Francesca Lantieri

Rare structural variants found in attention-deficit hyperactivity disorder are preferentially associated with neurodevelopmental genes.

Attention-deficit/hyperactivity disorder (ADHD) is a common and highly heritable disorder, but specific genetic factors underlying risk remain elusive. To assess the role of structural variation in ADHD, we identified 222 inherited copy number variations (CNVs) within 335 ADHD patients and their parents that were not detected in 2026 unrelated healthy individuals. Although no excess CNVs, either deletions or duplications, were found in the ADHD cohort relative to controls, the inherited rare CNV-associated gene set was significantly enriched for genes reported as candidates in studies of autism, schizophrenia and Tourette syndrome, including A2BP1, AUTS2, CNTNAP2 and IMMP2L. The ADHD CNV gene set was also significantly enriched for genes known to be important for psychological and neurological functions, including learning, behavior, synaptic transmission and central nervous system development. Four independent deletions were located within the protein tyrosine phosphatase gene, PTPRD, recently implicated as a candidate gene for restless legs syndrome, which frequently presents with ADHD. A deletion within the glutamate receptor gene, GRM5, was found in an affected parent and all three affected offspring whose ADHD phenotypes closely resembled those of the GRM5 null mouse. Together, these results suggest that rare inherited structural variations play an important role in ADHD development and indicate a set of putative candidate genes for further study in the etiology of ADHD.

CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant (IVS1, +37 G>C) of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations.

Molecular mechanisms of RET‐induced Hirschsprung pathogenesis

The RET proto‐oncogene is the major gene involved in the pathogenesis of Hirschsprung (HSCR), a complex genetic disease characterized by lack of ganglia along variable lengths of the gut. Here we present a survey of the different molecular mechanisms through which RET mutations lead to the disease development. Among these, loss of function, gain of function, apoptosis, aberrant splicing and decreased gene expression are exemplified and considered with respect to their pathogenetic impact. In particular, RET transcription regulation represents a new insight into the outline of HSCR susceptibility, and having reached important progress in the last few years, deserves to be reviewed. Notably, gene expression impairment seems to be at the basis of the association of HSCR disease with several RET polymorphisms, allowing us to define a predisposing haplotype spanning from the promoter to exon 2. Putative functional variants, in the promoter and in intron 1, and proposed as low penetrant predisposing alleles, are presented and discussed. Finally, based on the RET mutation effects thus summarized, we attempt to derive conclusions which may be useful for HSCR risk prediction and genetic counselling.

Haplotypes of the Human RET Proto‐oncogene Associated with Hirschsprung Disease in the Italian Population Derive from a Single Ancestral Combination of Alleles

The RET proto‐oncogene is the major gene involved in the complex genetics of Hirschsprung disease (HSCR), or aganglionic megacolon, showing causative loss‐of‐function mutations in 15–30% of the sporadic cases. Several RET polymorphisms and haplotypes have been described in association with the disease, suggesting a role for this gene in HSCR predisposition, also in the absence of mutations in the coding region. Finally, the presence of a functional variant in intron 1 has repeatedly been proposed to explain such findings. Here we report a case‐control study conducted on 97 Italian HSCR sporadic patients and 85 population matched controls, using 13 RET polymorphisms distributed throughout the gene, from the basal promoter to the 3′UTR. Linkage disequilibrium and haplotype analyses have shown increased recombination between the 5′ and 3′ portions of the gene and an over‐representation, in the cases studied, of two haplotypes sharing a common allelic combination that extends from the promoter up to intron 5. We propose that these two disease‐associated haplotypes derive from a single founding locus, extending up to intron 19 and successively rearranged in correspondence with a high recombination rate region located between the proximal and distal portions of the gene. Our results suggests the possibility that a common HSCR predisposing variant, in linkage disequilibrium with such haplotypes, is located further downstream than the previously suggested interval encompassing intron 1.

