ADCK3 mutations with epilepsy, stroke-like episodes and ataxia: a POLG mimic?

BACKGROUND AND PURPOSE: Defects of coenzyme Q10 (CoQ10) metabolism cause a variety of disorders ranging from isolated myopathy to multisystem involvement. ADCK3 is one of several genes associated with CoQ10 deficiency that presents with progressive cerebellar ataxia, epilepsy, migraine and psychiatric disorders. Diagnosis is challenging due to the wide clinical spectrum and overlap with other mitochondrial disorders. METHODS: A detailed description of three new patients and one previously reported patient from three Norwegian families with novel and known ADCK3 mutations is provided focusing on the epileptic semiology and response to treatment. Mutations were identified by whole exome sequencing and in two measurement of skeletal muscle CoQ10 was performed. RESULTS: All four patients presented with childhood-onset epilepsy and progressive cerebellar ataxia. Three patients had epilepsia partialis continua and stroke-like episodes affecting the posterior brain. Electroencephalography showed focal epileptic activity in the occipital and temporal lobes. Genetic investigation revealed ADCK3 mutations in all patients including a novel change in exon 15: c.T1732G, p.F578V. There was no apparent genotype-phenotype correlation. CONCLUSION: ADCK3 mutations can cause a combination of progressive ataxia and acute epileptic encephalopathy with stroke-like episodes. The clinical, radiological and electrophysiological features of this disorder mimic the phenotype of polymerase gamma (POLG) related encephalopathy and it is therefore suggested that ADCK3 mutations be considered in the differential diagnosis of mitochondrial encephalopathy with POLG-like features.

A common variant of the latrophilin 3 gene, LPHN3, confers susceptibility to ADHD and predicts effectiveness of stimulant medication.

Attention-Deficit/Hyperactivity Disorder (ADHD) has a very high heritability (0.8), suggesting that about 80% of phenotypic variance is due to genetic factors. We used the integration of statistical and functional approaches to discover a novel gene that contributes to ADHD. For our statistical approach, we started with a linkage study based on large multigenerational families in a population isolate, followed by fine mapping of targeted regions using a family-based design. Family- and population-based association studies in five samples from disparate regions of the world were used for replication. Brain imaging studies were performed to evaluate gene function. The linkage study discovered a genome region harbored in the Latrophilin 3 gene (LPHN3). In the world-wide samples (total n=6360, with 2627 ADHD cases and 2531 controls) statistical association of LPHN3 and ADHD was confirmed. Functional studies revealed that LPHN3 variants are expressed in key brain regions related to attention and activity, affect metabolism in neural circuits implicated in ADHD, and are associated with response to stimulant medication. Linkage and replicated association of ADHD with a novel non-candidate gene (LPHN3) provide new insights into the genetics, neurobiology, and treatment of ADHD.

Meta-analysis of brain-derived neurotrophic factor p.Val66Met in adult ADHD in four European populations.

Attention-deficit hyperactivity disorder (ADHD) is a multifactorial, neurodevelopmental disorder that often persists into adolescence and adulthood and is characterized by inattention, hyperactivity and impulsiveness. Before the advent of the first genome-wide association studies in ADHD, genetic research had mainly focused on candidate genes related to the dopaminergic and serotoninergic systems, although several other genes had also been assessed. Pharmacological data, analysis of animal models and association studies suggest that Brain-Derived Neurotrophic Factor (BDNF) is also a strong candidate gene for ADHD. Several polymorphisms in BDNF have been reported and studied in psychiatric disorders but the most frequent is the p.Val66Met (rs6265G > A) single nucleotide polymorphism (SNP), with functional effects on the intracellular trafficking and secretion of the protein. To deal with the inconsistency raised among different case-control and family-based association studies regarding the p.Val66Met contribution to ADHD, we performed a meta-analysis of published as well as unpublished data from four different centers that are part of the International Multicentre Persistent ADHD CollaboraTion (IMpACT). A total of 1,445 adulthood ADHD patients and 2,247 sex-matched controls were available for the study. No association between the p.Val66Met polymorphism and ADHD was found in any of the four populations or in the pooled sample. The meta-analysis also showed that the overall gene effect for ADHD was not statistically significant when gender or comorbidity with mood disorders were considered. Despite the potential role of BDNF in ADHD, our data do not support the involvement of p.Val66Met in the pathogenesis of this neuropsychiatric disorder.

