD score 10.12 at recombination fraction.0, for marker n66c4; maximum multipoint LOD score 17 for this interval) by genome scan of 13 Turkish families. Linkage analysis under the genetic-heterogeneity hypothesis showed no genetic heterogeneity. No abnormalities were found in three tested candidate genes (fibulin-1 and glutathione S-transferases 1 and 2).

Two new loci for autosomal recessive ichthyosis on chromosomes 3p21 and 19p12-q12 and evidence for further genetic heterogeneity.

Autosomal recessive ichthyosis (ARI) includes a heterogeneous group of disorders of keratinization characterized by desquamation over the whole body. Two forms largely limited to the skin have been defined: lamellar ichthyosis (LI) and nonbullous congenital ichthyosiform erythroderma (NCIE). A first gene for LI, transglutaminase TGM1, has been identified on chromosome 14, and a second one has been localized on chromosome 2. In a genomewide scan of nine large consanguineous families, using homozygosity mapping, two new loci for ARI were found, one for a lamellar form in a 6-cM interval on chromosome 19 and a second for an erythrodermic form in a 7.7-cM interval on chromosome 3. Linkage to one of the four loci could be demonstrated in more than half of 51 consanguineous families, most of them from the Mediterranean basin. All four loci could be excluded in the others, implying further genetic heterogeneity in this disorder. Multipoint linkage analysis gave maximal LOD scores of 11.25 at locus D19S566 and 8.53 at locus D3S3564.

Common deletion of mitochondrial DNA in a 5-year-old girl with failure to thrive, ptosis, ophthalmoplegia and ragged-red fibers.

A girl aged 4 years and 10 months presented with failure to thrive, ptosis, ragged-red fibers and the common 4.9 kb mitochondrial DNA deletion. She had elevated serum lactic and pyruvic acids. The onset was at around 18 months. There were no signs of retinitis, and abnormal renal, liver or pancreatic functions. She later developed mild ophthalmoplegia at 6 years of age. Additional features of chronic progressive external ophthalmoplegia (CPEO) or Kearns-Sayre syndrome (KSS) are the conditions that should be watched and investigated in the long-term follow-up of this girl.

Identification of mutations in the galactose-1-phosphate uridyltransferase (GALT) gene in 16 Turkish patients with galactosemia, including a novel mutation of F294Y. Mutation in brief no. 235. Online.

Classical galactosemia caused by deficiency of galactose-1-phosphate uridyltransferase (GALT) is a severe autosomal recessive disorder. We report here molecular analysis of 16 unrelated Turkish galactosemia index cases without GALT activity. Almost 84% of all mutant alleles were identified in this study. The most common molecular defect observed in the Turkish population was Q188R (replacement of glutamine-188 by arginine) (57%). In order to facilitate the determination of unknown mutations in the entire coding region of GALT, we established an approach based on GALT cDNA synthesis and direct sequencing. We have identified one novel candidate galactosemia mutation, a T-to-A transversion at the codon 294 (F294Y) in exon 9 in addition to previously reported three missense (M142K K285N, A320T), one stop codon (E340X), and one silent (L218L) mutations in galactosemia patients which reflect considerable genetic heterogeneity in the Turkish population.

Deletion analysis in Turkish patients with spinal muscular atrophy.

Childhood proximal spinal muscular atrophy (SMA) is an autosomal recessive disorder which presents as a severe, intermediate or mild condition. Here we present the molecular analysis of SMA candidate genes, the survival motor neuron gene (SMN), the neuronal apoptosis inhibitory protein gene (NAIP) and the p44 gene. Deletion frequency rate of these candidate genes is 93% in 106 Turkish SMA patients. Various deletion haplotypes by using genotypes of SMN, NAIP and p44 genes are constructed. Haplotype A, which is the deletion of all three involved genes, was found only in the most severe group with an early onset of usually less than 2 months of age.

Clinical observations in autosomal recessive spastic paraplegia in childhood and further evidence for genetic heterogeneity.

Among our 23 families (32 cases) with autosomal recessive hereditary spastic paraplegia (AR-HSP) all presenting in childhood, 9 families had the "pure" form. Occasional patients with this form had upper extremity hyperreflexia, pes cavus and sphincter disturbances, even at the early stages. Fourteen families were classified as the "complicated" types which manifested with mental retardation and cerebellar abnormalities. The evolution and severity was variable, but was generally consistent within families. Carriers (parents) did not manifest any signs. A total of 5 multiplex families with "complicated" type were used to test for a genetic heterogeneity to the region on chromosome 8p12-q13 where the "pure" AR-HSP has been mapped previously. No evidence in favor of linkage was detected in 3 of our families, thus we further supported genetic heterogeneity for AR-HSP.

mtDNA nt3243 mutation, external ophthalmoplegia, and hypogonadism in an adolescent girl.

A 14-year-old girl presented with a 3-month history of easy fatigue and exercise intolerance, especially when climbing stairs. She had a mild ptosis and mild limitation of upward gaze. Her puberty was delayed, and she manifested hypogonadotrophic hypogonadism. Serum lactic and pyruvic acids were elevated. Cranial magnetic resonance imaging was normal. Muscle biopsy documented typical ragged-red fibers. A point mutation at nucleotide 3243 in the tRNALeu(UUR) (typical mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) mutation) was detected in mitochondrial DNA from both blood and muscle tissues, indicating that our patient was suffering from a mitochondrial myopathy. Hypogonadism may be a manifestation of the MELAS nucleotide 3243 mutation.