Intra-individual variability in genetic and environmental models of attention-deficit/hyperactivity disorder.

The frequent observation of intra-individual variability (IIV) in the expression of ADHD symptoms suggest that IIV is an integral component of the disorder. We tested IIV in ADHD-like phenotype from five different studies of rodent models of ADHD, including studies with Spontaneous Hypertensive Rats (SHR/NCrl and SHR/N), Wistar-Kyoto Hyperactive Rats (WKHA/N), Wistar-Kyoto Hypertensive rat (WKHT), PCB-126 and -153-treated Lewis rats and behaviorally normal Wistar/Mol, Wistar-Kyoto (WKY/N and WKY/NMol), and untreated Lewis rats. Averages of the absolute residual deviation of ADHD-like behavior from individual means ("individual phenotypic dispersion," PD(i)) were used to represent IIV in the fixed-interval (FI) and extinction (EXT) phases of operant behavioral activity. Across all studies, SHR rats had higher PD(i) than WKY rats (P < 0.0001) for all ADHD-like traits, and higher PD(i) for hyperactivity than WKHT and WKHA/N rats. Male SHR rats in particular had higher PD(i) for hyperactivity than male or female WKYs, SHR females for EXT hyperactivity, and higher dispersion for inattention than WKY females. These findings strongly suggest the genetic control of IIV, and suggest that the SHR may be a useful model for the identification of genes for IIV in human ADHD. These findings also obliquely support the SHR as a useful model for ADHD overall.

Low red blood cell glutathione reductase and pyridoxine phosphate oxidase activities not related to dietary riboflavin: selection by malaria?

This study was designed to confirm that low dietary riboflavin does not contribute to the flavin-deficient red blood cells commonly found in subjects in Ferrara Province, northern Italy. In this area it is primarily an inherited characteristic believed to have been selected for by malaria, which was endemic from the 12th century. In parallel with assessment of daily riboflavin intake (DRI), flavin adenine dinucleotide-dependent glutathione reductase (EGR) and flavin mononucleotide-dependent pyridoxine phosphate oxidase (PPO) were measured in beta-thalassemic heterozygotes, their normal relatives, and normal spouses (representative of the normal population). In all of these groups there is a high incidence of deficiency of these flavin enzymes. We found that the majority had an adequate riboflavin intake and there was no significant correlation of EGR and PPO activities with DRI. Thus, interpretation of low EGR activity is discussed with reference to studies of EGR done to detect nutritional riboflavin deficiency in countries where there is malnutrition and endemic malaria.

Rapid uptake and clearance of pyridoxine by red blood cells in vivo.

There is rapid pyridoxine (PN) uptake in vitro into red cells where it is converted to pyridoxal (PL) forms. To assess uptake in vivo, the equivalent of 48.6 and 118 mumol PN were given intravenously to a healthy female subject. Vitamin B-6 compounds were measured by a Lactobacillus casei microbiological assay in blood taken 1-60 min after injection. After either injection there was a considerable amount of PN in the red cells at 1 min but by 3 min a large amount of that PN had disappeared, mostly unaccounted for by conversion to PL forms. Although there was considerably less PN at 1 min in both red cells and plasma after the smaller injection, in the next 2 min similar amounts had left the red cells (4.59 and 4.30 mumol) and plasma (9.37 and 10.09 mumol), respectively, after the injections. Red cells, as well as plasma, may be transporting PN to other sites of metabolism in tissues.

Pulpitis increases the proportion of atypical nodes of Ranvier in human dental pulp axons without a change in Nav1.6 sodium channel expression.

