Comprehensive Cystic Fibrosis Mutation Epidemiology and Haplotype Characterization in a Southern Italian Population

We screened the whole coding region of the cystic fibrosis transmembrane regulator (CFTR) gene in 371 unrelated cystic fibrosis (CF) patients from three regions of southern Italy. Forty‐three mutations detected 91.5% of CF mutated chromosomes by denaturing gradient gel electrophoresis analysis, and three intragenic CFTR polymorphisms predicted a myriad of rare mutations in uncharacterized CF chromosomes. Twelve mutations are peculiar to CF chromosomes from southern Italy: R1158X, 4016insT, L1065P and 711+1G>T are present in 6.3% of CF chromosomes in Campania; G1244E and 852del22 are present in 9.6% of CF chromosomes in Basilicata and 4382delA, 1259insA, I502T, 852del22, 4016insT, D579G, R1158X, L1077P and G1349D are frequent in Puglia (19.6% of CF alleles). Several mutations frequently found in northern Italy (e.g., R1162X, 711+5G>T) and northern Europe (e.g., G551D, I507del and 621+1G>T) are absent from the studied population. The I148T‐3195del6 complex allele was present in two CF chromosomes, whereas I148T was present in both alleles (as a single mutation) in another CF patient and in five CF carriers; this could result from crossover events. The haplotype analysis of three intragenic polymorphisms (IVS8CA, IVS17bTA and IVS17bCA) compared with data from other studies revealed that several mutations (3849+10kbC>T, 1717‐1G>A, E585X, 3272‐26G>A, L558S, 2184insA and R347P) originated from multiple events, whereas others (R1158X and S549R) could be associated with one or more intragenic recombinant events. Given the large population migration from southern Italy, knowledge of the CF molecular epidemiology in this area is an important contribution to diagnosis, counselling and interlaboratory quality control for molecular laboratories worldwide.     

Frequencies of gap- and tight-junction mutations in Turkish families with autosomal-recessive non-syndromic hearing loss

Mutations in genes encoding gap- and tight-junction proteins have been shown to cause distinct forms of hearing loss. We have now determined the GJB2[connexin 26 (Cx26)] mutation spectrum in 60 index patients from mostly large Turkish families with autosomal-recessive inherited non-syndromic sensorineural hearing loss (NSSHL). GJB2 mutations were found in 31.7% of the families, and the GJB2-35delG mutation accounted for 73.6% of all GJB2 mutations. The carrier frequency of GJB2-35delG in the normal Turkish population was found to be 1.17% (five in 429). In addition to the described W24X, 233delC, 120delE and R127H mutations, we also identified a novel mutation, Q80R, in the GJB2 gene. Interestingly, the Q80R allele was inherited on the same haplotype as V27I and E114G polymorphisms. As little is known about the mutation frequencies of most other recently identified gap- and tight-junction genes as a cause for hearing loss, we further screened our patients for mutations in GJB3 (Cx31), GJA1 (Cx43), DeltaGJB6-D13S1830 (Cx30) and the gene encoding the tight-junction protein, claudin 14 (CLDN14). Several novel polymorphisms, but no disease-associated mutations, were identified in the CLND14 and GJA1 genes, and we were unable to detect the DeltaGJB6-D13S1830 deletion. A novel putative mutation, P223T, was found in the GJB3 gene in heterozygous form in a family with two affected children. Our data shows that the frequency of GJB2 mutations in Turkish patients with autosomal-recessive NSSHL and the carrier rate of the GJB2-35delG mutation in the Turkish population, is much lower than described for other Mediterranean countries. Furthermore, mutations in other gap- and tight-junction proteins are not a frequent cause of hearing loss in Turkey.     

