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Rubinstein-Taybi syndrome is a rare autosomal dominant congenital disorder characterized by postnatal growth retardation, psychomotor developmental delay, skeletal anomalies, peculiar facial morphology, and tumorigenesis. Mutations in the gene encoding the cAMP response element-binding protein (CREB, also known as CREBBP or CBP) on chromosome 16p13.3 have been identified. In addition, some patients with low intelligence quotients and autistic features bear large deletions. Based on these observations, we used multiplex ligation-dependent probe amplification to search for large deletions affecting the CREBBP gene in a Rubinstein-Taybi syndrome patient. We identified a novel heterozygote deletion removing five exons (exons 17-21), encoding the histone acetyltransferase domain. We propose the use of multiplex ligation-dependent probe amplification as a fast, accurate and cheap test for detecting large deletions in the CREBBP gene in the sub-group of Rubinstein-Taybi syndrome patients with low intelligence quotients and autistic features.

A detailed study of the mutant phenylalanine hydroxylase (PAH) gene from the eastern part of the Czech Republic (Moravia) is reported. A total of 190 mutant alleles from 95 phenylketonuria (PKU) families were analyzed for 21 prevalent Caucasian mutations and restriction fragment length polymorphism/variable number of tandem repeats (RFLP/VNTR) haplotypes. Eighty per cent of all mutant alleles were found to carry 11 mutations. The most common molecular defect was the mutation R408W (55.3%), with a very high degree of homozygosity (34.6%). Each of four other mutations (R158Q, R243X, G272X, IVS12nt1) accounted for more than 3% of PKU alleles. Rarely present were mutations IVS10nt546 (2.6%), R252W (2.6%), L48S (2.1%), R261Q (1.6%), Y414C (1.0%) and 165T (0.5%). Mutations that have been predominantly described in southern Europe (IVS7nt1, A259V, Y277D, R241H, T278N) were not detected. A total of 14 different mutant haplotypes were observed. Three unusual genotype-haplotype associations were identified (R158Q on haplotypes 2.3 and 7.8 and R252W on haplotype 69.3). There was a strong association between the mutation R408W and haplotype 2.3 (54.7%). Heterogeneity was found at mutations R408W (haplotypes 2.3 and 5.9), R158Q (haplotypes 4.3, 2.3 and 7.8) and IVS10nt546 (haplotypes 6.7 and 34.7). The molecular basis of PKU in the Moravian area appears to be relatively homogeneous in comparison with other southern and western European populations, thus providing a good starting point for prenatal diagnosis and early clinical classification.

The results of the neonatal screening for phenylalanine hydroxylase (PAH) deficiency in Sicily show that its incidence is higher than previously reported for mainland Italians and that non-PKU HPA is in excess of classical and mild PKU. The latter finding suggests that a high number of non-PKU HPA mutations would occur in the Sicilian population compared to populations with an inverted PKU/non-PKU HPA ratio. Previous studies have identified 40 mutations accounting for the majority (98%) of mutant alleles underlying PAH deficiency in Sicily. In order to study the molecular basis of the distribution of PAH deficiency phenotypes in the Sicilian population, we have correlated 31 of those mutations with clinical and metabolic phenotypes in 12 mentally retarded patients, 14 treated patients with classic or mild PKU, and 13 subjects presenting the non-PKU HPA phenotype. The present study proposes a tentative classification for a large number (26) of PAH gene mutations which may represent an additional tool for establishing a differential diagnosis for PAH deficiency in the Sicilian population.

A polymorphic short tandem repeat (STR) in intron 3 (Goltsov et al., 1993) and a variable number of tandem repeats (Hind III-VNTR) flanked by two constant Hind III sites (Golstov et al., 1992) have been recently identified in the human phenylalanine hydroxylase (PAH) gene. These polymorphisms are easily detected by the polymerase chain reaction (PCR) and gel electrophoresis. We report on the use of these two novel polymorphisms in three Italian families with pregnancies at risk for classical phenylketonuria (PKU). A carrier status for PKU was ascertained in two fetuses; the third family refused prenatal diagnosis, although informativeness was shown to be complete.

