Carlo Lenti

Altered calcium homeostasis in autism-spectrum disorders: evidence from biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier AGC1

Autism is a severe developmental disorder, whose pathogenetic underpinnings are still largely unknown. Temporocortical gray matter from six matched patient–control pairs was used to perform post-mortem biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier (AGC), which participates in the aspartate/malate reduced nicotinamide adenine dinucleotide shuttle and is physiologically activated by calcium (Ca2+). AGC transport rates were significantly higher in tissue homogenates from all six patients, including those with no history of seizures and with normal electroencephalograms prior to death. This increase was consistently blunted by the Ca2+ chelator ethylene glycol tetraacetic acid; neocortical Ca2+ levels were significantly higher in all six patients; no difference in AGC transport rates was found in isolated mitochondria from patients and controls following removal of the Ca2+-containing postmitochondrial supernatant. Expression of AGC1, the predominant AGC isoform in brain, and cytochrome c oxidase activity were both increased in autistic patients, indicating an activation of mitochondrial metabolism. Furthermore, oxidized mitochondrial proteins were markedly increased in four of the six patients. Variants of the AGC1-encoding SLC25A12 gene were neither correlated with AGC activation nor associated with autism-spectrum disorders in 309 simplex and 17 multiplex families, whereas some unaffected siblings may carry a protective gene variant. Therefore, excessive Ca2+ levels are responsible for boosting AGC activity, mitochondrial metabolism and, to a more variable degree, oxidative stress in autistic brains. AGC and altered Ca2+ homeostasis play a key interactive role in the cascade of signaling events leading to autism: their modulation could provide new preventive and therapeutic strategies.

Clinical, morphological, and biochemical correlates of head circumference in autism

BACKGROUND Head growth rates are often accelerated in autism. This study is aimed at defining the clinical, morphological, and biochemical correlates of head circumference in autistic patients. METHODS Fronto-occipital head circumference was measured in 241 nonsyndromic autistic patients, 3 to 16 years old, diagnosed according to DSM-IV criteria. We assessed 1) clinical parameters using the Autism Diagnostic Observation Schedule, Autism Diagnostic Interview-Revised, Vineland Adaptive Behavioral Scales, intelligence quotient measures, and an ad hoc clinical history questionnaire; 2) height and weight; 3) serotonin (5-HT) blood levels and peptiduria. RESULTS The distribution of cranial circumference is significantly skewed toward larger head sizes (p < .00001). Macrocephaly (i.e., head circumference >97th percentile) is generally part of a broader macrosomic endophenotype, characterized by highly significant correlations between head circumference, weight, and height (p < .001). A head circumference >75th percentile is associated with more impaired adaptive behaviors and with less impairment in IQ measures and motor and verbal language development. Surprisingly, larger head sizes are significantly associated with a positive history of allergic/immune disorders both in the patient and in his/her first-degree relatives. CONCLUSIONS Our study demonstrates the existence of a macrosomic endophenotype in autism and points toward pathogenetic links with immune dysfunctions that we speculate either lead to or are associated with increased cell cycle progression and/or decreased apoptosis.

Principal pathogenetic components and biological endophenotypes in autism spectrum disorders

Autism is a complex neurodevelopmental disorder, likely encompassing multiple pathogenetic components. The aim of this study is to begin identifying at least some of these components and to assess their association with biological endophenotypes. To address this issue, we recruited 245 Italian patients with idiopathic autism spectrum disorders and their first‐degree relatives. Using a stepwise approach, patient and family history variables were analyzed using principal component analysis (“exploratory phase”), followed by intra‐ and inter‐component cross‐correlation analyses (“follow‐up phase”), and by testing for association between each component and biological endophenotypes, namely head circumference, serotonin blood levels, and global urinary peptide excretion rates (“biological correlation phase”). Four independent components were identified, namely “circadian & sensory dysfunction,” “immune dysfunction,” “neurodevelopmental delay,” and “stereotypic behavior,” together representing 74.5% of phenotypic variance in our sample. Marker variables in the latter three components are positively associated with macrocephaly, global peptiduria, and serotonin blood levels, respectively. These four components point toward at least four processes associated with autism, namely (I) a disruption of the circadian cycle associated with behavioral and sensory abnormalities, (II) dysreactive immune processes, surprisingly linked both to prenatal obstetric complications and to excessive postnatal body growth rates, (III) a generalized developmental delay, and (IV) an abnormal neural circuitry underlying stereotypies and early social behaviors.

