Screening for congenital hypothyroidism: the significance of threshold limit in false-negative results.

CONTEXT: In our neonatal program, a number of infants with congenital hypothyroidism (CH) had escaped diagnosis, when a spot RIA-TSH value of 20 mU/liter whole blood was used as a cutoff point. OBJECTIVE: The objective of the study was to find out prospectively the additional number of newborns with CH if the TSH cutoff point is lowered to 10 mU/liter. POPULATION AND METHODS: The study included 311,390 screened newborns. The children with CH were followed up for a period of 3 yr. RESULTS: Twenty-eight percent of infants diagnosed with CH had neonatal TSH values between 10 and 20 mU/liter (56 of 200). Forty of 47 infants, who were reevaluated later on (85.1%), suffered permanent CH. A thyroid scintiscan and/or echogram revealed that eight of 40 children (20.0%) had a structural defect, and the remaining (32 of 40) had a functional defect of the thyroid gland without anatomical abnormality; 14 of 32 cases were familial. Eighteen of the 47 reevaluated infants were prematurely born (38.3%) and 15 of these 18 had permanent CH (83.3%). The lowering of TSH cutoff point from 20 to 10 mU/liter resulted in a 10-fold increase of recall rate. CONCLUSIONS: A significant number of cases with permanent CH are missed when a TSH threshold of 20 mU/liter is applied. Almost 40% of the missed CH cases were premature. A mild increase of TSH at screening is not a predictor of transient CH. The increase in recall rate constitutes a serious drawback and should be balanced against the possible consequences of thyroid dysfunction at this important developmental stage.

Primary open angle glaucoma due to T377M MYOC: Population mapping of a Greek founder mutation in Northwestern Greece.

BACKGROUND: Mutations in the MYOC gene have been shown to explain 5% of unrelated primary open angle glaucoma (POAG) in different populations. In particular, the T377M MYOC mutation has arisen at least three separate times in history, in Great Britain, India, and Greece. The purpose of this study is to investigate the distribution of the mutation among different population groups in the northwestern region of Greece. MATERIALS AND METHODS: We explored the distribution of the "Greek" T377M founder mutation in the Epirus region in Northwestern Greece, which could be its origin. Genotyping was performed in POAG cases and controls by PCR amplification of the MYOC gene, followed by digestion with restriction enzyme. Statistical analyses were performed by an exact test, the Kaplan-Meier method and the t-test. RESULTS: In the isolated Chrysovitsa village in the Pindus Mountains, a large POAG family demonstrated the T377M mutation in 20 of 66 family members while no controls from the Epirus region (n = 124) carried this mutation (P < 0.001). Among other POAG cases from Epirus, 2 out of 14 familial cases and 1 out of 80 sporadic cases showed the mutation (P = 0.057). The probability of POAG diagnosis with advancing age among mutation carriers was 23% at age 40, and reached 100% at age 75. POAG patients with the T377M mutation were diagnosed at a mean age of 51 years (SD +/- 13.9), which is younger than the sporadic or familial POAG cases: 63.1 (SD +/- 11) and 66.8 (SD +/- 9.8) years, respectively. CONCLUSIONS: The T377M mutation was found in high proportion in members of the Chrysovitsa family (30.3%), in lower proportion in familial POAG cases (14.2%) and seems rare in sporadic POAG cases (1.2%), while no controls (0%) from the Epirus region carried the mutation. Historical and geographical data may explain the distribution of this mutation within Greece and worldwide.

Are GJB2 mutations an aggravating factor in the phenotypic expression of mitochondrial non-syndromic deafness?

Hearing impairment is a frequent condition, and genes have an important role in its etiology. The majority of hearing loss occurs in non-syndromic form, with deafness being the only clinically recognizable feature. More than 60 nuclear genes or loci have been shown to be involved in non-syndromic hearing loss, but mutations in mitochondrial DNA also cause hearing impairment. Mitochondrial DNA mutations usually lead to progressive hearing loss with an age of onset varying from childhood to early adulthood. It is interesting to note that there is a great variability among phenotypes between individuals harboring the same mitochondrial mutation, even within the same family, and the phenotype may range from profound deafness to completely normal hearing. In the past years, the debate on mitochondrial mutations has been about the penetrance, the tissue specificity and the mechanisms of modifier genes that can modulate the severity of the phenotypic expression of the deafness-associated mitochondrial DNA mutations. Here we summarize evidence regarding modifying genes, and we discuss the effect of the coexistence of mitochondrial and GJB2 mutations in families reported to date.