Hyperphenylalaninemia due to a deficiency of hepatic phenylalanine hydroxylase (PAH) is the most common inborn error of amino acid metabolism. Clinically, the disorder is highly heterogeneous, spanning from nonphenylketonuria hyperphenylalaninemia to classical phenylketonuria. Only little is known about the molecular defects underlying hyperphenylalaninemia in Southern Europe. In this study, we conducted a systematic analysis of 53 patients from the Sicilian population. Each patient included in the study had persistently elevated blood levels of phenylalanine and met the differential criteria for PAH deficiency. Genomic DNA was analysed by scanning all PAH-coding exons for mutations by PCR in combination with denaturing gradient gel electrophoresis (DGGE). 52 patients were completely genotyped. A spectrum of 40 different mutations was established including 17 novel PAH mutations. Our results explain the clinical heterogeneity of hyperphenylalaninemia in Southern Europe, and form the basis for the establishment of phenotype-genotype correlations in Sicily and surrounding countries.

The forensic application of mtDNA typing requires large databases which are regionally well defined. To further this aim, we have typed mtDNA in a sample of 111 French and 106 Sicilians. The French were typed for both hypervariable segments (HVR1 and HVR2) of the mtDNA control region, whereas the Sicilians were only typed for HVR1, but in addition for the coding region RFLP markers for mtDNA groups H, I, J, K, L, M, T, U, V and X. In both samples, the predominant sequence type by far was the Cambridge reference sequence. Comparing HVR1 sequences, we found that the French sample was twice as diverse as the Sicilian sample as measured by sequence matches. A further set of sequence match comparisons including the French, Sicilian, and the published British mtDNA samples, demonstrate that sequence matching probabilities within samples differ by less than a factor of 2 from the matching probabilities between samples.

Angelman syndrome (AS) is a severe neurobehavioural disorder caused by failure of expression of the maternal copy of the imprinted domain located on 15q11-q13. There are different mechanisms leading to AS: maternal microdeletion, uniparental disomy, defects in a putative imprinting centre, mutations of the E3 ubiquitin protein ligase (UBE3A) gene. However, some of suspected cases of AS are still scored negative to all the latter mutations. Recently, it has been shown that a proportion of negative cases bear large deletions overlapping one or more exons of the UBE3A gene. These deletions are difficult to detect by conventional gene-scanning methods due to the masking effect by the non-deleted allele. In this study, we have used for the first time multiplex ligation-dependent probe amplification (MLPA) and comparative multiplex dosage analysis (CMDA) to search for large deletions affecting the UBE3A gene. Using this approach, we identified a novel causative deletion involving exon 8 in an affected sibling. Based on our results, we propose the use of MLPA as a fast, accurate and inexpensive test to detect large deletions in the UBE3A gene in a small but significant percentage of AS patients.

We have used Gene Ontology (GO) and pathway analyses to uncover the common functions associated to the genes overlapping Copy Number Variants (CNVs) in autistic patients. Our source of data were four published studies [1- 4]. We first applied a two-step enrichment strategy for autism-specific genes. We fished out from the four mentioned studies a list of 2928 genes overall overlapping 328 CNVs in patients and we first selected a sub-group of 2044 genes after excluding those ones that are also involved in CNVs reported in the Database of Genomic Variants (enrichment step 1). We then selected from the step 1-enriched list a sub-group of 514 genes each of which was found to be deleted or duplicated in at least two patients (enrichment step 2). The number of statistically significant processes and pathways identified by the Database for Annotation, Visualization and Integrated Discovery and Ingenuity Pathways Analysis softwares with the step 2-enriched list was significantly higher compared to the step 1-enriched list. In addition, statistically significant GO terms, biofunctions and pathways related to nervous system development and function were exclusively identified by the step 2-enriched list of genes. Interestingly, 21 genes were associated to axon growth and pathfinding. The latter genes and other ones associated to nervous system in this study represent a new set of autism candidate genes deserving further investigation. In summary, our results suggest that the autism’s “connectivity genes” in some patients affect very early phases of neurodevelopment, i.e., earlier than synaptogenesis.

To investigate the male genetic legacy of the Arab rule in southern Europe during medieval times, we focused on specific Northwest African haplogroups and identified evolutionary close STR-defined haplotypes in Iberia, Sicily and the Italian peninsula. Our results point to a higher recent Northwest African contribution in Iberia and Sicily in agreement with historical data, southern Italian regions known to have experienced long-term Arab presence also show an enrichment of Northwest African types. The forensic and genomic implications of these findings are discussed.