Urinary p-cresol is elevated in small children with severe autism spectrum disorder

Several studies have described in autistic patients an overgrowth of unusual gut bacterial strains, able to push the fermentation of tyrosine up to the formation of p-cresol. We compared levels of urinary p-cresol, measured by high-performance liquid chromatography–ultraviolet, in 59 matched case-control pairs. Urinary p-cresol was significantly elevated in autistic children smaller than 8 years of age (p < 0.01), typically females (p < 0.05), and more severely affected regardless of sex (p < 0.05). Urinary cotinine measurements excluded smoking-related hydrocarbon contaminations as contributors to these differences. Hence, elevated urinary p-cresol may serve as a biomarker of autism liability in small children, especially females and more severely affected males.

Family-based association study of ITGB3 in Autism Spectrum Disorder and its endophenotypes.

The integrin-β 3 gene (ITGB3), located on human chromosome 17q21.3, was previously identified as a quantitative trait locus (QTL) for 5-HT blood levels and has been implicated as a candidate gene for autism spectrum disorder (ASD). We performed a family-based association study in 281 simplex and 12 multiplex Caucasian families. ITGB3 haplotypes are significantly associated with autism (HBAT, global P=0.038). Haplotype H3 is largely over-transmitted to the affected offspring and doubles the risk of an ASD diagnosis (HBAT P=0.005; odds ratio (OR)=2.000), at the expense of haplotype H1, which is under-transmitted (HBAT P=0.018; OR=0.725). These two common haplotypes differ only at rs12603582 located in intron 11, which reaches a P-value of 0.072 in single-marker FBAT analyses. Interestingly, rs12603582 is strongly associated with pre-term delivery in our ASD patients (P=0.008). On the other hand, it is SNP rs2317385, located at the 5′ end of the gene, that significantly affects 5-HT blood levels (Mann–Whitney U-test, P=0.001; multiple regression analysis, P=0.010). No gene–gene interaction between ITGB3 and SLC6A4 has been detected. In conclusion, we identify a significant association between a common ITGB3 haplotype and ASD. Distinct markers, located toward the 5′ and 3′ ends of the gene, seemingly modulate 5-HT blood levels and autism liability, respectively. Our results also raise interest into ITGB3 influences on feto–maternal immune interactions in autism.

Cluster analysis of autistic patients based on principal pathogenetic components.

We have recently described four principal pathogenetic components in autism: (I) circadian and sensory dysfunction, (II) immune abnormalities, (III) neurodevelopmental delay, and (IV) stereotypic behaviors. Using hierarchical and k‐means clustering, the same 245 patients assessed in our principal component analysis can be partitioned into four clusters: (a) 43 (17.6%) have prominent immune abnormalities accompanied by some circadian and sensory issues; (b) 44 (18.0%) display major circadian and sensory dysfunction, with little or no immune symptoms; (c) stereotypies predominate in 75 (31.0%); and (d) 83 (33.9%) show a mixture of all four components, with greater disruptive behaviors and mental retardation. The “immune” component provides the largest contributions to phenotypic variance (P = 2.7 x 10–45), followed by “stereotypic behaviors.” These patient clusters may likely differ in genetic and immune underpinnings, developmental trajectories, and response to treatment. Autism Res 2012,••:••–••.

Neuropsychological and neuroradiological study of a case of early-onset Huntington's chorea.

The authors report a case of Huntingtons disease in an II‐year‐old boy with onset at six years of age. The neurological signs and symptoms were midway between the hyperkinetic and rigid forms of chorea. Intellectual development was characterized by a medium‐grade deficit. MRI revealed marked atrophy of the head of the caudate nucleus, with diffuse hypcrintcnsitv of the putamen. The most characteristic neuropsychological feature was ideoniotor aprasia. Neuropsychological and neuroradiological data are discussed in relation to the role of the basal nuclei and frontal cortex in the oreanization of movement.

Crying Abnormalities in Congenital Hypothyroidism: Preliminary Spectrographic Study

The aim of this preliminary study was to evaluate the acoustic patterns of the cries of hypothyroid newborns at the time of diagnosis and after the beginning of therapy. Cries were recorded at the nursery of the San Raffaele Hospital, Milan, Italy from 12 full-term subjects (three boys and nine girls) affected by congenital hypothyroidism. Results show that untreated hypothyroid infants at first recording had fewer voiceless and partially voiced cries than normal controls. The percent distribution of this pattern did not change at the second recording after the onset of substitutive therapy. Also, untreated hypothyroid infants had many more cry units showing a vibrato contour than did controls, and this pattern did not change after the onset of treatment. Starting, maximum, minimum, and end frequencies measured on the fundamental were significantly lower in the hypothyroid sample. Four hypothyroid subjects recorded before therapy and within 4 weeks after therapy onset significantly augmented their fundamental frequency parameters; however, in 25% of the sample, sound parameters remained unaltered after 3 or more weeks of treatment. To our knowledge the present preliminary study is the first one performed on follow-up of hypothyroid newborns and indicates that both central and peripheral damage might influence the pattern of crying in untreated hypothyroid infants. (J Child Neurol 2000;15:603-608).

