Novel mutations in two unrelated Italian patients with SSADH deficiency.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive disorder of γ-aminobutyric acid (GABA) catabolism caused by mutations in the gene coding for succinic semialdehyde dehydrogenase (ALDH5A1). The abnormal levels of GHB detected in the brain and in all physiological fluids of SSADHD patients represent a diagnostic biochemical hallmark of the disease. Here we report on the clinical and molecular characterization of two unrelated Italian patients and the identification of two novel mutations: a 22 bp DNA duplication in exon 1, c.114_135dup, p.(C46AfsX97), and a non-sense mutation in exon 10, c.1429C > T, p.(Q477X). The two patients showed very different clinical phenotypes, coherent with their age. These findings enrich the characterization of SSADHD families and contribute to the knowledge on the progression of the disease.

Succinic semialdehyde dehydrogenase deficiency: The combination of a novel ALDH5A1 gene mutation and a missense SNP strongly affects SSADH enzyme activity and stability.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive metabolic disorder of GABA catabolism. SSADH is a mitochondrial homotetrameric enzyme encoded by ALDH5A1 gene. We report the molecular characterization of ALDH5A1 gene in an Italian SSADHD patient, showing heterozygosity for four missense mutations: c.526G>A (p.G176R), c.538C>T (p.H180Y), c.709G>T (p.A237S) and c.1267A>T (p.T423S), the latter never described so far. The patient inherited c.526A in cis with c.538T from the mother and c.709T in cis with c.1267T from the father. To explore the effects of the two allelic arrangements on SSADH activity and protein level, wild type, single or double mutated cDNA constructs were expressed in a cell system. The p.G176R change, alone or in combination with p.H180Y, causes the abolishment of enzyme activity. Western blot analysis showed a strongly reduced amount of the p.176R-p.180Y double mutant protein, suggesting increased degradation. Indeed, in silico analyses confirmed high instability of this mutant homotetramer. Enzyme activity relative to the other p.423S-p.237S double mutant is around 30% of wt. Further in silico analyses on all the possible combinations of mutant monomers suggest the lowest stability for the tetramer constituted by p.176R-p.180Y monomers and the highest stability for that constituted by p.237S-p.423S monomers. The present study shows that when a common SNP, associated with a slight reduction of SSADH activity, is inherited in cis with a mutation showing no consequences on the enzyme function, the activity is strongly affected. In conclusion, the peculiar arrangement of four missense mutations occurring in this patient is responsible for the SSADHD phenotype.

SSADH deficiency in an Italian family: a novel ALDH5A1 gene mutation affecting the succinic semialdehyde substrate binding site.

SSADH deficiency (SSADHD) is a rare autosomal recessively inherited metabolic disorder. It is associated with mutations of ALDH5A1 gene, coding for the homotetrameric enzyme SSADH. This enzyme is involved in γ-aminobutyric acid (GABA) catabolism, since it oxidizes succinic semialdehyde (SSA) to succinate. Mutations in ALDH5A1 gene result in the abnormal accumulation of γ-hydroxybutyrate (GHB), which is pathognomonic of SSADHD. In the present report, diagnosis of SSADHD in a three-month-old female was achieved by detection of high levels of GHB in urine. Sequence analysis of ALDH5A1 gene showed that the patient was a compound heterozygote for c.1226G > A (p.G409D) and the novel missense mutation, c.1498G > C (p.V500 L). By ALDH5A1 gene expression in transiently transfected HEK293 cells and enzyme activity assays, we demonstrate that the p.V500 L mutation, despite being conservative, produces complete loss of enzyme activity. In silico protein modelling analysis and evaluation of tetramer destabilizing energies suggest that structural impairment and partial occlusion of the access channel to the active site affect enzyme activity. These findings add further knowledge on the missense mutations associated with SSADHD and the molecular mechanisms underlying the loss of the enzyme activity.

Alcohol use disorder and GABAB receptor gene polymorphisms in an Italian sample: haplotype frequencies, linkage disequilibrium and association studies.

BACKGROUND: Alcohol use disorder (AUD) is a complex trait with genetic and environmental influences. Several gene variants have been associated with the risk for AUD, including genes encoding the sub-units of the γ-aminobutyric acid (GABA) receptors. AIM: This study evaluated whether specific single nucleotide polymorphisms (SNPs) in genes encoding GABAB receptor sub-units can be considered as candidates for the risk of AUD. SUBJECTS AND METHODS: Seventy-four AUD subjects and 128 Italian controls were genotyped for 10 SNPs in genes encoding GABA-B1 and GABA-B2 sub-units (GABBR1 and GABBR2). Allele, genotype, and haplotype frequencies were tested for the association with the AUD trait. RESULTS: A significant difference between AUD individuals and controls was observed at genotype level for rs2900512 of GABBR2 gene. The homozygous T/T genotype was not found in the controls, whereas it was over-represented in the AUD individuals. Under the recessive model (T/T vs C/T + C/C) this result was statistically significant, as well as the Odds Ratio for the association with the AUD trait. CONCLUSIONS: The results provide preliminary data on the association between GABAB receptor gene variation and risk of AUD. To confirm this finding, studies with larger samples and additional characterisation of the phenotypic AUD trait are required.