The presence or absence of genetic heterogeneity in Sicily has long been debated. Through the analysis of the variation of Y-chromosome lineages, using the combination of haplogroups and short tandem repeats from several areas of Sicily, we show that traces of genetic flows occurred in the island, due to ancient Greek colonization and to northern African contributions, are still visible on the basis of the distribution of some lineages. The genetic contribution of Greek chromosomes to the Sicilian gene pool is estimated to be about 37% whereas the contribution of North African populations is estimated to be around 6%. In particular, the presence of a modal haplotype coming from the southern Balkan Peninsula and of its one-step derivates associated to E3b1a2-V13, supports a common genetic heritage between Sicilians and Greeks. The estimate of Time to Most Recent Common Ancestor is about 2380 years before present, which broadly agrees with the archaeological traces of the Greek classic era. The Eastern and Western part of Sicily appear to be significantly different by the χ2-analysis, although the extent of such differentiation is not very high according to an analysis of molecular variance. The presence of a high number of different haplogroups in the island makes its gene diversity to reach about 0.9. The general heterogeneous composition of haplogroups in our Sicilian data is similar to the patterns observed in other major islands of the Mediterranean, reflecting the complex histories of settlements in Sicily.

A new RFLP marker U6.2 defining the locus DXS304 was recently mapped to the distal long arm of the X chromosome. In the present study we report the results of genetic linkage analysis of 13 fragile X [fra(X)] families that were informative for the new marker. Analysis of the recombinants for F9-FRAXA, DXS105-FRAXA, DXS98-FRAXA, DXS52-FRAXA, DXS15-FRAXA, and F8C-FRAXA, places DXS304 distal and near to the FRAXA locus. Combined with results from previous studies, our results support the order Xcen.-F9-DXS105-DXS98-FRAXA-DXS304-DXS5 2-DXS15-F8C-Xqter. Close linkage was observed between DXS304 and the disease locus with a peak lod score of 5.12 at theta = 0.04 from the present study and, with a peak lod score of 17.45 at theta = 0.035 when our data are combined with published data from 2 other studies. The present study confirms that U6.2 is useful for prenatal diagnosis and carrier testing in families affected by fra(X) syndrome.

The molecular basis of hereditary fructose intolerance (HFI) was studied in 50 subjects (41 pedigrees, 82 apparently independent mutant alleles of aldolase B) by direct analysis of aldolase B genes amplified by means of the polymerase chain reaction. The mutation A149P (ala 149----pro) was found in 67% of alleles but was significantly more common in patients from northern than from southern Europe. Two other point mutations of aldolase B were identified. A174D (C----A; ala 174----asp) was found in subjects from Italy, Switzerland, and Yugoslavia (overall frequency 16%) but not in those from the United Kingdom, France, or the United States. L288 delta C carried a single base-pair deletion causing frameshift at codon 288 and was restricted to Sicilian subjects. By testing for these mutations in amplified DNA with a limited panel of allele-specific oligonucleotides, more than 95% of HFI patients will be susceptible to genetic diagnosis.

Within the framework of a FISH screening protocol to detect cryptic subtelomeric rearrangements in autistic disorder (AD), a patient bearing three copies of the subtelomeric portion of the q arm of chromosome 13 has been identified. Beside AD, the patient also has severe mental retardation and displays several dysmorphic features. Further FISH analyses revealed that the trisomy was caused by the translocation of a 13q subtelomeric fragment to the acrocentric tip of one chromosome 21 [46,XY.ish der(21) t(13;21) (q34;p13)(D13S1825+)]. Gene dosage experiments carried out with three multiallelic polymorphisms of the subtelomeric region of chromosome 13q showed that the putative length of the triplicate region does not exceed 300 kb about, that is, the distance from telomere to the first normally inherited marker. In addition, gene dosage analysis performed on the derivative chromosome 21, did not reveal loss of the most telomeric protein-encoding genes on 21p. The potential relationship between a postulated increased expression of genes on 13q34 and the complex phenotype in this trisomic patient is discussed.

The Mediterranean region has been characterised by a number of pre-historical and historical demographic events whose legacy on the current genetic landscape is still a matter of debate. In order to investigate the degree of population structure across the Mediterranean, we have investigated Y chromosome variation in a large dataset of Mediterranean populations, 11 of which are first described here. Our analyses identify four main clusters in the Mediterranean that can be labelled as North Africa, Arab, Central-East and West Mediterranean. In particular, Near Eastern samples tend to separate according to the presence of Arab Y chromosome lineages, suggesting that the Arab expansion played a major role in shaping the current genetic structuring within the Fertile Crescent.