Dental care protocol based on visual supports for children with autism spectrum disorders.

Background Subjects with Autism Spectrum Disorders (ASDs) have often difficulties to accept dental treatments. The aim of this study is to propose a dental care protocol based on visual supports to facilitate children with ASDs to undergo to oral examination and treatments. Material and Methods 83 children (age range 6-12 years) with a signed consent form were enrolled; intellectual level, verbal fluency and cooperation grade were evaluated. Children were introduced into a four stages path in order to undergo: an oral examination (stage 1), a professional oral hygiene session (stage 2), sealants (stage 3), and, if necessary, a restorative treatment (stage 4). Each stage came after a visual training, performed by a psychologist (stage 1) and by parents at home (stages 2, 3 and 4). Association between acceptance rates at each stage and gender, intellectual level, verbal fluency and cooperation grade was tested with chi-square test if appropriate. Results Seventy-seven (92.8%) subjects overcame both stage 1 and 2. Six (7.2%) refused stage 3 and among the 44 subjects who need restorative treatments, only three refused it. The acceptance rate at each stage was statistically significant associated to the verbal fluency (p=0.02; p=0.04; p=0.01, respectively for stage 1, 3 and 4). In stage 2 all subjects accepted to move to the next stage. The verbal/intellectual/cooperation dummy variable was statistically associated to the acceptance rate (p<0.01). Conclusions The use of visual supports has shown to be able to facilitate children with ASDs to undergo dental treatments even in non-verbal children with a low intellectual level, underlining that behavioural approach should be used as the first strategy to treat patients with ASDs in dental setting. Key words:Autism spectrum disorders, behaviour management, paediatric dentistry, visual learning methods.

Candidate gene study of HOXB1 in autism spectrum disorder

BackgroundHOXB1 plays a major role in brainstem morphogenesis and could partly determine the cranial circumference in conjunction with HOXA1. In our sample, HOXA1 alleles significantly influence head growth rates both in autistic patients and in population controls. An initial report, suggesting that HOXB1 could confer autism vulnerability in interaction with HOXA1, was not confirmed by five small association studies.MethodsOur sample includes 269 autistic individuals, belonging to 219 simplex and 28 multiplex families. A mutational analysis of the two exons and flanking intronic sequences of the HOXB1 gene was carried out in 84 autistic patients by denaturing high performance liquid chromatography, followed by DNA sequencing. Identified rare variants were then searched by a restriction analysis in 236 autistic patients and 325-345 controls. Case-control and family-based association studies were performed on two common variants in 169 Italian patients versus 184 Italian controls and in 247 trios.ResultsWe identified three common polymorphisms, rs72338773 [c.82insACAGCGCCC (INS/nINS)], rs12939811 [c.309A>T (Q103H)], and rs7207109 [c.450G>A (A150A)] and three rare variants, namely IVS1+63G>A, rs35115415 [c.702G>A (V234V)] and c.872_873delinsAA (S291N). SNPs rs72338773 and rs12939811 were not associated with autism, using either a case-control (alleles, exact P = 0.13) or a family-based design [transmission/disequilibrium test (TDT)χ2 = 1.774, P = 0.183]. The rare variants, all inherited from one of the parents, were present in two Italian and in two Caucasian-American families. Autistic probands in two families surprisingly inherited a distinct rare variant from each parent. The IVS1+63A allele was present in 3/690 control chromosomes, whereas rare alleles at rs35115415 and c.872_873delinsAA (S291N) were not found in 662 and 650 control chromosomes, respectively. The INS-T309 allele influenced head size, but its effect appears more modest and shows no interaction with HOXA1 alleles. The INS-T309 allele is also associated with more severe stereotypic behaviours, according to ADI-R scores (N = 60 patients, P < 0.01).ConclusionsHOXB1 mutations do not represent a common cause of autism, nor do HOXB1 common variants play important roles in autism vulnerability. HOXB1 provides minor, albeit detectable contributions to head circumference in autistic patients, with HOXA1 displaying more prominent effects. HOXB1 variants may modulate the clinical phenotype, especially in the area of stereotypic behaviours.