Hypothesizing an ancient Greek origin of the GJB2 35delG mutation: can science meet history?

One specific mutation of the GJB2 gene that encodes the connexin 26 protein, the 35delG mutation, has become a major interest among scientists who focus on the genetics of nonsyndromic hearing loss. The mutation accounts for the majority of GJB2 mutations detected in Caucasian populations and represents one of the most frequent disease mutations identified so far. The debate was so far between the arguments whether or not the 35delG mutation constitutes a mutational hot-spot or a founder effect; however, it was recently clarified that the latter seems the most likely. In an attempt to explore the origin and propagation of the 35delG mutation, several groups have reported the prevalence of the mutation and the carrier rates in different populations worldwide. It is now certain that the theory of a common founder prevails and that the highest carrier frequencies of the 35delG mutation are observed in southern European populations, giving rise to a discussion regarding the origin of the 35delG mutation. In this study, we discuss data previously published by our and other groups and also compare the haplotype distribution of the mutation in southern Europe, trying to understand the pathways of science and history and the conflict between them.

Easy, rapid, and cost-effective methods for identifying carriers of recurrent GJB2 mutations causing nonsyndromic hearing impairment in the Greek population.

A variety of techniques have been developed for screening the GJB2 gene for known and unknown mutations, especially the most common mutation in the Caucasian population, the c.35delG. Other mutations that have been so far characterized in the GJB2 gene seem to have different geographical distributions, and therefore there is an interest in identifying recurrent mutations specific for each population and developing easy and rapid screening techniques. Here we present easy screening protocols for already identified recurrent mutations in the Greek population. Developing easy, rapid, and cost-effective screening methods will facilitate the detection of GJB2 recurrent mutation carriers, at large, in the Greek population.

Serum copper and zinc levels in healthy greek children and their parents.

The aim of this study was to investigate whether there is a correlation between copper (Cu) and zinc (Zn) levels in children and their parents, considering their nutritional habits. Cu and Zn concentrations were measured by flame atomic absorption spectrophotometry in the serum of 66 healthy children, aged 3-14 years, and their parents, residing in a region of Greece (Thrace). Cu levels were higher in mothers than those in fathers, but they were lower in both parents than those in children. They also tended to decrease with age in both parents and children, whereas Zn levels significantly increased with age in children. There was a positive correlation between children's and mothers' Zn levels, as well as children's and both parents' Cu levels. Children used to eat meat, fish, vegetables, and legumes as frequently as their parents, but they were consuming more eggs, milk, and fruits than the latest. Regarding parents' diet, higher Zn levels were depended on the consumption of meat and milk, whereas higher Cu levels were depended on the consumption of milk. Consequently, children's Cu and Zn levels are related to their parents' levels, which can be influenced by their nutritional habits.

The A1555G mitochondrial DNA mutation in Greek patients with non-syndromic, sensorineural hearing loss.

Mitochondrial DNA mutations are undoubtedly a factor that contributes to sensorineural, non-syndromic deafness. One specific mutation, the A1555G, is associated with both aminoglycoside-induced and non-syndromic hearing impairment. The mutation is considered to be the most common of all mitochondrial DNA deafness-causing mutations but its frequency varies between different populations. Here we report on the first large screening of the A1555G mitochondrial DNA mutation in the Greek population. The aim of this study was to determine the frequency of the A1555G mutation in Greek sensorineural, non-syndromic deafness patients, with childhood onset. We screened 478 unrelated Greek patients with hearing loss of any degree and found two individuals harboring the A1555G mutation (0.42%). Both cases had been subjected to aminoglycosides. They were prelingual, familial and homoplasmic for the A1555G mutation. One of the cases was also found heterozygous for the frequent GJB2 35delG mutation, while the other case was negative. The A1555G mutation seems to be less common than in other European populations.

Investigating the impact of the Down syndrome related common MTHFR 677C>T polymorphism in the Danish population.

Chromosomal aneuploidy consists the leading cause of fetal death in our species. Around 50% of spontaneous abortions until 15 weeks of gestational age are chromosomally aneuploid, with trisomies accounting for 50% of the abnormal abortions. Trisomy 21 is the most common chromosome abnormality in liveborns and is usually the result of nondisjunction of chromosome 21 in meiosis in either oogenesis or spermatogenesis. To investigate the relationship between folate metabolism and Down syndrome (DS) in a Danish population, we analyzed the common 677C>T genetic polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene. Our cohort consisted of 181 mothers of children with DS versus 1,084 healthy controls. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to examine the MTHFR 677C>T polymorphism. No significant association between the polymorphism and the risk for DS was found. We conclude that the common MTHFR 677C>T polymorphism is not likely to be a maternal risk factor for DS in our cohort and that the difference to previous studies can probably be explained by small sample size or geographic variation in gene polymorphisms involving gene-nutritional or gene-gene or gene-nutritional-environmental factors.