Studies show a change in sodium channel (NaCh) expression after inflammatory lesions, and this change is implicated in the generation of pain states. We are using the extracted human tooth to study NaCh expression and here examine the expression of the major NaCh isoform located at nodes of Ranvier, Na(v)1.6, in normal and painful samples. Pulpal sections were double-labeled with human-specific Na(v)1.6 antibody and caspr antibody (paranodal protein to identify nodes). Confocal microscopy was used to obtain a z-series of optically-sectioned images of axon bundles surrounded by inflammatory cells in painful samples and of similar regions within the coronal pulp of normal samples. Nodes contained within these images were classified as typical or atypical as based on caspr staining relationships, and NIH ImageJ software was used to quantify the size and immunofluorescence staining intensity of Na(v)1.6 accumulations at these nodal sites. Results show no significant difference in the size or immunofluorescence staining intensity of Na(v)1.6 nodal accumulations located at either typical or atypical nodal sites (heminodes and split nodes) within axons in normal samples when compared to painful samples (n=9/each group). In contrast, there was a highly significant decrease in the proportion of typical nodal sites and an increase in atypical nodal sites in painful samples when compared to normal samples. The unchanged expression of Na(v)1.6 contrasts to our previous finding that showed an increased expression of Na(v)1.7 at both typical and atypical nodal sites within painful samples. Together, these findings suggest there is not a simple replacement of one isoform with another, but rather an increased co-expression of multiple isoforms at both intact and remodeling/demyelinating (atypical) nodal sites within the painful dental pulp. The resultant heterogeneous population of isoforms may produce unique axonal excitability properties that could contribute to spontaneous pain sensations that are common in toothache.

Maternal genetic effects on adaptive divergence between anadromous and resident brook charr during early life history.

The importance of directional selection relative to neutral evolution may be determined by comparing quantitative genetic variation in phenotype (Q(ST)) to variation at neutral molecular markers (F(ST)). Quantitative divergence between salmonid life history types is often considerable, but ontogenetic changes in the significance of major sources of genetic variance during post-hatch development suggest that selective differentiation varies by developmental stage. In this study, we tested the hypothesis that maternal genetic differentiation between anadromous and resident brook charr (Salvelinus fontinalis Mitchill) populations for early quantitative traits (embryonic size/growth, survival, egg number and developmental time) would be greater than neutral genetic differentiation, but that the maternal genetic basis for differentiation would be higher for pre-resorption traits than post-resorption traits. Quantitative genetic divergence between anadromous (seawater migratory) and resident Laval River (Québec) brook charr based on maternal genetic variance was high (Q(ST) > 0.4) for embryonic length, yolk sac volume, embryonic growth rate and time to first response to feeding relative to neutral genetic differentiation [F(ST) = 0.153 (0.071-0.214)], with anadromous females having positive genetic coefficients for all of the above characters. However, Q(ST) was essentially zero for all traits post-resorption of the yolk sac. Our results indicate that the observed divergence between resident and anadromous brook charr has been driven by directional selection, and may therefore be adaptive. Moreover, they provide among the first evidence that the relative importance of selective differentiation may be highly context-specific, and varies by genetic contributions to phenotype by parental sex at specific points in offspring ontogeny. This in turn suggests that interpretations of Q(ST)-F(ST) comparisons may be improved by considering the structure of quantitative genetic architecture by age category and the sex of the parent used in estimation.

Sex-linked quantitative trait loci for thermotolerance and length in the rainbow trout.

We hypothesized that correlation between growth traits and upper thermal tolerance (UTT) in rainbow trout (Oncorhynchus mykiss) might be explained by quantitative trait loci (QTL) localized to the same linkage groups. Microsatellites on three autosomal linkage groups carrying UTT QTL in rainbow trout were tested for associations with fork length (FL) and condition factor (K) in half-sib families of outbred rainbow trout and in backcrosses of trout lines selected on UTT. Additionally, we used a sex-linked microsatellite (OmyFGT19TUF) to test for marker-trait associations at the sex chromosomes. The sex-linked marker OmyFGT19TUF was significantly associated with FL and UTT, accounting for up to 9.6% and 9.7% of variance in these traits, respectively. Male advantages in FL (and, to a lesser extent, UTT) relative to their female sibs were dependent on the origin of the Y chromosome and thus varied among grandsire lines. However, males had higher K in a manner unrelated to Y chromosomal origin, suggesting a partially sex-limited expression of this trait. Omy325UoG was significantly associated with K in one of the outbred half-sib families, but no other significant autosomal marker-trait associations were detected. Our findings illustrate minor evidence that correlation between UTT and FL is partially determined by one or more sex-chromosomal QTL.