Eight Y-chromosomal short tandem repeats (STRs)-DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393 and DYS385 - were typed in a population sample (n = 255) of unrelated Sicilian males from nine different towns on the main island and from the island of Pantelleria. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

International audience; Objective: Autism is a complex, largely genetic psychiatric disorder. In the majority of cases, the cause of autism is not known, but there is strong evidence for a genetic etiology. To identify candidate genes, the physical mapping of balanced chromosomal aberrations is a powerful strategy, since several genes have been characterized in numerous disorders. In this study, the authors analyzed a balanced reciprocal translocation arising de novo in a subject with autism and mental retardation. Method: The authors performed the physical mapping of the balanced 9q23/ 10q22 translocation by fluorescent in situ hybridization experiments using bacterial artificial chromosome clones covering the areas of interest. Results: Findings revealed that the KCNMA1 gene, which encodes the alpha- subunit of the large conductance Ca2+-activated K+ ( BKCa) channel, a synaptic regulator of neuronal excitability, is physically disrupted. Further molecular and functional analyses showed the haploinsufficiency of this gene as well as decreased activity of the coded BKCa channel. This activity can be enhanced in vitro by addition of a BKCa channel opener (BMS-204352). Further mutational analyses on 116 autistic subjects led to the identification of an amino acid substitution located in a highly conserved domain of the protein not found in comparison subjects. Conclusions: These results suggest a possible association between a functional defect of the BKCa channel and autistic disorder and raise the hypothesis that deficits in synaptic transmission may contribute to the physiopathology of autism and mental deficiency.

The sarcophagus containing the remains of Federico II, located in the Cathedral of Palermo (Sicily, Italy), was opened on 1998 to perform a multidisciplinary survey [1]. Next to the remains of Federico II and in close contact with them were laying two other skeletons belonging, according to historical records, to Pietro II di Aragona and to an anonymous person (“The Third Individual”), probably a woman. The bones appeared severely deteriorated. Chemical analysis performed on bone samples excluded that the bodies underwent some kind of embalming process. The analysis of mtDNA from bone samples taken from the three skeletons was successful in only one of the two labs involved. The HVR1-mtDNA sequence (region: from nt 16,035 to nt 16,395), obtained from the bone samples of Federico II and “The Third Individual” appear identical but bear double peaks at the same nucleotide positions, suggesting mixing (i.e. contamination) of different mtDNA types. The HVR1 sequence obtained from the bone sample of Pietro II di Aragona does not present double peaks and differ from the Cambridge Reference Sequence (CRS) at six nucleotide positions. Cloning experiment of the Federico II amplicon demonstrated that the mixed mtDNA types are only two: one identical to CRS, the other identical to the sequence of Pietro II di Aragona. A reconstruction of these data are proposed in the Discussion. Due to the problematic context in which this study was carried out (mixed and deteriorated biological material, failure to replicate results in two different labs), our results and reconstruction can only be offered on a tentative basis. It is hoped that the data presented in this study will reveal useful, for future comparison, if further molecular genetics research will be carried out on the royal dynasties that ruled Sicily in the early centuries of the past millennium.

DNA samples from 465 blood donors living in 7 towns of Sicily, the largest island of Italy, have been collected according to well defined criteria, and their genetic heterogeneity tested on the basis of 9 autosomal microsatellite and mitochondrial DNA polymorphisms for a total of 85 microsatellite allele and 10 mtDNA haplogroup frequencies. A preliminary account of the results shows that: a) the samples are genetically heterogeneous; b) the first principal coordinates of the samples are correlated more with their longitude than with their latitude, and this result is even more remarkable when one outlier sample (Butera) is not considered; c) distances among samples calculated from allele and haplogroup frequencies and from the isonymy matrix are weakly correlated (r = 0.43, P = 0.06) but such correlation disappears (r = 0.16) if the mtDNA haplogroups alone are taken into account; d) mtDNA haplogroups and microsatellite distances suggest settlements of people occurred at different times: divergence times inferred from microsatellite data seem to describe a genetic composition of the town of Sciacca mainly derived from settlements after the Roman conquest of Sicily (First Punic war, 246 BC), while all other divergence times take root from the second to the first millennium BC, and therefore seem to backdate to the pre-Hellenistic period. A more reliable association of these diachronic genetic strata to different historical populations (e.g. Sicani, Elymi, Siculi), if possible, must be postponed to the analysis of more samples and hopefully more informative uniparental DNA markers such as the recently available DHPLC-SNP polymorphisms of the Y chromosome.

