Vito Guarnieri

Paraoxonase gene variants are associated with autism in North America, but not in Italy: possible regional specificity in gene-environment interactions

Organophosphates (OPs) are routinely used as pesticides in agriculture and as insecticides within the household. Our prior work on Reelin and APOE delineated a gene–environment interactive model of autism pathogenesis, whereby genetically vulnerable individuals prenatally exposed to OPs during critical periods in neurodevelopment could undergo altered neuronal migration, resulting in an autistic syndrome. Since household use of OPs is far greater in the USA than in Italy, this model was predicted to hold validity in North America, but not in Europe. Here, we indirectly test this hypothesis by assessing linkage/association between autism and variants of the paraoxonase gene (PON1) encoding paraoxonase, the enzyme responsible for OP detoxification. Three functional single nucleotide polymorphisms, PON1 C−108T, L55M, and Q192R, were assessed in 177 Italian and 107 Caucasian-American complete trios with primary autistic probands. As predicted, Caucasian-American and not Italian families display a significant association between autism and PON1 variants less active in vitro on the OP diazinon (R192), according to case–control contrasts (Q192R: χ2=6.33, 1 df, P<0.025), transmission/disequilibrium tests (Q192R: TDT χ2=5.26, 1 df, P<0.025), family-based association tests (Q192R and L55M: FBAT Z=2.291 and 2.435 respectively, P<0.025), and haplotype-based association tests (L55/R192: HBAT Z=2.430, P<0.025). These results are consistent with our model and provide further support for the hypothesis that concurrent genetic vulnerability and environmental OP exposure may possibly contribute to autism pathogenesis in a sizable subgroup of North American individuals.

Association between the HOXA1 A218G polymorphism and increased head circumference in patients with autism

BACKGROUND The HOXA1 gene plays a major role in brainstem and cranial morphogenesis. The G allele of the HOXA1 A218G polymorphism has been previously found associated with autism. METHODS We performed case-control and family-based association analyses, contrasting 127 autistic patients with 174 ethnically matched controls, and assessing for allelic transmission disequilibrium in 189 complete trios. RESULTS A, and not G, alleles were associated with autism using both case-control (chi(2) = 8.96 and 5.71, 1 df, p <.005 and <.025 for genotypes and alleles, respectively), and family-based (transmission/disequilibrium test chi(2) = 8.80, 1 df, p <.005) association analyses. The head circumference of 31 patients carrying one or two copies of the G allele displayed significantly larger median values (95.0th vs. 82.5th percentile, p <.05) and dramatically reduced interindividual variability (p <.0001), compared with 166 patients carrying the A/A genotype. CONCLUSIONS The HOXA1 A218G polymorphism explains approximately 5% of the variance in the head circumference of autistic patients and represents to our knowledge the first known gene variant providing sizable contributions to cranial morphology. The disease specificity of this finding is currently being investigated. Nonreplications in genetic linkage/association studies could partly stem from the dyshomogeneous distribution of an endophenotype morphologically defined by cranial circumference.

Frequent epigenetics inactivation of KEAP1 gene in non-small cell lung cancer

The KEAP1/Nrf2 pathway is a master regulator of several redox-sensitive genes implicated in resistance of tumor cells against chemotherapeutic drugs. Recent data suggest that epigenetic mechanisms may play a pivotal role in the regulation of KEAP1 expression. We performed a comprehensive genetic and epigenetic analysis of the KEAP1 gene in 47 non-small cell lung cancer tissues and normal specimens. Promoter methylation analysis was performed using a quantitative methylation specific PCR assay in real time. Methylation at the KEAP1 promoter region was detected in 22 out of the 47 NSCLCs (47%) and in none of the normal tissues analyzed. Somatic mutations were detected in 7 out of the 47 tumors (15%) and loss of heterozygosity (LOH) in 10 out of the 47 (21%) of the cases. Overall, we found at least one molecular alteration in 57% of the cases. Approximately one third of the tumors had two alterations and this feature was associated with higher risk of disease progression in univariate COX regression analysis (HR = 3.62; 95% CI 1.24–10.65, p = 0.02). This result was confirmed by Kaplan-Meier analysis, which demonstrated an association between worst outcome and KEAP1 double alterations (p = 0.01, Log rank test). Our results further suggest that deregulation of the NRF2/KEAP1 system could play a pivotal role in the cancerogenesis of NSCLC. In addition identifying patients with KEAP1 genetic and epigenetic abnormalities may contribute to disease progression prediction and response to therapy in lung cancer patients.

