Leonardo D'Agruma

Connexin-26 mutations in sporadic and inherited sensorineural deafness

BACKGROUND Hearing impairment affects one infant in 1000 and 4% of people aged younger than 45 years. Congenital deafness is inherited or apparently sporadic. We have shown previously that DFNB1 on chromosome 13 is a major locus for recessive deafness in about 80% of Mediterranean families and that the connexin-26 gene gap junction protein beta2 (GJB2) is mutated in DFNB1 families. We investigated mutations in the GJB2 gene in familial and sporadic cases of deafness. METHODS We obtained DNA samples from 82 families from Italy and Spain with recessive non-syndromic deafness and from 54 unrelated participants with apparently sporadic congenital deafness. We analysed the coding region of the GJB2 gene for mutations. We also tested 280 unrelated people from the general populations of Italy and Spain for the frameshift mutation 35delG. FINDINGS 49% of participants with recessive deafness and 37% of sporadic cases had mutations in the GJB2 gene. The 35delG mutation accounted for 85% of GJB2 mutations, six other mutations accounted for 6% of alleles, and no changes in the coding region of GJB2 were detected in 9% of DFNB1 alleles. The carrier frequency of mutation 35delG among people from the general population was one in 31 (95% CI one in 19 to one in 87). INTERPRETATION Mutations in the GJB2 gene are a major cause of inherited and apparently sporadic congenital deafness. Mutation 35delG is the most common mutation for sensorineural deafness. Identification of 35delG and other mutations in the GJB2 gene should facilitate diagnosis and counselling for the most common genetic form of deafness.

Reelin gene alleles and haplotypes as a factor predisposing to autistic disorder

Autistic disorder (MIM 209850) is currently viewed as a neurodevelopmental disease. Reelin plays a pivotal role in the development of laminar structures including the cerebral cortex, hippocampus, cerebellum and of several brainstem nuclei. Neuroanatomical evidence is consistent with Reelin involvement in autistic disorder. In this study, we describe several polymorphisms identified using RNA-SSCP and DNA sequencing. Association and linkage were assessed comparing 95 Italian patients to 186 ethnically-matched controls, and using the transmission/disequilibrium test and haplotype-based haplotype relative risk in 172 complete trios from 165 families collected in Italy and in the USA. Both case-control and family-based analyses yield a significant association between autistic disorder and a polymorphic GGC repeat located immediately 5′ of the reelin gene (RELN) ATG initiator codon, as well as with specific haplotypes formed by this polymorphism with two single-base substitutions located in a splice junction in exon 6 and within exon 50. Triplet repeats located in 5′ untranslated regions (5′UTRs) are indicative of strong transcriptional regulation. Our findings suggest that longer triplet repeats in the 5′UTR of the RELN gene confer vulnerability to autistic disorder.

Molecular basis of childhood deafness resulting from mutations in the GJB2 (connexin 26) gene

Abstract. Mutations in the GJB2 gene have been identified in many patients with childhood deafness, 35delG being the most common mutation in Caucasoid populations. We have analyzed a total of 576 families/unrelated patients with recessive or sporadic deafness from Italy and Spain, 193 of them being referred as autosomal recessive, and the other 383 as apparently sporadic cases (singletons). Of the 1152 unrelated GJB2 chromosomes analyzed from these patients, 37% had GJB2 mutations. Twenty-three different mutations were detected (1 in-frame deletion, 4 nonsense, 5 frameshift, and 13 missense mutations). Mutation 35delG was the most common, accounting for 82% of all GJB2 deafness alleles. The relative frequency of 35delG in Italy and Spain was different, representing 88% of the alleles in Italian patients and only 55% in the Spanish cases. Eight non-35delG mutations were detected more than once (V37I, E47X, 167delT, L90P, 312del14, 334delAA, R143W, and R184P), with relative frequencies ranging between 0.5 and 1.6% of the GJB2 deafness alleles. The information based on conservation of amino acid residues, coexistence with a second GJB2 mutation or absence of the mutation in non-deaf control subjects, suggests that most of these missense changes should be responsible for the deafness phenotype.

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.

Osteoporosis in beta-thalassaemia major patients: analysis of the genetic background.

Regular blood transfusions from infancy until adulthood in β‐thalassaemia major patients have substituted severe bone deformities with less marked skeletal lesions as osteoporosis. Osteoporosis is characterized by low bone mass and disruption of bone architecture, resulting in reduced bone strength and increased risk of fractures. Genetic factors have an important role in determining bone mineral density (BMD). We have investigated the possible association between BMD and two polymorphisms in 135 β‐thalassaemic patients: (i) a substitution G→Τ in a regulatory region of the COLIA1 gene encoding for the major protein of bone (type 1 collagen), and (ii) a one‐base deletion in intron 4 (713–8del C) of transforming growth factor beta 1 (TGF‐β1) gene. We have found a remarkable incidence (90%) of osteopenia and osteoporosis among regularly transfused patients. Bone mass was lower in men than in women (P = 0·0023), with a more prevalent osteopenia/osteoporosis of the spine in men than in women (P = 0·001). The sample was stratified on the basis of BMD expressed as Z‐score, i.e. normal, osteopenic and osteoporotic patients, and genotype frequencies of each group were evaluated. TGF‐β1 polymorphism failed to demonstrate a statistical difference in BMD groups. However, subjects with heterozygous or homozygous polymorphism of the COLIA1 gene showed a lower BMD than subjects without the sequence variation (P = 0·012). The differences among genotypes were still present when the BMD was analysed as adjusted Z‐score and when men and women were analysed separately (P = 0·022 and 0·004 respectively), with men more severely affected. Analysis of COLIA1 polymorphism could help to identify those thalassaemic patients at risk of osteoporosis and fractures.