Variation of the 3'RR1 HS1.2 Enhancer and Its Genomic Context.

In humans, the HS1.2 enhancer in the Ig heavy-chain locus is modular, with length polymorphism. Previous studies have shown the following features for this variation: (i) strong population structuring; (ii) association with autoimmune diseases; and (iii) association with developmental changes in Ig expression. The HS1.2 region could then be considered as a contributor to inter-individual diversity in humoral response in adaptive immunity. We experimentally determined the HS1.2-length class genotype in 72 of the 1000 Genomes CEU cell lines and assigned the HS1.2 alleles to haplotypes defined by 18 landmark SNPs. We also sequenced the variable portion and ~200 bp of the flanking DNA of 34 HS1.2 alleles. Furthermore, we computationally explored the ability of different allelic arrangements to bind transcription factors. Non-random association between HS1.2 and Gm allotypes in the European population clearly emerged. We show a wealth of variation in the modular composition of HS1.2, with five SNPs further contributing to diversity. Longer alleles offer more potential sites for binding but, for same-length alleles, SNP variation creates/destroys potential binding sites. Altogether, the arrangements of modules and SNP alleles both inside and outside HS1.2 denote an organization of diversity far from randomness. In the context of the strong divergence of human populations for this genomic region and the reported disease associations, our results suggest that selective forces shaped the pattern of its diversity.

Genetic aspects underlying the normocalcemic and hypercalcemic phenotypes of primary hyperparathyroidism.

PURPOSE: Hypercalcemic primary hyperparathyroidism (PHPT) is a common endocrine disorder that has been very well characterized. In contrast, many aspects of normocalcemic primary hyperparathyroidism (NPHPT) such as natural history, organ damage, and management are still matter of debate. In addition, both the pathophysiology and molecular basis of NPHPT are unclear. We investigated whether PHPT and NPHPT patient cohorts share the same pattern of genetic variation in genes known to be involved in calcium and/or bone metabolism. RESEARCH DESIGN AND METHODS: Genotyping for 9 single nucleotide polymorphisms (SNPs) was performed by Real-Time PCR (TaqMan assays) on 27 NPHPT and 31 PHPT patients evaluated in a tertiary referral Center. The data of both groups were compared with 54 in house-controls and 503 subjects from the 1000 Genomes Project. All groups were compared for allele/haplotype frequencies, on a single locus, two loci and multi-locus basis. RESULTS: The NPHPT group differed significantly at SNPs in OPG and ESR1. Also, the NPHPT cohort was peculiar for pairwise associations of genotypes and for the overrepresentation of unusual multilocus genotypes. CONCLUSIONS: Our NPHPT patient set harbored a definitely larger quota of genetic diversity than the other samples. Specific genotypes may help in defining subgroups of NPHPT patients which deserve ad hoc clinical and follow-up studies.

Haemoglobin S and haemoglobin C: 'quick but costly' versus 'slow but gratis' genetic adaptations to Plasmodium falciparum malaria.

Haemoglobin S (HbS; beta6Glu-->Val) and HbC (beta6Glu-->Lys) strongly protect against clinical Plasmodium falciparum malaria. HbS, which is lethal in homozygosity, has a multi-foci origin and a widespread geographic distribution in sub-Saharan Africa and Asia whereas HbC, which has no obvious CC segregational load, occurs only in a small area of central West-Africa. To address this apparent paradox, we adopted two partially independent haplotypic approaches in the Mossi population of Burkina Faso where both the local S (S(Benin)) and the C alleles are common (0.05 and 0.13). Here we show that: both C and S(Benin) are monophyletic; C has accumulated a 4-fold higher recombinational and DNA slippage haplotypic variability than the S(Benin) allele (P = 0.003) implying higher antiquity; for a long initial lag period, the C alleles did apparently remain very few. These results, consistent with epidemiological evidences, imply that the C allele has been accumulated mainly through a recessive rather than a semidominant mechanism of selection. This evidence explains the apparent paradox of the uni-epicentric geographic distribution of HbC, representing a 'slow but gratis' genetic adaptation to malaria through a transient polymorphism, compared to the polycentric 'quick but costly' adaptation through balanced polymorphism of HbS.

Polymorphic Genetic Markers of the GABA Catabolism Pathway in Alzheimer's Disease.

BACKGROUND: The compilation of a list of genetic modifiers in Alzheimer's disease (AD) is an open research field. The GABAergic system is affected in several neurological disorders but its role in AD is largely understudied. OBJECTIVE/METHODS: As an explorative study, we considered variants in genes of GABA catabolism (ABAT, ALDH5A1, AKR7A2), and APOE in 300 Italian patients and 299 controls. We introduce a recent multivariate method to take into account the individual APOE genotype, thus controlling for the effect of the discrepant allele distributions in cases versus controls. We add a genotype-phenotype analysis based on age at onset and the Mini-Mental State Evaluation score. RESULTS: On the background of strongly divergent APOE allele distributions in AD versus controls, two genotypic interactions that represented a subtle but significant peculiarity of the AD cohort emerged. The first is between ABAT and APOE, and the second between some ALDH5A1 genotypes and APOE. Decreased SSADH activity is predicted in AD carriers of APOEɛ4, representing an additional suggestion for increased oxidative damage. CONCLUSION: We identified a difference between AD and controls, not in a shift of the allele frequencies at genes of the GABA catabolism pathway, but rather in gene interactions peculiar of the AD cohort. The emerging view is that of a multifactorial contribution to the disease, with a main risk factor (APOE), and additional contributions by the variants here considered. We consider genes of the GABA degradation pathway good candidates as modifiers of AD, contributing to energy impairment in AD brain.