Chapter 3 Calcium-Sensing Receptor and Associated Diseases

The calcium-sensing receptor (CASR) is expressed in parathyroid hormone (PTH)-secreting cells of the parathyroid gland and cells lining the renal tubule. The activated CASR modulates intracellular signaling pathways altering PTH secretion and renal cation and water handling. Inherited abnormalities of the CASR gene give rise to a variety of disorders of mineral ion homeostasis. Heterozygous loss-of-function mutations cause familial (benign) hypocalciuric hypercalcemia (FHH) in which the lifelong mild hypercalcemia is generally asymptomatic. Homozygous inactivating mutations give rise to neonatal severe hyperparathyroidism (NSHPT) with extreme hypercalcemia and marked skeletal changes. Heterozygous activating mutations of the CASR cause autosomal dominant hypocalcemia (ADH) that may be asymptomatic or present with seizures in the neonatal period or childhood or later in life. Phenocopies of FHH or ADH are due to circulating CASR inactivating or activating autoantibodies, respectively. The CASR is the target of small molecule allosteric modifiers, either activators, calcimimetics, or inhibitors, calcilytics.

Hemangioblastomas of Central Nervous System: Molecular Genetic Analysis and Clinical Management

OBJECTIVE: Hemangioblastomas of the central nervous system (CNS) are benign neoplasms that may occur sporadically or in association with von Hippel-Lindau (VHL) disease. The proportion of primary symptomatic hemangioblastomas associated with VHL disease is estimated to be from 10 to 40%, but it seems to be underestimated. We investigated the frequency of VHL germline mutation in patients with symptomatic CNS hemangioblastoma without evidence of VHL disease to define the role of molecular genetic analysis in the management of such patients and their relatives. METHODS: We analyzed 14 patients (6 female and 8 male; mean age, 43.5 yr) with no family history and no other clinical manifestations of VHL disease who had been operated on for symptomatic CNS hemangioblastoma. Exons 1, 2, and 3 of the VHL gene and their immediately flanking sequences were amplified by use of polymerase chain reaction followed by analysis with denaturing high-performance liquid chromatography and sequencing the anomalous samples. RESULTS: Germline mutations of the VHL gene were identified in 2 (14%) of 14 patients. VHL gene mutation analysis was performed in both patients’ family members, which showed another affected asymptomatic subject for VHL disease. The affected subjects were recommended for VHL disease surveillance protocol. CONCLUSION: Molecular genetic analysis is a safer and more specific instrument to confirm or exclude VHL disease in patients with CNS hemangioblastoma, a negative family history, or absence of other known manifestations of the disease. Early identification of VHL mutation gene carriers is important for reducing disease morbidity and mortality. Nonsymptomatic family members will benefit from early VHL disease diagnosis or by being excluded as at-risk subjects, reducing the psychological and economic burden of screening and surveillance protocols.

The microRNA cluster C19MC is deregulated in parathyroid tumours.