Pah(enu2) mice, created by chemically induced genetic mutation, are characterized by biochemical phenotypes closely resembling untreated human phenylketonuria (PKU). However, studies conducted in adult Pah(enu2) mice have shown no indices of the severe mental retardation that characterizes untreated PKU. The present experiments explored recognition of novel spatial and non-spatial information in Pah(enu2) mice by two nonassociative tests that do not use explicit reinforcement and avoid lengthy training. Moreover, we evaluated emotional reactivity by the Elevated Plus Maze. Finally, the performance of affected mutants was compared with that of their unaffected and heterozygous littermates and also with that of mice of the C57BL/6 (C57) inbred strain, an increasingly used background for genetic targeted organisms, and with DBA/2 (DBA) mice, known for their nonpathological deficits in spatial learning. The results demonstrated that mutant Pah(enu2) mice are characterized by deficits involving both spatial and nonspatial recognition, that are not related to motor impairment or to high emotional reactivity to novelty. These results indicate that Pah(enu2) mice show pathological cognitive deficits and support their use to test hypotheses about neurodevelopmental disturbances involved in mental retardation.

The R408W phenylketonuria mutation in Europe has arisen by recurrent mutation in the human phenylalanine hydroxylase (PAH) locus and is associated with two major PAH haplotypes. R408W-2.3 exhibits a west-to-east cline of relative frequency reaching its maximum in the Balto–Slavic region, while R408W-1.8 exhibits an east-to-west cline peaking in Connacht, the most westerly province of Ireland. Spatial autocorrelation analysis has demonstrated that the R408W-2.3 cline, like that of R408W-1.8, is consistent with a pattern likely to have been established by human dispersal. Genetic diversity within wild-type and R408W chromosomes in Europe was assessed through variable number tandem repeat (VNTR) nucleotide sequence variation and tetranucleotide short tandem repeat (STR) allelic associations. Wild-type VNTR-8 chromosomes exhibited two major cassette sequence organizations: (a1)5-b3-b2-c1 and (a1)5-b5-b2-c1. R408W-1.8 was predominantly associated with (a1)5-b5-b2-c1. Both wild-type VNTR-3 and R408W-2.3 chromosomes exhibited a single invariant cassette sequence organization, a2-b2-c1. STR allele distributions associated with the cassette variants were consistent with greater diversity in the wild-type VNTR-8 lineage and were suggestive of different levels of diversity between R408W-1.8 and R408W-2.3. The finding of greater genetic diversity within the wild-type VNTR-8 lineage compared to VNTR-3 suggests that VNTR-8 may be older within the European population. However, in the absence of a more extensive STR data-set, no such conclusions are possible for the respective R408W mutant lineages. Hum Mutat 21:387–393, 2003. r 2003 Wiley-Liss, Inc.

The molecular basis of PAH deficiency in the Sicilian population is characterized by a marked heterogeneity, with 44 mutations at a single locus identified by a "gene-scanning" approach and accounting for a detection rate of 91%. The remaining 9% of PAH alleles does not bear mutations in any of the 13 exons and 24 exon/intron junctions. Three mutations IVS10nt-11 G > A, R261Q, and A300S accounted for 30.5%, whereas the remaining mutations were found at relative frequencies of less than 5% and 20 mutations were observed once only. Five mutations have been detected only in Sicilians so far. By studying the association of mutations with intragenic STR-VNTR haplotypes ("minihaplotypes"), "identity by descent" has been established for 24 mutations also detected in other populations. This finding supports the hypothesis of a multipolar origin for a large proportion of PAH mutant alleles currently detected in Sicilians. In order to improve our understanding of the clinical heterogeneity of PAH deficiency in this population, we have for the first time analyzed three missense mutations L41F, T92I, and P211T in vitro by the pCDNA3/COS-7 eukaryotic expression system and found an activity of 10, 76, and 72%, respectively, compared to normal PAH. In two HPA patients with mild PKU and mild hyperphenylalaninemia (MHP), harboring respectively L41F/R261Q and T92I/P281L genotypes, the predicted biochemical effect of these genotypes appeared to be consistent with the metabolic phenotypes. In contrast, discordant metabolic phenotypes (mild PKU and MHP) were observed in two unrelated patients bearing the same R261Q/P211T genotype, a finding which underscores the complex relationship linking genotype to phenotype in PAH deficiency. Hypotheses on the possible mechanisms responsible for the observed discordance are discussed. The spectrum of PAH gene mutations in Sicily reflects the complex demographic history of this island at the crossroad of prehistoric and historical migrations in the Mediterranean sea. The data presented in this study also add to the present knowledge on the relationship between PAH genotypes and HPA phenotype and are expected to improve PAH genotyping among individuals with hyperphenylalaninemia.