A coding polymorphism in NALP1 confers risk for autoimmune Addison's disease and type 1 diabetes.

Variants in the gene encoding NACHT leucine-rich-repeat protein 1 (NALP1), an important molecule in innate immunity, have recently been shown to confer risk for vitiligo and associated autoimmunity. We hypothesized that sequence variants in this gene may be involved in susceptibility to a wider spectrum of autoimmune diseases. Investigating large patient cohorts from six different autoimmune diseases, that is autoimmune Addison's disease (n=333), type 1 diabetes (n=1086), multiple sclerosis (n=502), rheumatoid arthritis (n=945), systemic lupus erythematosus (n=156) and juvenile idiopathic arthritis (n=505), against 3273 healthy controls, we analyzed four single nucleotide polymorphisms (SNPs) in NALP1. The major allele of the coding SNP rs12150220 revealed significant association with autoimmune Addison's disease compared with controls (OR=1.25, 95% CI: 1.06-1.49, P=0.007), and with type 1 diabetes (OR=1.15, 95% CI: 1.04-1.27, P=0.005). Trends toward the same associations were seen in rheumatoid arthritis, systemic lupus erythematosus and, although less obvious, multiple sclerosis. Patients with juvenile idiopathic arthritis did not show association with NALP1 gene variants. The results indicate that NALP1 and the innate immune system may be implicated in the pathogenesis of many autoimmune disorders, particularly organ-specific autoimmune diseases.

AIRE variations in Addison's disease and autoimmune polyendocrine syndromes (APS): partial gene deletions contribute to APS I.

Autoimmune Addison's disease (AAD) is often associated with other components in autoimmune polyendocrine syndromes (APS). Whereas APS I is caused by mutations in the AIRE gene, the susceptibility genes for AAD and APS II are unclear. In the present study, we investigated whether polymorphisms or copy number variations in the AIRE gene were associated with AAD and APS II. First, nine SNPs in the AIRE gene were analyzed in 311 patients with AAD and APS II and 521 healthy controls, identifying no associated risk. Second, in a subgroup of 25 of these patients, AIRE sequencing revealed three novel polymorphisms. Finally, the AIRE copy number was determined by duplex quantitative PCR in 14 patients with APS I, 161 patients with AAD and APS II and in 39 healthy subjects. In two Scandinavian APS I patients previously reported to be homozygous for common AIRE mutations, we identified large deletions of the AIRE gene covering at least exon 2 to exon 8. We conclude that polymorphisms in the AIRE gene are not associated with AAD and APS II. We further suggest that DNA analysis of the parents of patients found to be homozygous for mutations in AIRE, always should be performed.

Genetic analyses of dopamine related genes in adult ADHD patients suggest an association with the DRD5-microsatellite repeat, but not with DRD4 or SLC6A3 VNTRs.

Attention deficit hyperactivity disorder (ADHD) is a common and highly heritable psychiatric disorder in children and adults. Recent meta-analyses have indicated an association between genes involved in dopaminergic signaling and childhood ADHD, but little is known about their possible role in adult ADHD. In this study of adults with ADHD, we evaluated the three most commonly studied ADHD candidate genetic polymorphisms; the dopamine receptor D4 (DRD4) exon 3 VNTR repeat, a microsatellite repeat 18.5 kb upstream of the DRD5 locus and the 3'UTR dopamine transporter SLC6A3 (DAT 1) VNTR. We examined 358 clinically diagnosed adult Norwegian ADHD patients (51% males) and 340 ethnically matched controls. We found a nominally significant overall association with adult ADHD for the DRD5 microsatellite marker (P = 0.04), and a trend toward increased risk associated with the 148-bp allele consistent with recent meta-analyses. The strongest overall association (P = 0.02) and increased risk for the 148-bp allele [odds ratio (OR) = 1.27 (95% CI: 1.00-1.61)] were seen in the inattentive and combined inattentive/hyperactive group as previously reported for childhood ADHD. No association was found for the DRD4 or SLC6A3 polymorphisms in this patient sample. In conclusion, our results among adults with a clinical diagnosis of ADHD support an association between ADHD and the DRD5 locus, but not the DRD4 or SLC6A3 loci. It is possible that the latter polymorphisms are associated with a transient form of ADHD with better long-term clinical outcome.