A subset of over-expressed microRNAs (miRNAs) identified in parathyroid carcinomas (Ca) compared to normal glands belongs to C19MC, a cluster on chromosome 19q13.4 involved in stem cell biology and tumourigenesis. In this study, the expression of C19MC-MIR371-3 clusters and the molecular mechanisms presiding their modulation were investigated in a series of six normal parathyroids, 24 adenomas (Ad), 15 Ca and five matched metastases. The general expression levels of C19MC or MIR371-3 clusters in Ad lesions did not differ from normal glands, while they distinguished Ad from Ca at unsupervised hierarchical cluster analysis (P=0.0008). MIR517C showed the most significant difference in expression between Ca and Ad (P=0.0003) and it positively correlated with serum calcium, parathormone and tumour weight. In regard to the molecular mechanism determining C19MC cluster activation, we could detect C19MC copy number (CN) gain in ten Ca (67%) extending distal to the MIR371-3 cluster in almost all samples. Conversely, only four Ad (16%) showed C19MC amplification, with one case presenting distal genomic aberration to MIR371-3. Globally, CN variations of 19q13.4 loci were significantly associated with MIR517C up-regulation (P=0.006). Opposite to normal glands where C19MC promoter was methylated, hypomethylation occurred in 15 out of 30 analysed tumours. Though the epigenetic status did not correlate with C19MC miRNA expression levels, loss of C19MC promoter methylation was significantly associated with Ca and metastatic disease (P=0.01). In conclusion, C19MC cluster aberrations are a characteristic of Ca with respect to Ad. Altogether, these evidences point towards a role for 19q13.4 miRNA clusters as oncogenes in parathyroid tumourigenesis.

Polymorphisms at the regulatory regions of the CASR gene influence stone risk in primary hyperparathyroidism

BACKGROUND AND OBJECTIVE Single nucleotide polymorphisms (SNPs) of the calcium-sensing receptor (CASR) gene at the regulatory region were associated with idiopathic calcium nephrolithiasis. To confirm their association with nephrolithiasis, we tested patients with primary hyperparathyroidism (PHPT). DESIGN A genotype-phenotype association study. METHODS In all, 332 PHPT patients and 453 healthy controls were genotyped for the rs7652589 (G>A) and rs1501899 (G>A) SNPs sited in the noncoding regulatory region of the CASR gene. Allele, haplotype, and diplotype distribution were compared between PHPT patients and controls, and in stone forming and stone-free PHPT patients. RESULTS The allele frequency at rs7652589 and rs1501899 SNPs was similar in PHPT patients and controls. The A minor alleles at these two SNPs were more frequent in stone forming (n=157) than in stone-free (n=175) PHPT patients (rs7652589: 36.9 vs 27.1%, P=0.007; rs1501899: 37.1 vs 26.4%, P=0.003). Accordingly, homozygous or heterozygous PHPT patients for the AA haplotype (n=174, AA/AA or AA/GG diplotype) had an increased stone risk (odds ratio 1.83, 95% confidence interval 1.2-2.9, P=0.008). Furthermore, these PHPT patients had higher serum concentrations of ionized calcium and parathyroid hormone (1.50 ± 0.015 mmol/l and 183 ± 12.2 pg/ml) than patients with the GG/GG diplotype (n=145, 1.47 ± 0.011 mmol/l (P=0.04) and 150 ± 11.4 pg/ml (P=0.049)). Using a logistic regression model, the increase in stone risk in PHPT patients was predicted by AA/AA or AA/GG diplotype, the highest tertile of serum ionized calcium values and the lowest tertile of age. CONCLUSIONS Polymorphisms located in the regulatory region of the CASR gene may increase susceptibility of the PHPT patients to kidney stone production.