The elucidation of the molecular basis of hyperphenylalaninemia in various world populations (PKU Consortium Database: http://www.mcgill.ca/pahdb/) has revealed a remarkable molecular heterogeneity at the locus encoding for phenylalanine hydroxylase. As a consequence, genotyping of HPA patients has prompted the establishment of an impressive number of mutation detection protocols. In spite of the large variety of methods proposed so far, no comprehensive strategy has been yet developed for the detection of PAH gene mutations. Therefore, new approaches, combining the advantages of individual methods are required, especially in populations with a high number of PAH gene mutations. In this study, we propose the use of Reverse Dot Blot Analysis within a general mutation detection protocol to simplify the genotyping of hyperphenylalaninemics in the very heterogeneous population of Sicily (Italy).

Y-chromosome variation was analyzed in a sample of 1127 males from the Western Mediterranean area by surveying 16 biallelic and 4 multiallelic sites. Some populations from Northeastern Europe and the Middle East were also studied for comparison. All Y-chromosome haplotypes were included in a parsimonious genealogic tree consisting of 17 haplogroups, several of which displayed distinct geographic specificities. One of the haplogroups, HG9.2, has some features that are compatible with a spread into Europe from the Near East during the Neolithic period. However, the current distribution of this haplogroup would suggest that the Neolithic gene pool had a major impact in the eastern and central part of the Mediterranean basin, but very limited consequences in Iberia and Northwestern Europe. Two other haplogroups, HG25.2 and HG2.2, were found to have much more restricted geographic distributions. The first most likely originated in the Berbers within the last few thousand years, and allows the detection of gene flow to Iberia and Southern Europe. The latter haplogroup is common only in Sardinia, which confirms the genetic peculiarity and isolation of the Sardinians. Overall, this study demonstrates that the dissection of Y-chromosome variation into haplogroups with a more restricted geographic distribution can reveal important differences even between populations that live at short distances, and provides new clues to their past interactions.

summary We typed 1801 males from 55 locations for the Y-specific binary markers YAP, DYZ3, SRY "!)$" and the (CA)n microsatellites YCAII and DYS413. Phylogenetic relationships of chromosomes with the same binary haplotype were condensed in seven large one-step networks, which accounted for 95% of all chromosomes. Their coalescence ages were estimated based on microsatellite diversity. The three largest and oldest networks undergo sharp frequency changes in three areas. The more recent network 3‐1A clearly discriminates between Western and Eastern European populations. Pairwise Fst showed an overall increment with increasing geographic distance but with a slope greatly reduced when compared to previous reports. By sectioning the entire data set according to geographic and linguistic criteria, we found higher Fst-on-distance slopes within Europe than in West Asia or across the two continents.

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The genes for human cytokeratins 4 and 15 (KRT4 and 15) are assigned to the p11.2→q12 region of chromosome 12 (cytokeratin 4) and to the q21→q23 region of chromosome 17 (cytokeratin 15), respectively.

IVS10nt546 (IVS10nt-11g→a) is the most common molecular defect of the phenylalanine hydroxylase gene causing phenylketonuria in Mediterranean populations. Previous studies have proposed various and alternative hypotheses concerning the geographical origin and pattern of diffusion of this mutation in this area. In this study, this issue was re-examined on a large sample (149) of “Mediterranean” IVS10nt546 mutant alleles analysed with multiallelic intragenic polymorphisms. The analysis of intragenic microsatellite (STR) and minisatellite (VNTR) polymorphisms shows allelic heterogeneity of the IVS10nt546 mutation. Eight STR and three VNTR alleles were found in association with the splicing defect. Of the ten detected STR–VNTR combinations (“minihaplotypes”), we identified a predominant allelic association (VNTR7 – STR252) embedded in a RFLP-haplotype 6 background, which seems to correspond to the ancestral gene originating in the Turkey–Israel area. Analysis of both absolute and relative gene frequencies of the STR252 – IVS10nt546 – VNTR7 minihaplotypes, shows statistically significant (P < 0.02) variations and may suggest gene flow from Turkey and/or Israel to Italy and Spain. The associated migratory events need not be unique in time (and people) but seem to suggest they may be traced back to the expansion of the Neolithic culture and people, thus allowing dating of the origin of this mutation to at least 5000–10 000 years ago. Alternative hypotheses are discussed to explain, in light of the available historical and pre-historical evidence, the pattern of diffusion of the IVS10nt546 mutation in the Mediterranean basin.

Mutation analysis at the phenylalanine hydroxylase (PAH) locus was undertaken in 56 Egyptian hyperphenylalaninemic patients. Selected screening for 11 known mutations and denaturing Gradient gel electrophoresis (DGGE) analysis of the entire coding sequence and exon/intron boundaries led to the identification of a new mutation (I224T), four previously described mutations, and several polymorphisms. Overall, 18 mutant alleles could thus be characterized. In contrast to the high mutation detection rate typical of the DGGE-based scanning approach, only 6 of 16 mutant alleles tested were identified. Since BH4 deficiency could not be excluded in any of these patients, the latter results may be explained by the occurrence of mutations affecting the genes controlling the synthesis and recycling of tetrahydrobiopterin: the cofactor of PAH. An alternative hypothesis is also discussed.