Identification of 25 new mutations in 40 unrelated Spanish Niemann-Pick type C patients: genotype-phenotype correlations

To better characterize Niemann-Pick type C (NPC) in Spain and improve genetic counselling, molecular analyses were carried out in 40 unrelated Spanish patients. The search identified 70/80 alleles (88%) involving 38 different NPC1 mutations, 26 of which are described for the first time. No patient with NPC2 mutations was identified. The novel NPC1 mutations include 14 amino acid substitutions [R372W (c.1114C>T), P434L (c.1301C>T), C479Y (c.1436G>A), K576R (c.1727G>A), V727F (c.2179G>T), M754K (c.2261T>A), S865L (c.2594C>T), A926T (c.2776G>A), D948H (c.2842G>C), V959E (c.2876T>A), T1036K (c.3107C>A), T1066N (c.3197C>A), N1156I (c.3467A>T) and F1224L (c.3672C>G)], four stop codon [W260X (c.780G>A), S425X (c.1274C>A), C645X (c.1935T>A) and R1059X (c.3175C>T)], two donor splice-site mutations [IVS7+1G>A (g.31432G>A) and IVS21+2insG (g.51871insG)], one in-frame mutation [N961_F966delinsS (c.2882del16bpins1bp)] and five frameshift mutations [V299fsX8 (c.895insT), A558fsX11 (c.1673insG), C778fsX10 (c.2334insT), G993fsX3 (c.2973_78delG) and F1221fsX20 (c.3662delT)]. We also identified three novel changes [V562V (c.1686G>A), A580A (c.1740C>G) and A1187A (c.3561G>T)] in three independent NPC patients and five polymorphisms that have been described previously. The combination of these polymorphisms gave rise to the establishment of different haplotypes. Linkage disequilibrium was detected between mutations C177Y and G993fsX3 and specific haplotypes, suggesting a unique origin for these mutations. In contrast, I1061T mutation showed at least two different origins. The most prevalent mutations in Spanish patients were I1061T, Q775P, C177Y and P1007A (10, 7, 7 and 5% of alleles, respectively). Our data in homozygous patients indicate that the Q775P mutation correlates with a severe infantile neurological form and the C177Y mutation with a late infantile clinical phenotype.     

Mutation R184Q of connexin 26 in hearing loss patients has a dominant-negative effect on connexin 26 and connexin 30

Hearing impairment is the most common sensory disorder worldwide. In a recent study, the authors have shown that a heterozygous missense mutation, p.R184Q, in the connexin 26 (Cx26) is causally related to hearing loss. However, the functional change in the Cx26R184Q mutant remains unknown. This study compared the intracellular distribution and assembly of mutant Cx26R184Q with that of the wild-type (WT) Cx26 and Cx30WT in tet-on HeLa cells and the effect that the mutant protein had on those cells. Fluorescent localization assay of WT Cx26 showed the typical punctuate pattern of gap junction channel between neighboring expression cells. Conversely, the p.R184Q missense mutation resulted in accumulation of the Cx26 mutant protein in the Golgi apparatus rather than in the cytoplasmic membrane. Cx26R184Q coexpressed with either Cx26WT or Cx30WT showed perinuclear localization by bidirectional tet-on expression system, suggesting the impairment of the ability of both WT proteins to intracellular trafficking and targeting to the plasma membrane. Therefore, we proposed that Cx26R184Q has a dominant-negative effect on the function of WT Cx26 and Cx30.     

McArdle disease: the mutation spectrum of PYGM in a large Italian cohort

Deficiency of the muscle isozyme of glycogen phosphorylase is causative of McArdle disease or Glycogen storage disease type V (GSD-V), the most common autosomal recessive disorder of glycogen metabolism. The typical clinical presentation is characterized by exercise intolerance with cramps, and recurrent myoglobinuria. To date, 46 mutations in the PYGM gene have been detected in GSD-V patients. We report the mutational spectrum in 68 Italian patients. We identified 30 different mutations in the PYGM gene, including 19 mutations that have not been reported previously. The novel mutations include: eight missense mutations (c.475G>A, p.G159R; c.689C>G, p.P230R; c.1094C>T, p.A365E; c.1151C>A, p.A384D; c.1182C>T, p.R428C; c.1471C>T, p.R491C; c.2444A>C, p.D815A; c.2477G>C, p.W826S), two nonsense mutations (c.1475G>A, p.W492X; c.1627A>T, p.K543X), five splice site mutations (c.855 +1G>C; c.1092 +1G>A; c. 1093-1G>T; c.1239 +1G>A; c.2380 +1G>A), and four deletions (c.715_717delGTC, p.V239del; c.304delA, p.N102DfsX4; c.1970_2177del, p.V657_G726; c.2113_2114delGG, p.G705RfsX16). Whereas we confirmed lack of direct correlation between the clinical phenotype and the genotype, we also found that the so-called 'common mutation' (p.R50X) accounted for about 43% of alleles in our cohort and that no population-related mutations are clearly identified in Italian patients.     