Cold-induced sweating syndrome: a report of two cases and demonstration of genetic heterogeneity.

OBJECTIVES: To characterize the specific autonomic disturbances underlying the cold-induced sweating syndrome (CISS), and to describe a novel genetic variant of this rare recessive disorder. The two not previously reported patients had similar dysmorphic features: abnormal facial appearance, high arched palate, low set rotated ears, flexion deformities of elbows and fingers and scoliosis. Most noticeable were their paradoxical sweat responses: cold ambient temperature induced a profuse sweating over the face, arms and trunk but not over the lower limbs; while in the heat very little sweating occurred primarily on the legs. Testing of autonomic functions demonstrated normal cardiovascular reflexes and postganglionic sympathetic efferent functions. Sural nerve morphology and number of unmyelinated fibers was normal and skin biopsies showed normal appearing eccrine sweat glands. MRI scans revealed no structural brain abnormalities. Oral clonidine, prescribed in one patient, completely suppressed cold-induced sweating. Observed clinical features matched those of two sisters reported from Israel and of two brothers reported from Norway. All six cases presented a similar phenotype. The Norwegian, Israeli and Canadian cases were homozygous or compound heterozygous, respectively, for mutations in the CRLF1 gene on chromosome 19p12 (CISS1). The Australian case, however, had no pathogenic sequence variants in the CRLF1 gene, but was compound heterozygous for mutations in the CLCF1 gene on chromosome 11q13.3 (CISS2). CONCLUSION: The rare cold-induced sweating syndrome is genetically heterogeneous and is probably caused by central and peripheral impairment of sudomotor functions. This is the first detailed report on the clinical consequences of mutations in the CLCF1 gene in humans. Directions for medical therapies are outlined to achieve long term symptom control.

Relative frequency, heterogeneity and geographic clustering of PKU mutations in Norway.

We have analysed 236 Norwegian phenylketonuria (PKU) alleles by a combination of mutation scanning methods, restriction enzyme-based assays and DNA sequencing. Thirty-three different mutations constituted 99.6% of all mutant alleles (only 1 allele remains unidentified), 23 of these have been identified also in other European countries. Twenty were predicted missense mutations, 6 splice mutations, 4 nonsense mutations and 2 deletion mutations and 1 mutation disrupted the start codon. The 8 most common mutations represented 83.5% of the PKU alleles, with single allele frequencies ranging from 5.9 to 15.7%. Four of these mutations (R261Q, R408W, Y414C, and 1VS12nt1) are commonly occurring also in PKU patients in other European countries, while the other 4 (G46S, G272X, F299C, and R408Q) have higher frequencies in Norway than in any other country studied. Six mutations (I65T, L249F, P281L, Y356X, R158Q, and R252W) have frequencies between 0.8% and 2.1%, and 19 mutations were encountered only once. The majority of PKU mutations were found on the same RFLP/VNTR haplotype backgrounds in Norway as in other European populations, suggesting that only a few of the mutations may represent recurrent mutations (< 3.4%). Among 10 mutations only reported for our population, we detected 2 de novo mutations (0.8%) arisen in Norway. From the birthplaces of the probands' grandparents, each mutation seemed to have an individual geographic distribution within Norway, with patterns of local mutation clustering. Our observations are compatible with multiple founder effects and genetic drift for the distribution of PKU mutations within Norway.

Effect of mutations at Cys237 on the activation state and activity of human phenylalanine hydroxylase.

Wild-type recombinant human phenylalanine hydroxylase (wt-hPAH) is activated about 1.5-fold by exposure to alkaline pH (pH 8.5-9.0). In order to study whereas this activation might be related to the activation of the rat enzyme by N-ethylamaleimide-modification of Cys237 [Gibbs and Benkovic (1991) Biochemistry 30, 67951, mutant proteins of hPAH with Cys237 changed to Ser (S) or Glu (D) have been prepared. The mutant forms have high specific activity at pH 7.0 and high affinity for L-Phe, notably for hPAH-C237D, which shows a 3-fold higher activity than L-Phe-activated wt-hPAH and it is not further activated by pre-incubation with L-Phe. Moreover, the emission maximum of the intrinsic fluorescence of hPAH-C237D (lambda(maxem) = 347 nm) resembles that of activated forms of wt-hPAH. However, the activity of this mutant at neutral pH is further activated by exposure to alkaline pH, indicating that activation of wt-hPAH by alkaline pH is not restricted to ionization of Cys237.