Lack of association of HOXA1 and HOXB1 mutations and autism in Sicilian (Italian) patients

The study of the molecular basis of phenylalanine hydroxylase (PAH) deficiency performed on 59 Sicilian PKU families has led to the identification of 40% of the mutant genotypes (Romano et al 1993). Of the more than 60 mutations affecting the PAH locus in PKU, 10 have been identified so far in Sicily, thus uncovering a remarkable heterogeneity of PKU at the molecular level in this population

In the present study the human cytokeratin 13 gene (KRT13), encoding a polypeptide characteristic of internal stratified epithelia, has been mapped with the help of the polymerase chain reaction and somatic cell hybrids to chromosome 17. In situ hybridization of a KRT13 cDNA probe to metaphase chromosomes allowed the assignment of the KRT13 gene within the q12–q21.2 region of chromosome 17.

To study the regulation of the expression of cyto-keratins characteristic of simple epithelia, i.e., human cytokeratins nos. 7, 8, 18, and 19, we prepared several cDNA clones coding for these proteins and their bovine counterparts. In the present study, we describe a cDNA clone of the mRNA coding for human cytokeratin no. 18, which was isolated from an expression library using the monoclonal antibody, K G 8.13. This clone (756 nucleotides, excluding the poly A portion), encodes approximately one-half of the mRNA (approximately 1.4 kb), identifies one mRNA band in Northern-hybridization blots, and specifically selects one mRNA species coding for cytokeratin no. 18, as demonstrated by translation in vitro. Comparison of the deduced amino acid sequence - confirmed by direct amino-acid-sequence analyses of some polypeptide fragments produced by cleavage with cyanogen bromide - indicated that cytokeratin no. 18 is a member of the acidic (type I) subfamily of cytokeratins. It has only limited sequence homologies in common with other intermediate-sized filament proteins, and these are essentially restricted to certain domains of the a-helical rod portion. The carboxyterminal tail sequence does not contain glycine-rich elements, thus distinguishing this cytokeratin from those acidic (type I) cytokeratins that are characterized by this feature. The similarities and differences between cytokeratin no. 18 and previously described epidermal cytokeratins are discussed in relation to the differences in the stability of the complexes which this cytokeratin forms with basic (type 11) cytokeratins, as well as in relation to possible functional differences of cytokeratins in simple and stratified epithelia.

We describe cDNA clones of mRNAs encoding human cytokeratins nos. 8 and 18, and the amino acid sequences deduced from their nucleotide sequences. Human cytokeratin no. 8 is a typical cytokeratin of the basic (type II) subfamily, which is highly homologous to the corresponding bovine and amphibian (Xenopus laevis) proteins; however, unlike the amphibian protein, it does not contain glycine-rich oligopeptide repeats in its carboxyterminal 'tail' domain. Comparison with the reported amino acid sequences of two fragments of human 'tissue polypeptide antigen' (TPA), a widely used serodiagnostic carcinoma marker, revealed sequence identity, indicating that this serum component is derived from the intracellular cytokeratin no. 8 present in diverse kinds of epithelia and epithelium-derived tumors. Human cytokeratin no. 18 is very similar to the corresponding murine protein but contains two additional blocks of 4 and 5 amino acids in the 'head' portion. These cDNA clones and the RNA probes derived therefrom were used to detect specifically mRNAs by Northern-blot assays of RNAs from various carcinomas and cultured carcinoma cells. Using in situ hybridization on frozen sections of tumor-containing tissues, notably lymph nodes containing metastatic breast carcinoma, we were able to demonstrate the specificity and sensitivity of this procedure. The potential value for cell-biological research and pathology of being able to detect a mRNA encoding a given cytokeratin polypeptide in situ is discussed.

The chromosomal location of representative members of the type I and type II subfamilies of the cytokeratin multigene family was determined using specific cDNA probes in Southern blot hybridization with DNA from somatic cell hybrids. Our results show that the gene encoding human type II cytokeratin 4 resides on chromosome 12 and that encoding type I cytokeratin 15 is located on chromosome 17. The results indicate that cytokeratins are not concentrated in only one cluster. The possibility of the existence of separate type I and type II cytokeratin gene clusters is discussed.