Different functional consequences of two missense mutations in the GJB2 gene associated with non‐syndromic hearing loss

Mutations in the GJB2 gene, which encodes the gap junction (GJ) protein connexin26 (Cx26), are the most common cause of inherited non‐syndromic hearing loss (NSHL). We identified two missense mutations, p.D46E (c.138T>G) and p.T86R (c.257C>G), of GJB2 in Korean HL families. The novel p.D46E mutation exhibited autosomal dominant inheritance, while the p.T86R mutation, which is exclusively found in Asians, segregated with an autosomal recessive pattern. Thus, we sought to elucidate the pathogenic nature of such different inherited patterns of HL. We studied protein localization and gap junction functions in cells transfected with wild‐type or mutant Cx26 tagged with fluorescent proteins, which allowed visual confirmation of homozygous or heterozygous mutant GJs. The Cx26‐D46E mutant was targeted to the plasma membrane, but this mutant protein failed to transfer Ca²⁺ or propidium iodide intercellularly, suggesting disruption of both ionic and biochemical coupling. Heterozygous GJs also showed dysfunctional intercellular couplings and hemichannel opening, confirming the dominant‐negative nature of the p.D46E mutation. The Cx26‐T86R mutant protein did not form GJs, since the mutated protein was confined in the cytoplasm and not transported to the cell membrane. When Cx26‐T86R was co‐expressed with Cx26‐WT, ionic and biochemical coupling was normal, consistent with the recessive nature of the mutation. These studies revealed distinct pathogenic mechanisms of two GJB2 mutations identified in Korean families. © 2009 Wiley‐Liss, Inc.     

Mutation spectrum of phenylketonuria in Iranian population

Identification of molecular basis of phenylketonuria (PKU) in Iran has been accomplished through the analysis of 248 unrelated chromosomes from 124 Iranian classic PKU subjects. Phenylalanine hydroxylase (PAH) gene mutations were analyzed through a combined approach in which p.S67P, p.R252W, p.R261Q, p.R261X, p.L333F, IVS10-11G>A, IVS11+1G>C, p.L364del, p.R408Q and p.R408W mutations were first screened by PCR of PAH gene exons 3, 7, 10, 11 and 12, followed by digestion with the appropriate digestion enzymes. Subsequently SSCP analysis for exons 2, 6, 7 and 11 of the PAH gene and finally, sequencing of 13 PAH gene exons have been used to study uncharacterized PKU chromosomes. 26 different mutations were found. The predominant mutation in this population sample was IVS10-11G>A, with a frequency of 24.6%. Nine mutations (IVS10-11G>A, p.R261Q, p.P281L, IVS11+1G>C, p.K363>NFS, p.R243X, IVS2+5G>C, p.R261X and p.R252W) represent almost 84% of all PKU chromosomes studied. IVS10-11G>A mutation is the major PKU-causing mutation throughout the Mediterranean region. The finding of the high prevalence of this mutation in Iranian population is consistent with the historical and geographical links between Iranian and Mediterranean populations.     

Mucopolysaccharidosis I mutations in Chinese patients: identification of 27 novel mutations and 6 cases involving prenatal diagnosis

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease that results from the deficiency of α-l-iduronidase and is transmitted in an autosomally recessive manner. This report describes the first systematic screening for mutations in Chinese MPS I patients from mainland China, wherein we have summarized the phenotype/genotype correlation of the individuals in Chinese MPS I patients. Mutational analyses were performed in 57 unrelated Chinese MPS I patients. Overall, 105 mutant alleles were identified from a set of 41 different mutations. Notably, of these 41 mutations, 27 were novel mutations that consisted of 8 splicing mutations (c.1-2C>G, c.296+4G>A, c.300-1G>C, c.792+1G>C, c.973-4G>A, c.1189+5G>T, c.1402+1G>T and c.1402+2T>G), 1 nonsense mutation (p.W41X), 1 insertion (c.668-670ins GCG), 5 duplications (c.531dupT, c.657dupG, c.883dupC, c.1147dupG and c.1225dupG), 3 deletions (c.349delT, c.1593delG and c.1244-1271del27),1 nucleotide substitution c.2T>C and 8 missense mutations (p.H33P,p.F52L, p.G168V,p.T179R,p. E182D, p.L237R, p.L238R and p.L421P). The missense mutations p.A79V and p.L346R, which accounted for 16.7% (19/114) and 12.3% (14/114) respectively, were the common mutations in Chinese patients but were rare in the mutational profiles reported for other populations. These results indicate that Chinese MPS I patients may have a different mutational spectrum compared to those of other populations. Moreover, for the first time in China, molecular genetic methods were used for prenatal diagnosis of six cases in five families.     