Utilization of red-cell FAD by methaemoglobin reductases at the expense of glutathione reductase in heterozygous beta-thalassaemia.

FAD-dependent methaemoglobin reductases (MHR) were studied in red cells in heterozygous beta-thalassaemia to investigate how they related to low FAD-dependent glutathione reductase (GR). In contrast to GR, MHR activities were usually normal or increased. In particular, whether expressed in relation to haemoglobin or number of red cells, NADPH-MHR activity was markedly increased in most subjects, probably being a response to increased oxidative stress. Oral riboflavin had no effect on MHR activities, indicating saturation with FAD even though GR was deficient. A strong correlation between percent stimulation of GR by FAD and NADPH-MHR activity indicates that FAD is utilized by MHR at the expense of GR. This could be an important influence on GR in heterozygous beta-thalassaemia. Thus, the low activity resulting from an inherited deficiency of FAD is decreased further.

The effect of riboflavin on red-cell vitamin B6 metabolism and globin synthesis.

Red-cell conversion rate of pyridoxine to pyridoxal phosphate, and globin synthesis were measured before and after oral riboflavin in a patient with heterozygous beta-thalasaemia in 3 separate trials. In this patient a very slow B6 conversion rate increased to normal on each occasion after riboflavin, and there was a marked increase in synthesis of alpha and beta globin chains but no change in beta/alpha ratio. This was confirmed in a similar patient after a single trial of oral riboflavin. In 5 control subjects after a single trial of riboflavin the red-cell pyridoxine conversion rate increased whether the initial rate was fast or slow, and there was a considerable increase in globin synthesis in 3 of these.

Low red cell activity of pyridoxine (pyridoxamine) phosphate oxidase and glutathione reductase associated with thalassaemia.

It was demonstrated in heterozygous alpha 1- and beta-thalassaemia, that the slow rate of red-cell metabolism of vitamin B6, previously shown to be inherited, is regulated by the FMN-dependent pyridoxine (pyridoxamine) phosphate oxidase, as in control subjects. In this study, 60% of the patients with thalassaemia had a low B6 oxidase activity. An inverse correlation with the stimulation of the FAD-dependent glutathione reductase activity by FAD confirmed that red-cell riboflavin status was responsible. The inherited nature and lack of signs of nutritional riboflavin deficiency led to the conclusion that this was the result of a slow rate of red-cell metabolism of riboflavin. Stimulation of glutathione reductase activity by FAD correlated inversely with its basic activity in thalassaemia and control subjects. There was a high incidence of a low activity of this enzyme per red cell in patients with thalassaemia. The possibility that a low activity of glutathione reductase and a slow metabolism of B6 and riboflavin in the red-cell might play a part in the degree of severity of the thalassaemic disease is discussed.

Deficiency of two red-cell flavin enzymes in a population in Sardinia: was glutathione reductase deficiency specifically selected for by malaria?

In two areas in Italy where malaria was endemic--in the Po delta and Maremma on the west coast--we have found a high prevalence of an inherited flavin-deficient red cell in the normal population, suggesting selection by malaria. This study in Sardinia enabled a direct comparison of red-cell activities of FAD-dependent glutathione reductase (EGR) and FMN-dependent pyridoxine phosphate (PNP) oxidase in an ethnically homogeneous population, between two coastal villages where malaria was endemic from 300 B.C. and two mountain villages with no history of malaria. Both enzyme activities were significantly lower on the coast, and it did not seem that this could be explained by possible small differences in dietary riboflavin. As was thought to be the case in Ferrara and Grosseto, it is probable that a genetically controlled flavin-deficient red cell was selected for by malaria. Low EGR apoenzyme activity was more common on the coast, usually explaining the accompanying low basic EGR activity, and may also have been selected for by malaria. This adds to evidence from others that the mechanism of defence of a flavin-deficient red cell against malaria may be through EGR deficiency. It could also play a part in the protection given by heterozygous beta-thalassemia. The multifactorial protection of the population against malaria is discussed.

Red-cell GSH regeneration and glutathione reductase activity in G6PD variants in the Ferrara area.