Diverse PAH transcripts in lymphocytes of PKU patients with putative nonsense (G272X, Y356X) and missense (P281L, R408Q) mutations.

The majority of mutations in the human phenylalanine hydroxylase (PAH) gene that lead to the recessive disease phenylketonuria (PKU) are believed to affect the activity or stability of the PAH enzyme. In this study we have performed in vivo analyses of lymphocyte PAH mRNA from PKU patients homozygous for the PKU missense mutations P281L and R408Q as well as the nonsense mutations G272X and Y356X. The mutations G272X, P281L and R408Q, which are located outside the consensus splice site sequence, result in transcripts with one or more exons skipped in addition to full-length transcripts. The mutation Y356X results in transcripts with one or more exons skipped, but no full-length transcripts. Our findings question the value of functional and structural predictions of mutations at the protein level without analyses of the corresponding transcript.

Crystallization and preliminary diffraction analysis of a truncated homodimer of human phenylalanine hydroxylase.

A recombinant truncated form (delta1-102/delta428-452) of the non-heme iron-dependent metalloenzyme human phenylalanine hydroxylase (hPAH, phenylalanine 4-monooxygenase; EC 1.14.16.1) was expressed in E. coli, purified to homogeneity as a homodimer (70 kDa) and crystallized using the hanging drop vapour diffusion method. The crystals are orthorhombic, space group C222 with cell dimensions of a = 66.6 A, b = 108.4 A, c = 125.7 A. The calculated packing parameter (Vm) is 3.24 A3/Da with four 2-fold symmetric dimers (or eight momomers) in the unit cell. Data have been collected to 2.0 A resolution.

Domain structure and stability of human phenylalanine hydroxylase inferred from infrared spectroscopy.

We have studied the conformation and thermal stability of recombinant human phenylalanine hydroxylase (hPAH) and selected truncated forms, corresponding to distinct functional domains, by infrared spectroscopy. The secondary structure of wild-type hPAH was estimated to be 48% alpha-helix, 28% extended structures, 12% beta-turns and 12% non-structured conformations. The catalytic C-terminal domain (residues 112-452) holds most of the regular secondary structure elements, whereas the regulatory N-terminal domain (residues 2-110) adopts mainly an extended and disordered, flexible conformation. Thermal stability studies of the enzyme forms indicate the existence of interactions between the two domains. Our results also demonstrate that the conformational events involved in the activation of hPAH by its substrate (L-Phe) are mainly related to changes in the tertiary/quaternary structure. The activating effect of phosphorylation, however, affects the secondary structure of the N-terminal domain of the protein.

A structural approach into human tryptophan hydroxylase and its implications for the regulation of serotonin biosynthesis.

Tryptophan hydroxylase (TPH) catalyzes the 5-hydroxylation of tryptophan, which is the first step in the biosynthesis of indoleamines (serotonin and melatonin). Serotonin functions mainly as a neurotransmitter, whereas melatonin is the principal hormone secreted by the pineal gland. TPH belongs to the family of the aromatic amino acid hydroxylases, including phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH), which all have a strict requirement for dioxygen, non-heme iron (II) and tetrahydrobiopterin (BH4). During the last three years there has been a formidable increase in the amount of structural information about PAH and TH, which has provided new insights into the active site structure, the binding of substrates, inhibitors and pterins, as well as on the effect of disease-causing mutations in these hydroxylases. Although structural information about TPH is not yet available, the high sequence homology between the three mammalian hydroxylases, notably at the catalytic domains, and the similarity of the reactions that they catalyze, indicate that they share a similar 3D-structure and a common catalytic mechanism. Thus, we have prepared a model of the structure of TPH based on the crystal structures of TH and PAH. This structural model provides a frame for understanding the specific interactions of TPH with L-tryptophan and substrate analogues, BH4 and cofactor analogues, L-DOPA and catecholamines. The interactions of these ligands with the enzyme are discussed focusing on the physiological and pharmacological regulation of serotonin biosynthesis, notably by tryptophan supplementation therapy and substitution therapy with tetrahydrobiopterin analogues (positive effects), as well as the effect of catecholamines on TPH activity in L-DOPA treated Parkinson's disease patients (enzyme inhibition).