Identification of the bruton tyrosine kinase (BTK) gene mutations in 20 Australian families with X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the Bruton tyrosine kinase (BTK) gene. Twenty Australian patients with an XLA phenotype, from 15 unrelated families, were found to have 14 mutations. Five of the mutations were previously described c.83G>A (p.R28H), c.862C>T (p.R288W), c.904G>A (p.R302G), c.1535T>C (p.L512P), c.700C>T (p.Q234X), while nine novel mutations were identified: four missense c.82C>A (p.R28S), c.494G>A (p.C165Y), c.464G>A (p.C155Y), c.1750G>A (p.G584E), one deletion c.142_144delAGAAGA (p.R48_G50del), and four splice site mutations c.241-2A>G, c.839+4A>G, c.1350-2A>G, c.1566+1G>A. Carrier analysis was performed in 10 mothers and 11 female relatives. The results of this study further support the notion that molecular genetic testing represents an important tool for definitive and early diagnosis of XLA and may allow accurate carrier status and prenatal diagnosis.     

GJB2 mutations in Iranians with autosomal recessive non-syndromic sensorineural hearing loss

Hereditary hearing loss (HHL) is an extremely common disorder. About 70% of HHL is non-syndromic, with autosomal recessive forms accounting for approximately 85% of the genetic load. Although very heterogeneous, the most common cause of HHL in many different world populations is mutations of GJB2, a gene that encodes the gap junction protein connexin 26 (Cx26). This study investigates the contribution of GJB2 to the autosomal recessive non-syndromic deafness (ARNSD) load in the Iranian population. One hundred sixty eight persons from 83 families were studied. GJB2-related deafness was diagnosed in 9 families (4, 35delG homozygotes; 3, 35delG compound heterozygotes; 1, W24X homozygote; 1, non-35delG compound heterozygote). The carrier frequency of the 35delG allele in this population was approximately 1% (1/83). Because the relative frequency of Cx26 mutations is much less than in the other populations, it is possible that mutations in other genes play a major role in ARNSD in Iran.     

Mutational analysis of mucopolysaccharidosis type VI patients undergoing a trial of enzyme replacement therapy

Mucopolysaccharidosis type VI (MPS VI), or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Seven MPS VI patients were chosen for the initial clinical trial of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Nine substitutions (c.289C>T [p.Q97X], c.629A>G [p.Y210C], c.707T>C [p.L236P], c.936G>T [p.W312C], c.944G>A [p.R315Q], c.962T>C [p.L321P], c.979C>T [p.R327X], c.1151G>A [p.S384N], and c.1450A>G [p.R484G]), two deletions (c.356_358delTAC [p.Y86del] and c.427delG), and one intronic mutation (c.1336+2T>G) were identified. A total of 7 out of the 12 mutations identified were novel (p.Y86del, p.Q97X, p.W312C, p.R327X, c.427delG, p.R484G, and c.1336+2T>G). Two of these novel mutations (p.Y86del and p.W312C) were expressed in Chinese hamster ovary cells and analyzed for residual ARSB activity and mutant ARSB protein. The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified among the patients, along with the silent mutation c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient, and, together with genotype information, were used to predict the expected clinical severity of each MPS VI patient.     