Early effects of sodium valproate monotherapy on serum paraoxonase/arylesterase activities.

OBJECTIVE: Valproic acid (VPA) treatment and paraoxonase1/arylesterase (PON1/Aryl) activities are related to the production of free radicals. Our aim was to study the PON1/Aryl activities in children on VPA therapy. MATERIAL AND METHODS: Thirty-two children with seizures and 30 healthy child volunteers took part. Ill children underwent the common laboratory tests, as well as total antioxidant status (TAS), total oxidant status (TOS), lipid profile, liver enzymes and PON1/Aryl activities pre- and post-60 days on VPA therapy (30 mg/kg/24 h), whereas the healthy children were tested just once. RESULTS: None of the studied biochemical parameters differed between volunteers and children with seizures pretreatment. Liver enzymes, lipids and TOS levels (124+/-30 versus 580+/-40 micromol/L; p<0.001) were significantly elevated, whereas the activities of PON1/Aryl (146+/-43 versus 118+/-40 U/mL/min 120+/-42 versus 98+/-38 KU/mL/min; p<0.01) and TAS levels (436+/-42 versus 288+/-39 micromol/L; p<0.001) were decreased in children after treatment. Additionally, strong negative correlations were found between PON1/Aryl activities, liver enzymes, TOS (r = -0.69) and VPA levels (r = -0.57), whereas PON1/Aryl activities correlated positively with TAS, HDL and Apo A-I in all groups. CONCLUSIONS: Serum PON1/Aryl activities were decreased after 60 days on VPA treatment, probably due to liver dysfunction and free radicals production by VPA, without excluding the possibility of a direct action of the drug on the enzymes.

Strong linkage disequilibrium for the frequent GJB2 35delG mutation in the Greek population.

Approximately one in 1,000 children is affected by severe or profound hearing loss at birth or during early childhood (prelingual deafness). Up to 40% of congenital, autosomal recessive, severe to profound hearing impairment cases result from mutations in a single gene, GJB2, that encodes the connexin 26 protein. One specific mutation in this gene, 35delG, accounts for the majority of GJB2 mutations detected in Caucasian populations. Some previous studies have assumed that the high frequency of the 35delG mutation reflects the presence of a mutational hot spot, while other studies support the theory of a common founder. Greece is among the countries with the highest carrier frequency of the 35delG mutation (3.5%), and a recent study raised the hypothesis of the origin of this mutation in ancient Greece. We genotyped 60 Greek deafness patients homozygous for the 35delG mutation for six single nucleotide polymorphisms (SNPs) and two microsatellite markers inside or flanking the GJB2 gene. The allele distribution in the patients was compared to 60 Greek normal hearing controls. A strong linkage disequilibrium was found between the 35delG mutation and markers inside or flanking the GJB2 gene. Furthermore, we found a common haplotype with a previous study, suggesting a common founder for the 35delG mutation.

Sudden hearing loss in a family with GJB2 related progressive deafness.

Mutations of GJB2, the gene encoding connexin 26, have been associated with prelingual, sensorineural hearing loss of mild to profound severity. One specific mutation, the 35delG, has accounted for the majority of mutations detected in the GJB2 gene in Caucasian populations. Recent studies have described progression of hearing loss in a proportion of cases with GJB2 deafness. We report an unusual family with four 35delG homozygous members, in which the parents were deaf-mute whilst both children had a postlingual progressive hearing loss. Furthermore, the son suffered from sudden hearing loss.

Cohen syndrome resulting from a novel large intragenic COH1 deletion segregating in an isolated Greek island population.

Cohen syndrome, caused by mutations in the COH1 gene, is an autosomal recessive disorder consisting of mental retardation, microcephaly, growth delay, severe myopia, progressive chorioretinal dystrophy, facial anomalies, slender limbs with narrow hands and feet, tapered fingers, short stature, kyphosis and/or scoliosis, pectus carinatum, joint hypermobility, pes calcaneovalgus, and, variably, truncal obesity. Here, we describe the clinical and molecular findings in 14 patients from an isolated Greek island population. The clinical phenotype was fairly homogeneous, although microcephaly was not constant, and some patients had severe visual disability. All patients were homozygous for a novel intragenic COH1 deletion spanning exon 6 to exon 16, suggesting a founder effect. The discovery of this mutation has made carrier detection and prenatal diagnosis possible in this population.