A forensic DNA profiling system for Northern European brown bears (Ursus arctos).

A set of 13 dinucleotide STR loci (G1A, G10B, G1D, G10L, MU05, MU09, MU10, MU15, MU23, MU26, MU50, MU51, MU59) were selected as candidate markers for a DNA forensic profiling system for Northern European brown bear (Ursus arctos). We present results from validation of the markers with respect to their sensitivity, species specificity and performance (precision, heterozygote balance and stutter ratios). All STRs were amplified with 0.6ng template input, and there were no false bear genotypes in the cross-species amplification tests. The validation experiments showed that stutter ratios and heterozygote balance was more pronounced than in the tetranucleotide loci used in human forensics. The elevated ratios of stutter and heterozygote balance at the loci validated indicate that these dinucleotide STRs are not well suited for interpretation of individual genotypes in mixtures. Based on the results from the experimental validations we discuss the challenges related to genotyping dinucleotide STRs in single source samples. Sequence studies of common alleles showed that, in general, the size variation of alleles corresponded with the variation in number of repeats. The samples characterized by sequence analysis may serve as standard DNA samples for inter laboratory calibration. A total of 479 individuals from eight Northern European brown bear populations were analyzed in the 13 candidate STRs. Locus MU26 was excluded as a putative forensic marker after revealing large deviations from expected heterozygosity likely to be caused by null-alleles at this locus. The remaining STRs did not reveal significant deviations from Hardy-Weinberg equilibrium expectations except for loci G10B and MU10 that showed significant deviations in one population each, respectively. There were 9 pairwise locus comparisons that showed significant deviation from linkage equilibrium in one or two out of the eight populations. Substantial genetic differentiation was detected in some of the pairwise population comparisons and the average estimate of population substructure (F(ST)) was 0.09. The average estimate of inbreeding (F(IS)) was 0.005. Accounting for population substructure and inbreeding the total average probability of identity in each of the eight populations was lower than 1.1×10(-9) and the total average probability of sibling identity was lower than 1.3×10(-4). The magnitude of these measurements indicates that if applying these twelve STRs in a DNA profiling system this would provide individual specific evidence.

Cold-induced sweating syndrome: CISS1 and CISS2: manifestations from infancy to adulthood. Four new cases.

Cold-induced sweating syndrome (CISS), a rare autosomal recessive disorder, is genetically heterogeneous. Deficiency of the CRLF1 and the CLCF1 gene functions results in CISS1 and CISS2, respectively. So far, only a single patient with CISS2 has been reported. Here we describe four new cases of CISS, two additional patients with CISS2 (confirming locus heterogeneity) and two patients with CISS1. Their case histories are given in detail to emphasize the striking similarity of their presentation, which makes a clinical differentiation impossible. All four cases had a uniform presentation in the neonatal period, much like Crisponi syndrome - inability to suckle and swallow due to facial and bulbar weakness; excessive startle and trismus-like facial contractions when crying or being handled; apnoeic spells; episodic unexplained fevers (up to 41 degrees C) and associated seizures or even sudden death; erythematous skin rashes; and camptodactyly. Thus it is evident that Crisponi syndrome is the pediatric manifestation of both CISS1 and CISS2. Signs abate during infancy and most children have a normal psychomotor development. During the first decade all children develop scoliosis and abnormal sweating which is the most disabling symptom in adulthood. We report that cold-induced sweating can be effectively treated. Detailed clinical observations, correlated with the findings from basic science research, may serve to elucidate the role(s) of this important cytokine complex in embryonic and postnatal development.

An international multicenter association study of the serotonin transporter gene in persistent ADHD.