Liver glycogen storage diseases due to phosphorylase system deficiencies: diagnosis thanks to non invasive blood enzymatic and molecular studies

Glycogen storage disease (GSD) due to a deficient hepatic phosphorylase system defines a genetically heterogeneous group of disorders that mainly manifests in children. We investigated 45 unrelated children in whom a liver GSD VI or IX was suspected on the basis of clinical symptoms including hepatomegaly, increased serum transaminases, postprandial lactatemia and/or mild fasting hypoglycemia. Liver phosphorylase and phosphorylase b kinase activities studied in peripheral blood cells allowed to suspect diagnosis in 37 cases but was uninformative in 5. Sequencing of liver phosphorylase genes was useful to establish an accurate diagnosis. Causative mutations were found either in the PYGL (11 patients), PHKA2 (26 patients), PHKG2 (three patients) or in the PHKB (three patients) genes. Eleven novel disease causative mutations, five missense (p.N188K, p.D228Y, p.P382L, p.R491H, p.L500R) and six truncating mutations (c.501_502ins361pb, c.528+2T>C, c.856-29_c.1518+614del, c.1620+1G>C, p.E703del and c.2313-1G>T) were identified in the PYGL gene. Seventeen novel disease causative mutations, ten missense (p.A42P, p.Q95R, p.G131D, p.G131V, p.Q134R, p.G187R, p.G300V, p.G300A, p.C326Y, p.W820G) and seven truncating (c.537+5G>A, p.G396DfsX28, p.Q404X, p.N653X, p.L855PfsX87, and two large deletions) were identified in the PHKA2 gene. Four novel truncating mutations (p.R168X, p.Q287X, p.I268PfsX12 and c.272-1G>C) were identified in the PHKG2 gene and three (c.573_577del, p.R364X, c.2427+3A>G) in the PHKB gene. Patients with PHKG2 mutations evolved towards cirrhosis. Molecular analysis of GSD VI or IX genes allows to confirm diagnosis suspected on the basis of enzymatic analysis and to establish diagnosis and avoid liver biopsy when enzymatic studies are not informative in blood cells.     

Molecular analysis of 30 mucopolysaccharidosis type I patients: evaluation of the mutational spectrum in Italian population and identification of 13 novel mutations

Mucopolysaccharidosis type I (MPS-I orMPS1) is an autosomal recessive condition characterized by a broad range of clinical symptoms. Molecular diagnosis of MPS-I is important for analyzing genotype-phenotype correlation and for selecting patients for innovative therapies. In this study we analyzed 30 Italian MPS-I patients with different phenotypes (20 severe, 6 intermediate, 4 mild) in an attempt to recognize the mutational spectrum in our population and to identify major DNA alterations specific to our country. We identified 93% of mutated alleles (56 out of 60) with the reconstruction of the complete genotype in 26 patients out of 30. Twenty-three different mutations were found, 13 of which are novel while the remaining 10 have been already described. Among the novel mutations we found 5 non conservative missense mutations (A160D, E178K, P183R, G197D, D349Y), one nonsense mutation (C53X), 6 deletions (468-470del3, 486-491del6, 755-759del5, 1251delC, 1839-1867del29, 1902-1903del2), and one splice site mutation (IVS11+5G>A). No common mutation for MPS-I is present in our country. Frequently (40% of the alleles), mutations were found in just one or two patients. However, Q70X, P533R, G51D, and W402X mutations were present in several patients (15%, 13.3%, 13.3%, and 11.6% of the alleles respectively) suggesting a Mediterranean origin of the P533R and G51D mutations. In most cases the patients' genotypes were unique combinations of mutations. The great heterogeneity found in our MPS-I population hampers mutation detection and hinders the genotype-phenotype correlation.     

Fabry disease: 45 novel mutations in the alpha-galactosidase A gene causing the classical phenotype

The nature of the molecular lesions in the alpha-galactosidase A (alpha-Gal A) gene causing Fabry disease was determined in 50 unrelated families with the classic phenotype of this X-linked recessive lysosomal storage disease. Genomic DNA was isolated from affected males or obligate carrier females, and the entire alpha-Gal A coding region as well as the flanking and intronic sequences were analyzed by PCR amplification and automated sequencing. Forty-five new mutations were identified including 38 single base substitutions (32 missense and four nonsense) and nine gene rearrangements: MIR, M42T, G43D, G43V, H46Y, F50C, L68F, G132R, T141I, Y152X, K168R, G183S, V199M, P205R, Y207S, Q221X, C223R, C223Y, D234Y, G271C, A288P, P293A, R301G, I303N, I317T, E341D, P362L, R363C, R363H, G373D, I384N, T385P, Q396X, E398K, S401X, P409A, g7325insC, g7384del13, g8341delG, g8391del4/ins3, g10511delTAGT, g10704delACAG, g11019insG, g11021insG, and g11048delAGG. In the remaining five Fabry families, four previously reported mutations were detected (W81X, R112C, g11011delTC, and g11050delGAG) of which the R112C substitution was found in two families who were unrelated by haplotyping. These studies further define the heterogeneity of mutations in the alpha-Gal A gene causing the classical Fabry disease phenotype, and permit precise carrier detection and prenatal diagnosis in these families.     