Pathologic, radiographic and molecular findings in three fetuses diagnosed with HEM/Greenberg skeletal dysplasia.

BACKGROUND: Greenberg skeletal dysplasia is a very rare, autosomal recessive, in utero, lethal chondrodystrophy for which only eight index cases of diverse ethnic origin have been reported so far. The defect is associated with a defect in cholesterol biosynthesis and due to mutations in the gene encoding the lamin B receptor (LBR). METHODS: A familial case of three fetuses of a consanguineous Greek couple is presented including prenatal, physical, radiographic, histopathologic, and molecular genetic findings. RESULTS: The tentative diagnosis of Greenberg skeletal dysplasia based on pathological findings was confirmed by the identification of a homozygous, N547D amino acid substitution in the LBR gene in the third affected fetus. CONCLUSION: The present case represents the ninth described case of Greenberg dysplasia and the second case of Greek origin. The characteristic 'moth-eaten' radiographic appearance is already seen at 13 weeks' gestational age.

Melatonin and immunomodulation: connections and potential clinical applications.

Melatonin is the main hormone secreted by the pineal gland in the human brain. It has a strong impact on the sleep-wake cycle and is considered a general modulator of the human circadian rhythm. Apart from these well-established properties, melatonin possesses immunomodulatory, antioxidative and antiinflammatory properties. The potential ability of this hormone to act synergistically with several cytokines by enhancing their antitumoral activity and dramatically decreasing their adverse effects has placed melatonin among the new and promising agents in cancer immunotherapy. The use of the neurohormone alone or in combination with cytokines and traditional chemotherapeutic drugs is currently under vigorous investigation. Experimental and clinical trials have already depicted some of the immunomodulatory and antitumor effects of melatonin, delineating the need for further research in this field.

Erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in subjects with methylenetetrahydrofolate reductase (MTHFR) 677 C-->T genotype and moderate hyperhomocysteinaemia. The role of L-phenylalanine and L-alanine.

BACKGROUND: Increased homocysteine (Hcy) blood levels are correlated with vascular and neurological problems. The aim of our study was to investigate erythrocyte membrane Na(+),K(+)-ATPase and Mg(2+)-ATPase activities in patients with methylenetetrahydrofolate reductase (MTHFR) 677 C-->T genotype. METHODS: Blood was obtained from 25 patients before and after folic acid supplementation and from controls (n=30) once. Plasma folate, vitamin B(12) and total antioxidant status (TAS) were measured using commercial kits, Hcy was determined by HPLC and membrane enzyme activities were measured spectrophotometrically. RESULTS: Mg(2+)-ATPase remained unaltered. Membrane Na(+),K(+)-ATPase activity was remarkably increased in patients (0.77+/-0.06 micromol Pi/h x mg protein) and decreased to normal levels (0.52+/-0.05 micromol Pi/h x mg protein; p<0.001) after therapy. TAS did not differ significantly before and after treatment. Hcy levels were significantly higher before therapy (25.4+/-2.8 micromol/L) than levels after therapy (12.1+/-2.0 micromol/L; p<0.001) and in controls (10.5+/-2.5 micromol/L, p<0.001). In vitro, L-phenylalanine (Phe) reversed to normal the stimulated enzyme from patients before therapy. In addition, Phe incubation of the Hcy activated membrane Na(+),K(+)-ATPase from controls resulted in restoration of its activity, whereas L-alanine (Ala) incubation protected the enzyme from Hcy activation. CONCLUSIONS: The increased membrane Na(+),K(+)-ATPase activity may be due to high -SH group Hcy levels. In vitro, Phe reversed the increase in enzyme activity induced by Hcy in controls, as well as the stimulated membrane enzyme in untreated patients. Ala protected the enzyme from Hcy action.

Calcium and vitamin D metabolism in hypocalcemic vitamin D-resistant rickets carriers.