Induction of RET Dependent and Independent Pro-Inflammatory Programs in Human Peripheral Blood Mononuclear Cells from Hirschsprung Patients

Hirschsprung disease (HSCR) is a rare congenital anomaly characterized by the absence of enteric ganglia in the distal intestinal tract. While classified as a multigenic disorder, the altered function of the RET tyrosine kinase receptor is responsible for the majority of the pathogenesis of HSCR. Recent evidence demonstrate a strong association between RET and the homeostasis of immune system. Here, we utilize a unique cohort of fifty HSCR patients to fully characterize the expression of RET receptor on both innate (monocytes and Natural Killer lymphocytes) and adaptive (B and T lymphocytes) human peripheral blood mononuclear cells (PBMCs) and to explore the role of RET signaling in the immune system. We show that the increased expression of RET receptor on immune cell subsets from HSCR individuals correlates with the presence of loss-of-function RET mutations. Moreover, we demonstrate that the engagement of RET on PBMCs induces the modulation of several inflammatory genes. In particular, RET stimulation with glial-cell line derived neurotrophic factor family (GDNF) and glycosyl-phosphatidylinositol membrane anchored co-receptor α1 (GFRα1) trigger the up-modulation of genes encoding either for chemokines (CCL20, CCL2, CCL3, CCL4, CCL7, CXCL1) and cytokines (IL-1β, IL-6 and IL-8) and the down-regulation of chemokine/cytokine receptors (CCR2 and IL8-Rα). Although at different levels, the modulation of these “RET-dependent genes” occurs in both healthy donors and HSCR patients. We also describe another set of genes that, independently from RET stimulation, are differently regulated in healthy donors versus HSCR patients. Among these “RET-independent genes”, there are CSF-1R, IL1-R1, IL1-R2 and TGFβ-1, whose levels of transcripts were lower in HSCR patients compared to healthy donors, thus suggesting aberrancies of inflammatory responses at mucosal level. Overall our results demonstrate that immune system actively participates in the physiopathology of HSCR disease by modulating inflammatory programs that are either dependent or independent from RET signaling.

TNF-α gene polymorphisms: Association with disease susceptibility and response to anti-TNF-α treatment in psoriatic arthritis

The tumor necrosis factor-α (TNF-α) gene has been proposed as a major candidate gene in psoriatic arthritis (PsA). TNF-α is a therapeutic target for patients responding poorly to conventional treatments. We investigated the role of single-nucleotide polymorphisms (SNPs) at positions -238, -308, and +489 of the TNF-α gene in the genetic susceptibility to PsA, in the severity of the disease, and, finally, in the response to TNF-α inhibitors (adalimumab, etanercept, or infliximab). Fifty-seven Caucasian PsA patients and 155 healthy matched controls were studied. The SNP +489 variant allele A was significantly associated with PsA susceptibility (P=0.0136) and severity of clinical (Psoriasis Area and Severity Index score, American College of Rheumatology criteria, Disease Activity Score 28, and Disability Index Health Assessment Questionnaire) and laboratory (C-reactive protein and erythrocyte sedimentation rate) parameters (P-values ranging from 0.016 to 2.908 × 10(-12)). The difference in severity was accounted for by the differences between the AA and GA genotypes with respect to the GG genotype. The SNP +489A allele shows a trend of association with the response to PsA treatment with etanercept. These findings suggest a role of the SNP +489A allele in the susceptibility and severity of PsA.

Nocturnal enuresis: a suggestive endophenotype marker for a subgroup of inattentive attention-deficit/hyperactivity disorder.

OBJECTIVE Attention-deficit/hyperactivity disorder (ADHD) and enuresis co-occur at a higher rate than expected; the cause for this is unclear. STUDY DESIGN Diagnostic and demographic variables were compared in 344 children ages 6 to 12 years, with and without enuresis, recruited in an ADHD genetic study. Sleep variables were investigated in a subgroup of 44 enuretic children with age- and sex-matched nonenuretic controls. The association of enuresis with single nucleotide polymorphisms located in regions reported in linkage with enuresis was explored. RESULTS The prevalence rate of nocturnal enuresis was 16.9% for the entire cohort. There were no differences in sex, age, socioeconomic status, intelligence quotient, medication treatment, or comorbidities. The enuresis group had a higher likelihood of inattentive symptoms than the nonenuretic group. Night wakings and ability of children to wake themselves in the morning were both significantly decreased in children with enuresis compared with control children in the Child Sleep Habits Questionnaire Night Wakings subscale. No significant association was found with chromosomal regions previously reported in linkage with enuresis. CONCLUSIONS Deficits in arousal may contribute to both enuresis and inattentive ADHD. Nocturnal enuresis may be a useful clinical marker in identifying a subgroup of the inattentive phenotype in ADHD genetic studies.