Acid sphingomyelinase: identification of nine novel mutations among Italian Niemann Pick type B patients and characterization of in vivo functional in-frame start codon

Niemann Pick disease (NPD) is an autosomal recessive disorder due to the deficit of lysosomal acid sphingomyelinase, which results in intracellular accumulation of sphingomyelin. In the present work we studied 18 patients with NPD type B, including five individuals who presented an intermediate phenotype characterised by different levels of neurological involvement. We identified nine novel mutations in the SMPD1 gene including six single base changes c.2T>G, c.96G>A, c.308T>C, c.674T>C, c.732G>C, c.841G>A (p.M1_W32del, p.W32X, p.L103P, p.L225P, p.W244C, p.A281T) and three frameshift mutations c.100delC, c.565dupC, c.575dupC (p.G34fsX42, p.P189fsX1 and p.P192fsX14). The novel c.2T>G (p.M1_W32del) mutation inactivates the first in-frame translation start site of the SMPD1 gene and in the homozygous status causes NPD type B indicating that in'vivo translation of wild type SMPD1 initiates from the first in-frame ATG. Moreover, the new c.96G>A (p.W32X) introduces a premature stop codon before the second in-frame ATG. As a consequence of either c.2T>G (p.M1_W32del) or c.96G>A (p.W32X), impaired translation from the first in-frame ATG results in a mild NPD-B phenotype instead of the severe phenotype expected for a complete deficiency of the enzyme, suggesting that when the first ATG is not functional, the second initiation codon (ATG33) still produces a fairly functional sphingomyelinase. Analysis of the patients'clinical and molecular data demonstrated that all five patients with the intermediate phenotype carried at least one severe mutation. No association between the onset of pulmonary symptoms and genotype was observed. Finally, the presence of c.96G>A (p.W32X), the most frequent allele among Italian NPD type B population, and c.1799G>C (p.R600P) as compound heterozygotes in association with severe mutations suggested a beneficial effect for both mutations.     

Prevalence of Deafness‐Associated Connexin‐26 (GJB2) and Connexin‐30 (GJB6) Pathogenic Alleles in a Large Patient Cohort from Eastern Sicily

Mutations in the gene encoding the gap junction protein connexin 26 (GJB2) and connexin 30 (GJB6) have been shown to be a major contributor to prelingual, sensorineural, nonsyndromic deafness. The aim of this study was to characterize and establish the prevalence of GJB2 and GJB6 gene alterations in 196 patients affected by sensorineural, nonsyndromic hearing loss, from Eastern Sicily. We performed sequence analysis of GJB2 and identified sequence variants in 68 out of 196 patients (34.7%); (28 homozygous for c.35delG, 22 compound heterozygous and 11 with only one variant allele). We found 12 different allelic variants, the most prevalent being c.35delG, which was found on 89 chromosomes (65.5%), followed by other alleles with different frequencies (p.E47X, c.‐23+1G>A, p.L90P, p.R184W, p.M34T, c.167delT, p.R127H, p.M163V, p.V153I, p.W24X, and p.T8M). Importantly, for the first time we present the frequency and spectrum of GJB2 mutations in NSHL patients from Eastern Sicily. No alterations were found in the GJB6 gene, confirming that alterations in this gene are uncommon in our geographic area. Note that 65.3% and 23.5% of our patients, respectively were found to be negative or carriers by GJB2 molecular screening. This emphasizes the need to broaden the genetic analysis to other genes involved in hearing loss.     