Red-cell studies were carried out on three groups of G6PD-deficient subjects with different G6PD variants from the Ferrara area of Northern Italy. Red-cell GSH and activities of G6PD, glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. A method was developed to measure red-cell GSH regeneration after oxidation of endogenous GSH in whole blood by diamide and only this clearly distinguished the variants from each other and from normal. Regeneration by 1 h was lowest in the Mediterranean variant, 0-10.2% in contrast to 93-98% in normal. A predisposition to a haemolytic crisis after ingestion of fava beans was not clearcut, but subjects appeared to be at risk if GSH regeneration at 1 h was less than 30% of the endogenous level, and red-cell FAD+ was very high indicated by high in vitro GR activity and inhibition by added FAD+. It is suggested that the most informative tests in G6PD deficiency are measurements of GSH regeneration in intact red cells plus GR activity and/or red-cell flavin compounds.

Red-cell metabolism of pyridoxine in controls and beta-Thalassaemia in Ferrara, Northern Italy.

The rate of red-cell metabolism of pyridoxine to pyridoxal phosphate was measured in control subjects and patients with homozygous and heterozygous beta-thalassaemia from Ferrara, Northern Italy, and in British control subjects of Anglo-Saxon origin. A high incidence of a slow rate of B6 metabolism was found in beta-thalassaemia in Ferrara similar to that found previously in Cypriots living in London. Of particular interest was a much slower rate in control subjects from Ferrara than in British control subjects of Anglo-Saxon origin. The suggestion that a high incidence of a slow red-cell metabolism of B6 is the result of selection by malaria, whether associated with thalassaemia or not, is considered.

Quantitative trait loci for upper thermal tolerance in outbred strains of rainbow trout (Oncorhynchus mykiss).

The expression of three putative QTL for upper thermal tolerance (UTT) was examined in two strains of outbred rainbow trout unselected for this trait using simple-sequence repeat (SSR; microsatellite) markers associated with UTT in backcrosses of lines selected on this trait. Two-way diallel lots in the third generation of an outbred pedigree were exposed to an acute thermal challenge. QTL detection was performed separately by each second-generation parent within each diallel lot, incorporating the effects of full sib families and correlated traits. Inheritance of different alleles at the SSR Ssa20.19NUIG from the sire 93-32-1 was strongly associated with the thermal tolerance of his half sib progeny, explaining 7.5% of their phenotypic variance in this trait. A hierarchical linear model incorporating allelic inheritance from all four grandsires of the experimental diallels (in addition to family specific and covariate trait effects) was also used to detect associations between the SSR and thermal tolerance in their third-generation grandprogeny. Ssa20.19NUIG was strongly associated with thermal tolerance in the grandprogeny of the grandsire G(0)SVM2. The generally stronger marker-trait associations found in male parents may be partially due to reduced chromosomal recombination rates in male salmonids compared to females. These results indicate the effects of a QTL on a fitness-related trait in unselected populations of rainbow trout.

Effects of genetic sex and genomic background on epistasis in rainbow trout (Oncorhynchus mykiss).

Epistasis among quantitative trait loci (QTL) for survival (upper thermal tolerance, UTT) and morphological (fork length, FL and condition factor, K) traits was detected in purestrain and interstrain rainbow trout (Oncorhynchus mykiss) families. One sex-linked (OmyFGT19TUF) and three autosomal (Omy325UoG, Ssa14DU and Ssa20.19NUIG; linkage groups B, D and S, respectively) microsatellite loci linked to UTT QTL in this species were used. Within half sib families, significant effects of full sib family on epistasis involving Omy325UoG and OmyFGT19TUF were detected at a rate significantly higher than expected for UTT (p < 0.001*) and FL (p < 0.01*), using results significant at comparisonwise significance thresholds derived from permutational analysis. Measured across half sib families, the phenotype of female genotypic classes was more divergent from the family trait mean than that of males where epistasis involved the sex-linked locus OmyFGT19TUF (p = 0.0176*), and also for means over all families (p = 0.0355*). Female genotypic classes were also more divergent (p = 0.0011 **) from the full sib trait mean where three-way interaction between OmyFGT19TUF, one of the autosomal loci and full sib family was significant, and marginally more divergent for trait means of genotypic classes across all full sib families (p = 0.0856+). There was no evidence that these effects were more pronounced in hybrid F1 families than purestrains.