Bone involvement in aldosteronism

In rats with aldosteronism, a reduction of bone mineral density (BMD) and cortical bone strength has been reported. Our study was aimed to evaluate bone involvement in patients with primary aldosteronism (PA). A total of 188 consecutive subjects with adrenal incidentaloma, observed between November 2009 and October 2011, were screened for PA with aldosterone‐to‐renin ratio. After confirmatory tests, in those who screened positive, 11 patients were diagnosed as PA and 15 patients were not (nPA). A serum/urinary biochemical profile, parathyroid hormone (PTH), BMD measured at lumbar spine (LS) and total and femoral neck (TN and FN) by dual X‐ray absorptiometry, and conventional spinal radiographs (T4–L4) were obtained in all subjects. PA patients had a significantly higher 24‐hour urinary calcium (6.28 ± 1.85 versus 4.28 ± 1.18 mmol/d; p < 0.01), and PTH (9.8 [5.8‐14.6], median [range] versus 5.3 [2.5‐10.8] pmol/L; p < 0.01) than nPA patients. BMD expressed as Z‐value at LS (−1.18 ± 0.99 versus 0.22 ± 1.12), FN (−0.85 ± 0.73 versus 0.01 ± 0.82), and TN (−0.49 ± 0.61 versus 0.39 ± 0.93) was lower in PA than in nPA (p = 0.003, p = 0.011, and p = 0.012, respectively). The prevalence of osteoporosis was higher in PA than in nPA (8/11, 72.7% versus 3/15, 20.0%; Fishers exact test: p = 0.015). Vertebral fractures tended to be more prevalent in PA than in nPA (5/11, 45.5% versus 2/15, 13.3%; Fishers exact test: p = 0.095). Logistic regression analysis showed that osteoporosis and morphometric vertebral fractures were associated with PA (odds ratio [OR], 15.4; 95% confidence interval [CI] = 1.83–130, p = 0.012; and OR, 30.4; 95%CI, 1.07–862, p = 0.045, respectively) regardless of age, body mass index (BMI), and LS‐BMD. In 9 of 11 PA patients, 6 months after beginning of treatment (surgery or spironolactone) there was a significant reduction of urinary calcium excretion (p < 0.01) and PTH (p < 0.01), whereas in 5 of 11 PA patients, 1 year after beginning of treatment, BMD was significantly increased at LS, p < 0.01). In conclusion, PA is associated with osteoporosis, vertebral fractures, and increased urinary calcium excretion.

HOXA1 gene variants influence head growth rates in humans

We previously described a significant association between the HOXA1 G218 allele and increased head circumference in autism [Conciatori et al. (2004); Biol Psychiatry 55:413–419]. The present study reveals identical effects also in normal children. HOXA1 A218G alleles and sex explain as much as 10.9 and 6.8% of the variance in head circumference in 142 pediatric controls and in 191 autistic children, aged 3–16 years (F = 6.777, 3 and 141 df, P < 0.001 and F = 5.588, 3 and 190 df, P < 0.01, respectively). Instead, no association is found in 183 adult controls and in 35 pediatric fragile‐X patients. Therefore HOXA1 A218G alleles significantly influence head growth rates, but not final head size, in normal human development. This influence does not differ between normal and autistic children, whereas the lack of FMRP seemingly overwhelms HOXA1 effects in fragile‐X patients.

Factors associated with vertebral fracture risk in patients with primary hyperparathyroidism

OBJECTIVE To examine factors, in addition to bone mineral density (BMD), such as the common calcium-sensing receptor (CASR) gene polymorphisms, associated with vertebral fracture (VFx) risk in primary hyperparathyroidism (PHPT). DESIGN AND METHODS A cross-sectional analysis of 266 Caucasian PHPT seen as outpatients. Serum calcium (sCa) phosphate metabolism parameters were measured. BMD was assessed by dual-energy X-ray absorptiometry (expressed as Z-score) at lumbar spine (Z-LS) and femoral neck, morphometric VFx by radiograph, and CASR A986S/R990G genotypes by PCR amplification and genomic DNA sequencing. RESULTS Fractured patients (n=100, 37.6%) had lower sCa (10.8±0.7 mg/dl) and Z-LS BMD (-1.0±1.44), higher age (61±10 years), and prevalence (51%) of ≥1 S alleles of the CASR A986S single-nucleotide polymorphism (SNP; AS/SS), than those not fractured (n=166, 11.2±1.0 mg/dl, -0.57±0.97, 58±13 years, and 38% AS/SS, respectively, P<0.05 for all comparisons). Logistic regression, with VFx as dependent variable, showed independent risks associated with increased age (OR 1.03, 95% CI 1.01-1.06, P=0.006), decreased sCa (OR 1.86, 95% CI 1.28-2.7, P=0.001), and Z-LS BMD (OR 1.4, 95% CI 1.12-1.7, P=0.002) and presence of AS/SS (OR 1.8, 95% CI 1.1-2.9, P=0.05). The presence of two out of three factors (age ≥58 years, sCa <10.8 and Z-LS BMD≤-1.0, and AS/SS genotype) gave an overall OR of 4.2 (95% CI 2.25-7.85, P<0.0001). CONCLUSIONS In PHPT, VFx is associated positively with age, negatively with sCa and spinal BMD, and presence of at least one copy of the CASR A986S SNP.