The major allele of the alanine:glyoxylate aminotransferase gene: nine novel mutations and polymorphisms associated with primary hyperoxaluria type 1

We describe nine novel mutations and polymorphisms occurring on the major allele of the human alanine:glyoxylate aminotransferase gene in patients with primary hyperoxaluria type 1, an autosomal recessive disease resulting from a deficiency of the liver peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44). The PH1 mutations include two small frameshift mutations, 327delG and 117_118insCA, a large deletion spanning exon 9 and portions of the flanking introns, a splice junction mutation, IVS6+5G>C, and two missense mutations, G161R and S218L. Expression studies of the two missense mutations indicated very little enzymatic activity associated with either of them. Three polymorphisms in the coding sequence were also identified, I279T, A280V, and T235T. Expression studies of I279T and A280V suggested essentially normal AGT activity. I279T, found in two cases, was located on a 33_34insC allele. A280V and T235T were both located on the same allele as IVS6+5G>C. We have also identified recurrences of previously reported rare mutations, 33delC, IVS7-1G>C, and IVS4-1G>A. Five of the six novel PH1 mutations occurred in a compound heterozygous state with either of two common PH1 mutations, G170R or 33_34insC. S218L was apparently homozygous in two individuals. These findings contribute to our overall picture of heterogeneity of mutations in PH1 and the AGT major allele.     

Analysis of the glucocerebrosidase gene and mutation profile in 144 Italian gaucher patients

Gaucher disease (GD), the most prevalent lysosomal storage disease characterized by a remarkable degree of clinical variability, results from deleterious mutations in the glucocerebrosidase gene (GBA). In this paper we report the molecular characterization of 144 unrelated Italian GD patients with the three types of the disease. The allelic frequencies of Italians are reported and the mutation profile is analyzed. Besides the common N370S, L444P, RecNciI, G202R, IVS2+1G>A, D409H, F213I mutations, the different molecular strategies, used for the mutation detection, identified the rare N107L, R131C, R170C, R170P, N188S, S196P, R285C, R285H, W312C, D399N, A446P, IVS10-1G>A, RecDelta55, total gene deletion, as well as 12 mutant alleles that were exclusively present in the Italian population until now: the previously reported R353G, N370S+S488P mosaicism, IVS8(-11delC)-14T>A), Rec I, Y418C, and the seven novel alleles D127X, P159T, V214X, T231R, L354X, H451R, and G202R+M361I. The wide phenotypic differences observed within the genotypic groups as well as between siblings implicate a significant contribution of other modifying genetic and/or non-genetic factors and claim a comprehensive valuation of the patient including clinical., biochemical and molecular investigations for prognosis, appropriate interventive therapy and reliable genetic counseling.     

Functional analysis of 13 GBA mutant alleles identified in Gaucher disease patients: Pathogenic changes and "modifier" polymorphisms

Gaucher disease, the most prevalent sphingolipidosis, is caused by the deficient activity of acid beta-glucosidase, mainly due to mutations in the GBA gene. Over 200 mutations have been identified worldwide, more than 25 of which were in Spanish patients. In order to demonstrate causality for Gaucher disease, some of them: c.662C>T (p.P182L), c.680A>G (p.N188S), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1093G>A (p.E326K), c.1289C>T (p.P391L), c.1292A>T (p.N392I), c.1322T>C (p.I402T), and the double mutants [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), were expressed in Sf9 cells using a baculovirus expression system. Other well-established Gaucher disease mutations, namely c.1226A>G (p.N370S), c.1342G>C (p.D409H), and c.1448T>C (p.L444P), were also expressed for comparison. The levels of residual acid beta-glucosidase activity of the mutant enzymes produced by the cDNAs carrying alleles c.662C>T (p.P182L), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1289C>T (p.P391L), and c.1292A>T (p.N392I) were negligible. The c.1226A>G (p.N370S), c.1322T>C (p.I402T), c.1342G>C (p.D409H), c.1448T>C (p.L444P), and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]) alleles produced enzymes with levels ranging from 6 to 14% of the wild-type. The three remaining alleles, c.680A>G (p.N188S), c.1093G>A (p.E326K), and [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]), showed higher activity (66.6, 42.7, and 23.2%, respectively). Expression studies revealed that the c.1093G>A (p.E326K) change, which was never found alone in a Gaucher disease-causing allele, when found in a double mutant such as [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), decreases activity compared to the activity found for the other mutation alone. These results suggest that c.1093G>A (p.E326K) should be considered a "modifier variant" rather than a neutral polymorphism, as previously considered. Mutation c.680A>G (p.N188S), which produces a mutant enzyme with the highest level of activity, is probably a very mild mutation or another "modifier variant."