Is the flavin-deficient red blood cell common in Maremma, Italy, an important defense against malaria in this area?

There is a high prevalence of a familial flavin-deficient red blood cell in Ferrara province in the Po delta in northern Italy, believed to have been selected for by malaria which was endemic from the 12th century. In the present study, activities of FAD-dependent red-cell glutathione reductase (EGR) in the Grosseto area of Maremma on the west coast of Italy where malaria was endemic from 300 B.C. are compared both with activities in the Ferrara area and with activities where there was no history of endemic malaria--in the Florence area and in London in people of Anglo-Saxon origin. EGR activities were similar in Grosseto and Ferrara and were significantly lower than in Florence and London. As previously found in Ferrara, low EGR activity in Grosseto was shown to be unrelated to low dietary riboflavin intake. These findings in Grosseto, suggesting selection by malaria, are particularly interesting because, unlike the situation in Ferrara and most other malarial areas, the prevalence of thalassemia and glucose-6-phosphate dehydrogenase deficiency is very low, and they do not appear to have been selected for in Maremma. It is possible that a flavin-deficient red cell, known to inhibit growth of the malaria parasite, was an important protecting factor in the population of this area over the centuries.

Glutathione reductase activity and its relationship to pyridoxine phosphate activity in G6PD deficiency.

Per cent stimulation of GR activity by FAD in vitro and PNP oxidase activity were measured in G6PD deficiency, heterozygous beta-thalassaemia and controls. It is confirmed that, in contrast to the high stimulation of GR by FAD commonly found in in thalassaemia indicating red-cell deficiency of FAD, and shown here to be greater in the Italian subjects, GR is usually saturated with FAD in G6PD deficiency, leading to high in vitro activity. Unexpectedly, on the other hand, low FMN-dependent PNP oxidase activity due to red-cell deficiency of FMN, confirmed by response to oral riboflavin, was found in the majority of subjects with G6PD deficiency, similar to that found in heterozygous beta-thalassaemia. Whereas this is explained in thalassaemia by an inherited slow red-cell metabolism of riboflavin to FMN, it is suggested that in G6PD deficiency an increased rate of red-cell metabolism of FMN to FAD leads to the low FMN and high FAD. When G6PD deficiency occurs with heterozygous beta-thalassaemia, GR is usually saturated with FAD as in G6PD deficiency alone, unless there is an inherited, very slow red-cell metabolism of riboflavin to FMN. The part played by GR in haemolytic crises in G6PD deficiency is discussed.

Genetic and other influences on red-cell flavin enzymes, pyridoxine phosphate oxidase and glutathione reductase in families with beta-thalassaemia.

In 18 beta-thalassaemia families from the Ferrara area the incidence of an inherited low flavin mononucleotide (FMN)-dependent pyridoxine phosphate (PNP) oxidase activity, a sensitive indicator of red-cell FMN deficiency, is higher in related members in these families than in the unrelated spouses and controls subjects without family history of thalassaemia. This suggests slower red-cell riboflavin metabolism in thalassaemia families, which may have resulted from selection in combination with thalassaemia by malaria. However, there was a markedly higher incidence of red-cell flavin adenine dinucleotide (FAD) deficiency in thalassaemia heterozygotes than in their normal relatives. This was indicated by higher stimulation of FAD-dependent glutathione reductase (GR) activity by FAD and lower GR activity per red cell, and suggests a marked additive effect by thalassaemia on the red cell FAD deficiency that results from the inherited slow riboflavin metabolism. There is evidence that diversion of FAD to other FAD-dependent enzymes might be an important factor.

Family studies of red-cell metabolism of pyridoxine in beta-thalassaemia.

Red-cell conversion of pyridoxine to pyridoxal phosphate was measured in 12 families with thalassaemia and 2 normal families. A strong familial pattern was demonstrated and the evidence suggested that the rate of red-cell conversion of pyridoxine is an independently inherited characteristic.