Signs of continental ancestry in urban populations of Peru through autosomal STR loci and mitochondrial DNA typing.

The human genetic diversity around the world was studied through several high variable genetic markers. In South America the demic consequences of admixture events between Native people, European colonists and African slaves have been displayed by uniparental markers variability. The mitochondrial DNA (mtDNA) has been the most widely used genetic marker for studying American mixed populations, although nuclear markers, such as microsatellite loci (STRs) commonly used in forensic science, showed to be genetically and geographically structured. In this work, we analyzed DNA from buccal swab samples of 296 individuals across Peru: 156 Native Amazons (Ashaninka, Cashibo and Shipibo from Ucayali, Huambiza from Loreto and Moche from Lambayeque) and 140 urban Peruvians from Lima and other 33 urban areas. The aim was to evaluate, through STRs and mtDNA variability, recent migrations in urban Peruvian populations and to gain more information about their continental ancestry. STR data highlighted that most individuals (67%) of the urban Peruvian sample have a strong similarity to the Amazon Native population, whereas 22% have similarity to African populations and only ~1% to European populations. Also the maternally-transmitted mtDNA confirmed the strong Native contribution (~90% of Native American haplogroups) and the lower frequencies of African (~6%) and European (~3%) haplogroups. This study provides a detailed description of the urban Peruvian genetic structure and proposes forensic STRs as a useful tool for studying recent migrations, especially when coupled with mtDNA.

Haplotype block structure study of the CFTR gene. Most variants are associated with the M470 allele in several European populations.

An average of about 1700 CFTR (cystic fibrosis transmembrane conductance regulator) alleles from normal individuals from different European populations were extensively screened for DNA sequence variation. A total of 80 variants were observed: 61 coding SNSs (results already published), 13 noncoding SNSs, three STRs, two short deletions, and one nucleotide insertion. Eight DNA variants were classified as non-CF causing due to their high frequency of occurrence. Through this survey the CFTR has become the most exhaustively studied gene for its coding sequence variability and, though to a lesser extent, for its noncoding sequence variability as well. Interestingly, most variation was associated with the M470 allele, while the V470 allele showed an 'extended haplotype homozygosity' (EHH). These findings make us suggest a role for selection acting either on the M470V itself or through an hitchhiking mechanism involving a second site. The possible ancient origin of the V allele in an 'out of Africa' time frame is discussed.

A large-scale study of the random variability of a coding sequence: a study on the CFTR gene.

Coding single nucleotide substitutions (cSNSs) have been studied on hundreds of genes using small samples (n(g) approximately 100-150 genes). In the present investigation, a large random European population sample (average n(g) approximately 1500) was studied for a single gene, the CFTR (Cystic Fibrosis Transmembrane conductance Regulator). The nonsynonymous (NS) substitutions exhibited, in accordance with previous reports, a mean probability of being polymorphic (q > 0.005), much lower than that of the synonymous (S) substitutions, but they showed a similar rate of subpolymorphic (q < 0.005) variability. This indicates that, in autosomal genes that may have harmful recessive alleles (nonduplicated genes with important functions), genetic drift overwhelms selection in the subpolymorphic range of variability, making disadvantageous alleles behave as neutral. These results imply that the majority of the subpolymorphic nonsynonymous alleles of these genes are selectively negative or even pathogenic.

Confirmation of the potential usefulness of two human beta globin pseudogene markers to estimate gene flows to and from sub-Saharan Africans.

Two polymorphic sites, -107 and -100 with respect to the "cap" site of the human beta globin pseudogene, recently discovered in our laboratory, turned out to have an ethnically complementary distribution. The first site is polymorphic in Europeans, North Africans, Indians (Hindu), and Oriental Asians, and monomorphic in sub-Saharan Africans. Conversely, the second site is polymorphic in sub-Saharan African populations and monomorphic in the aforementioned populations. Here we report the gene frequencies of these two polymorphic sites in nine additional populations (Egyptians, Spaniards, Japanese, Chinese, Filipinos, Vietnamese, Africans from Togo and from Benin, and Pygmies), confirming their ethnospecificity and, through the analysis of these two markers in Oromo and Amhara of Ethiopia (two mixed populations), their usefulness in genetic admixture studies. Moreover, we studied another marker polymorphic in sub-Saharan African populations only, a TaqI restriction fragment length polymorphism located in the same region as the present markers, demonstrating the absence of linkage disequilibrium between it and the -100 site, so that we can exclude that the information they provide is redundant.