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.

Sporadic and MEN1-related primary hyperparathyroidism: differences in clinical expression and severity.

Primary hyperparathyroidism (PHPT) is a common endocrine disease that is associated with multiple endocrine neoplasia type 1 (MEN1) in ∼2% of PHPT cases. Lack of a family history and other specific expressions may lead to underestimated MEN1 prevalence in PHPT. The aim of this study was to identify clinical or biochemical features predictive of MEN1 and to compare the severity of the disease in MEN1‐related versus sporadic PHPT (sPHPT). We performed a 36‐mo cross‐sectional observational study in three tertiary referral centers on an outpatient basis on 469 consecutive patients with sporadic PHPT and 64 with MEN1‐related PHPT. Serum calcium, phosphate, PTH, 25(OH)D3, and creatinine clearance were measured, and ultrasound examination of the urinary tract/urography was performed in all patients. In 432 patients, BMD was measured at the lumbar spine (LS) and femoral neck (FN). MEN1 patients showed lower BMD Z‐scores at the LS (−1.33 ± 1.23 versus −0.74 ± 1.4, p = 0.008) and FN (−1.13 ± 0.96 versus −0.6 ± 1.07, p = 0.002) and lower phosphate (2.38 ± 0.52 versus 2.56 ± 0.45 mg/dl, p = 0.003) and PTH (113.8 ± 69.5 versus 173.7 ± 135 pg/ml, p = 0.001) levels than sPHPT patients. Considering probands only, the presence of MEN1 was more frequently associated with PTH values in the normal range (OR, 3.01; 95% CI, 1.07–8.50; p = 0.037) and younger age (OR, 1.61; 95% CI, 1.28–2.02; p = 0.0001). A combination of PTH values in the normal range plus age <50 yr was strongly associated with MEN1 presence (OR, 13.51; 95% CI, 3.62–50.00; p = 0.0001). In conclusion, MEN1‐related PHPT patients show more severe bone but similar kidney involvement despite a milder biochemical presentation compared with their sPHPT counterparts. Normal PTH levels and young age are associated with MEN1 presence.

Transforming Growth Factor-β1 Gene Polymorphism, Bone Turnover, and Bone Mass in Italian Postmenopausal Women

Transforming growth factor β1 (TGF‐β1) is abundant in bone and is an important regulator of the osteoclastic‐osteoblastic interaction (coupling). The sequence variation, 713–8delC in the TGF‐β1 gene has previously been found to be associated with very low bone mass in osteoporotic women and with increased bone turnover in both osteoporotic and normal women. The possible association of this polymorphism with bone mass and bone turnover has now been investigated in 256 postmenopausal Italian women. A significant association of TGF‐β1 with bone mass was detected in the populations. Subjects carrying the sequence variation 713–8delC (Tt) genotype showed a significantly lower bone mineral density (BMD) at the hip than those without sequence variation in the genotype (TT). Individuals carrying the tt genotype have a more severe osteoporosis (P = 0.0001 vs. TT and Tt genotypes). The frequency of the fragility fractures was significantly lower in individuals with TT genotype than in those with the Tt and tt genotypes (χ2 = 21.9; P = 0.006). Furthermore a significant association was found between 713–8delC and bone turnover. The results suggest a strong evidence for an association among the 713–8delC allele of the TGF‐β1 gene and the femoral BMD, the prevalence of osteoporotic fractures, and finally a high bone turnover in a sample of Italian postmenopausal women.

3p14.1 de novo microdeletion involving the FOXP1 gene in an adult patient with autism, severe speech delay and deficit of motor coordination

Interstitial deletion of chromosome region 3p14.1, including FOXP1 gene, is relatively rare and, until recently, there were no strong evidences to support the hypothesis that this microdeletion could play a role in the etiology of genomic disorders. Here, we report on an adult patient with a recognizable phenotype of autism, severe speech delay, deficit of motor coordination and typical dysmorphic features. Analysis of a dense whole genome single-nucleotide polymorphism (SNP) array showed a 1Mb interstitial deletion of chromosome region 3p14.1 including the entire coding region of FOXP1 (MIM 605515) gene. In order to study the parental origin of the deletion, we analyzed selected SNPs in the deleted area in the proband and his parents showing Mendelian incompatibilities suggesting a de novo deletion on the chromosome of paternal origin. Despite the frequency of this genomic alteration has not been estimated, our patient confirm the hypothesis that microdeletion of 3p14.1 seems to be a rare cause of cognitive disorders and that haploinsufficiency of FOXP1 may play a role in neurological and language deficits in patients carrying a 3p14.1 deletion. Finally, our patient is also important because useful to further delineate the clinical spectrum secondary to the 3p14.1 microdeletions.