BACKGROUND/AIMS: Hypocalcemic vitamin D-resistant rickets (HVDRR) is a rare monogenic autosomal recessive disorder associated with mutations in the gene of the vitamin D receptor (VDR), the mediator of 1,25(OH)2D3 action. Although many investigations have discussed the clinical manifestations and molecular etiology of this disease, only a few have investigated the biochemical and hormonal status of heterozygous HVDRR. The aim of the current work was to investigate the profile of selected biochemical and hormonal parameters related to the vitamin D endocrine system in a large number of HVDRR heterozygotes. METHODS: 67 relatives of 2 HVDRR patients, all members of an extended Greek kindred of five generations with a common ancestor, were included in the study. Direct sequencing was used to identify VDR gene mutations. Serum Ca, P, 25(OH)D, iPTH, and 1,25(OH)2D levels were determined in all members of the kindred. RESULTS: DNA analysis of the participants led to the design of two study groups: the HVDRR carriers (24) and the control subjects (43). Our results showed elevated circulating serum levels of 1,25(OH)2D3 and lower levels of PTH than their age- and sex-matched controls. No hypocalcemia or hypophosphatemia were detected in HVDRR carriers. CONCLUSIONS: Our findings suggest that HVDRR carriers may have compensatory elevated serum levels of 1,25(OH)2D3 through which they restrain PTH secretion. The study of HVDRR carriers could be a useful tool for the investigation of the vitamin D endocrine system.

Erythrocyte membrane acetylcholinesterase activity in subjects with MTHFR 677C-->T genotype.

BACKGROUND: Increased homocysteine (Hcy) blood levels are correlated with vascular and neurological problems. AIM: The aim of the present study was to investigate erythrocyte membrane acetylcholinesterase (AChE) activity in subjects with the MTHFR C677T genotype in relation to Hcy. METHODS: Blood was obtained from 22 individuals with the MTHFR C677T genotype before and after folic acid supplementation and once from controls (n = 30). Plasma folate, vitamin B(12) and total antioxidant status (TAS) were measured with commercial kits, Hcy by a HPLC method and membrane enzyme activity spectrophotometrically. RESULTS: In MTHFR C677T carriers, AChE activity was significantly higher (4.20 +/- 0.12 x mg protein) and decreased to normal levels (3.14 +/- 0.10 x mg protein; p < 0.001) after therapy. TAS differed slightly before and after treatment. Hcy levels were significantly higher before (22.4 +/- 2.8 microM) compared to after (12.1 +/- 2.0 microM; p < 0.001) therapy and compared to controls (10.5 +/- 2.5 micromol/L; p < 0.001). In an in vitro study, incubation of Hcy-activated membrane AChE from controls with phenylalanine resulted in restoration of activity, but failed to reverse the stimulated enzyme from hyperhomocysteinaemic MTHFR C677T subjects before therapy. Alanine incubation protected the enzyme from Hcy activation in controls. CONCLUSIONS: Increased membrane AChE activity may be due to high Hcy levels. In vitro, phenylalanine reversed the Hcy activation of the membrane enzyme from controls and alanine protected it from Hcy action.

The effect of aspartame metabolites on human erythrocyte membrane acetylcholinesterase activity.

Studies have implicated aspartame (ASP) with neurological problems. The aim of this study was to evaluate acetylcholinesterase (AChE) activity in human erythrocyte membranes after incubation with the sum of ASP metabolites, phenylalanine (Phe), methanol (met) and aspartic acid (aspt), or with each one separately. Erythrocyte membranes were obtained from 12 healthy individuals and were incubated with ASP hydrolysis products for 1 h at 37 degrees C. AChE was measured spectrophotometrically. Incubation of membranes with ASP metabolites corresponding with 34 mg/kg, 150 mg/kg or 200 mg/kg of ASP consumption resulted in an enzyme activity reduction by -33%, -41%, and -57%, respectively. Met concentrations 0.14 mM, 0.60 mM, and 0.80 mM decreased the enzyme activity by -20%, -32% or -40%, respectively. Aspt concentrations 2.80 mM, 7.60 mM or 10.0 mM inhibited membrane AChE activity by -20%, -35%, and -47%, respectively. Phe concentrations 0.14 mM, 0.35 mM or 0.50mM reduced the enzyme activity by -11%, -33%, and -35%, respectively. Aspt or Phe concentrations 0.82 mM or 0.07 mM, respectively, did not alter the membrane AChE activity. It is concluded that low concentrations of ASP metabolites had no effect on the membrane enzyme activity, whereas high or toxic concentrations partially or remarkably decreased the membrane AChE activity, respectively. Additionally, neurological symptoms, including learning and memory processes, may be related to the high or toxic concentrations of the sweetener metabolites.