Attention deficit hyperactivity disorder (ADHD) is a common behavioral disorder affecting children and adults. It has been suggested that gene variants related to serotonin neurotransmission are associated with ADHD. We tested the functional promoter polymorphism 5-HTTLPR and seven single nucleotide polymorphisms in SLC6A4 for association with ADHD in 448 adult ADHD patients and 580 controls from Norway. Replication attempts were performed in a sample of 1454 Caucasian adult ADHD patients and 1302 controls from Germany, Spain, the Netherlands and USA, and a meta-analysis was performed also including a previously published adult ADHD study. We found an association between ADHD and rs140700 [odds ratio (OR ) = 0.67; P = 0.01] and the short (S) allele of the 5-HTTLPR (OR = 1.19; P = 0.06) in the Norwegian sample. Analysis of a possible gender effect suggested that the association might be restricted to females (rs140700: OR = 0.45; P = 0.00084). However, the meta-analysis of 1894 cases and 1878 controls could not confirm the association for rs140700 [OR = 0.85, 95% confidence interval (CI) = 0.67-1.09; P = 0.20]. For 5-HTTLPR, five of six samples showed a slight overrepresentation of the S allele in patients, but meta-analysis refuted a strong effect (OR = 1.10, 95% CI = 1.00-1.21; P = 0.06). Neither marker showed any evidence of differential effects for ADHD subtype, gender or symptoms of depression/anxiety. In conclusion, our results do not support a major role for SLC6A4 common variants in persistent ADHD, although a modest effect of the 5-HTTLPR and a role for rare variants cannot be excluded.

Tryptophan fluorescence of human phenylalanine hydroxylase produced in Escherichia coli.

Human phenylalanine hydroxylase (hPAH) contains three tryptophan residues (W120, W187, and W326). All three tryptophan residues were mutated to phenylalanine either as single mutants or in combination, and one tryptophan was also mutated to isoleucine. The mutant enzymes were expressed in Escherichia coli and purified as fusion proteins with maltose-binding protein and a linker region containing a recognition site for the serine protease factor Xa. After cleavage by factor Xa, all mutants were purified to homogeneity, and the kinetic and spectroscopic properties of the proteins were studied. All the proteins had high catalytic activities, but the affinity for phenylalanine was increased for the W1201 and W120F mutants, and decreased for the W187F and W326F mutants. Using steady-state fluorescence spectroscopy, the contributions of the individual tryptophan residues to the total intrinsic fluorescence of the protein were estimated. On the basis of measurements of mutants containing only one tryptophan, it was calculated that W120, W187, and W326 account for approximately 61, 13, and 26% of the total tryptophan fluorescence of hPAH, respectively, while the positions of the emission maxima (335.5-336.5 nm) and the widths at half-height (55-60 nm) of the emission spectra of the individual tryptophans were rather similar. After incubation with phenylalanine, the quantum yield of wild-type hPAH increases by 15%, and the emission maximum is shifted from 336.5 to 347 nm. This effect is mainly due to changes in the W120 emission. On the basis of fluorescence quenching studies, this amino acid is the most surface-exposed of the tryptophan residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Urea-induced denaturation of human phenylalanine hydroxylase.

Human phenylalanine hydroxylase was expressed and purified from Escherichia coli as a fusion protein with maltose-binding protein. After removal of the fusion partner, the effects of increasing urea concentrations on enzyme activity, aggregation, unfolding, and refolding were examined. At pH 7.50, purified human phenylalanine hydroxylase is transiently activated in the presence of 0-4 M urea but slowly inactivated at higher denaturant concentrations. Intrinsic tryptophan fluorescence spectroscopy showed that the enzyme is denatured through at least two distinct transitions. The presence of phenylalanine (L-Phe) shifts the transition midpoint of the first transition from 1.4 to 2.7 M urea, whereas the second transition is unaffected by this substrate. Apparently the free energy of denaturation was almost identical for the free enzyme and for the enzyme-substrate complex, but significant differences in dDeltaG(D)/d[urea] (m(D) values) were observed for the first denaturation transition. In the absence of substrate, a high rate of non-covalent aggregation was observed for the enzyme in the presence of 1-4 M urea. All three tryptophan residues in the enzyme (Trp-120, Trp-187, and Trp-326) were mutated to phenylalanine, either as single mutations or in combination, in order to identify the residues involved in the spectroscopic transitions. A gradual dissociation of the native tetrameric enzyme to increasingly denatured dimeric and monomeric forms was demonstrated by size exclusion chromatography in the presence of denaturants.