Candidate gene analysis in an on-going genome-wide association study of attention-deficit hyperactivity disorder: suggestive association signals in adra1a

Objectives Attention-deficit hyperactivity disorder (ADHD) is a highly heritable, common developmental disorder. Although a few confirmed associations have emerged from candidate gene studies, these have shown the same limitations that have become evident in the study of other complex diseases, often with inconsistent and nonreplicated results across different studies. Methods In this report, 27 ADHD candidate genes were explored in greater depth using high-density tag single nucleotide polymorphism (SNP) genotyping. Association with 557 SNPs was tested using the transmission disequilibrium test in 270 nuclear pedigrees selected from an ongoing ADHD genetic study that includes all disease subtypes. Results SNPs in seven genes including SLC1A3, SLC6A3, HTR4, ADRA1A, HTR2A, SNAP25, and COMT showed a nominal level of association with ADHD (P values <0.05), but none remained significant after a stringent correction for the total number of tests performed. Conclusion The strongest signal emerged from SNPs in the promoter region (rs3808585) and in an intron (rs17426222, rs4732682, rs573514) of ADRA1A, all located within the same haplotype block. Some of the SNPs in HTR2A and COMT have already been reported by others, whereas other SNPs will need confirmation in independent samples.

Analysis of GWAS top hits in ADHD suggests association to two polymorphisms located in genes expressed in the cerebellum

Attention deficit/hyperactivity disorder (ADHD) is a common psychiatric disorder influenced by genetic factors. Several chromosomal regions with potential linkage and candidate genes associations have been reported, but findings are often inconsistent and non‐replicated. The few genome‐wide association studies (GWAS) carried out so far differ for study design and phenotypes analyzed, and did not detect any association significant at the genome‐wide level. In the present study we examined the top SNPs reported in the GWAS by Neale et al. 2008 in an independent cohort. Although our sample size is smaller (415 trios vs. 909), the power was sufficient to confirm the role of candidate markers in ADHD if a true association exists. Two out of 36 top SNPs were significant at α = 0.05 in our sample, although none was still significant after correction for multiple tests. These two SNPs are both located in genes coding for as yet uncharacterized proteins expressed in the cerebellum, XKR4 in 8q12.1, and FAM190A in 4q22.1. Three other FAM190A SNPs have TDT P‐values of <10−5 in our sample, a level of significance only reached by a total of five SNPs in our genome‐wide data. While these findings could be due to chance, we cannot exclude that these markers are indeed associated to disease risk. Remarkably, brain imaging studies have shown reduction of the posterior inferior cerebellar lobules volume of ADHD boys and girls compared to controls, persistent with age and not present in unaffected siblings, suggesting that the cerebellum may be directly related to pathophysiology of ADHD.

Behavior of non-classical soluble HLA class G antigens in human immunodeficiency virus 1-infected patients before and after HAART: Comparison with classical soluble HLA-A, -B, -C antigens and potential role in immune-reconstitution

We have reported that serum level of soluble HLA-A, -B, -C (sHLA-A,-B,-C) antigens is elevated in HIV-infected subjects and decreases after antiretroviral therapy (HAART). In this study, we measured the levels of soluble HLA-G (sHLA-G) antigens in a cohort of HIV-infected patients before and during HAART. sHLA-G and sHLA-A, -B, -C levels were significantly elevated in HIV-infected subjects as compared with controls before antiretroviral treatment and significantly decreased after 36 months of HAART. sHLA-G levels were correlated with sHLA-A, -B, -C levels, the decrease of plasma HIV-RNA level, the increase of CD4+ T-lymphocyte number and the decrease of CD8+ T-lymphocyte number. These results suggest that the measurement of sHLA-G and sHLA-A, -B, -C antigen serum levels might represent a useful surrogate marker to monitor virological response and immune reconstitution in HIV-positive subjects undergoing HAART treatment.