The fabrication of electrochemical double-layer capacitors (EDLCs) with high areal capacitance relies on the use of elevated mass loadings of highly porous active materials. Herein, we demonstrate a high-throughput manufacturing of graphene/nanotubes hybrid EDLCs. Wet-jet milling (WJM) method is exploited to exfoliate the graphite into single/few-layer graphene flakes (WJM-G) in industrial volume (production rate ~0.5 kg/day). Commercial single/double walled carbon nanotubes (SDWCNTs) are mixed with graphene flakes in order to act as spacers between the graphene flakes during their film formation. The latter is obtained by one-step vacuum filtration, resulting in self-standing, metal- and binder-free flexible EDLC electrodes with high active material mass loadings up to 30 mg cm-2. The corresponding symmetric WJM-G/SDWCNTs EDLCs exhibit electrode energy densities of 539 uWh cm-2 at 1.3 mW cm-2 and operating power densities up to 532 mW cm-2 (outperforming most of the EDLC technologies). The EDCLs show excellent cycling stability and outstanding flexibility even under highly folded states (up to 180 degrees). The combination of industrial-like production of active materials, simplified manufacturing of EDLC electrodes, and ultrahigh areal performance of the as-produced EDLCs are promising for novel advanced EDLC designs.

Efficient ionic transport in nanoporous carbon electrodes is pivotal for the development of high-rate electrochemical capacitive energy storage in supercapacitors (SCs). Over the past decade, the understanding of the charging/discharging mechanisms in nanostructured carbon electrodes and the elucidation of confinement and desolvation of ions in electrically charged carbon nanopores have spurred the development of advanced SCs holding ever-increasing energy density. Crucially, these advancements have to be accomplished without sacrificing the extraordinary power handling and cycling lifetime of the SC. In this work, we investigated the interaction between single-/few-layer graphene (SLG/FLG) flakes or activated carbon (AC) films and 1 M tetraethylammonium tetrafluoroborate (TEABF4) in propylene carbonate (PC) by lateral force microscopy (LFM) measurements. We unravel that the electrolyte nanotribology on SLG/FLG flakes incorporated into AC-based electrodes is effective at boosting the power performance of commercial-like SCs (active material mass loading ∼10 mg cm−2), thus maximizing the advantage of using nanoporous nanocarbon electrodes with high energy density. At a charge/discharge (CD) current density of 0.1 A g−1, our hybrid AC:SLG/FLG-based SC shows a 30% increase of specific capacitance (Cg) compared to the reference device, i.e., the AC-based one (Cg from 76.8 F g−1 to 98.2 F g−1). At higher CD current densities (>1 A g−1), the AC-based device displays a resistive behaviour, while the AC:SLG/FLG-based SCs still exhibit a Cg of 23.9 and 5.2 F g−1 at CD current densities as high as 2.5 and 5 A g−1, respectively. Beyond the control of the nanoporosity of carbon materials and the inter-ionic interaction of the electrolytes, the rationalization of the relationship between the nanotribology of nanocarbons and the performance of the corresponding SCs offers new opportunities to optimize the SC design compatibly with established high-throughput industrial manufacturing.

Monoclonal antibodies which recognize different epitopes on either titin or nebulin show normal staining patterns on frozen sections of three muscle biopsies of Duchenne muscular dystrophy (DMD). Gel electrophoresis and immunoblotting performed on two of these muscle biopsies show the normal pattern of titin and nebulin polypeptides. Since the donor of one of these biopsies has a large deletion of the 5′-region of the DMD gene, our results argue against the recent proposal that nebulin is the gene mutated in DMD.

Clinical data suggest that brain catecholamines and serotonin are deficient in phenylketonuria (PKU), an inherited metabolic disorder that causes severe mental retardation and neurological disturbances. To test this hypothesis, brain tissue levels of dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT) and their metabolites were evaluated in the genetic mouse model of PKU (Pah(enu2)). Results indicated a significant reduction of 5-HT levels and metabolism in prefrontal cortex (pFC), cingulate cortex (Cg), nucleus accumbens (NAc), caudate putamen (CP), hippocampus (HIP) and amygdala (AMY). NE content and metabolism were reduced in pFC, Cg, AMY and HIP. Finally, significantly reduced DA content and metabolism was observed in pFC, NAc, CP and AMY. In pFC, NAc and CP there was also a marked reduction of DA release.

In this study we report previously undescribed associations between mutations, haplotypes and a minisatellite polymorphism (Hind III VNTR) of the PAH gene in the Sicilian population. Analysis of the association between mutations and linked polymorphisms between Sicilians and other Mediterranean populations may be a useful tool to study the time-space origin of mutant PAH genes in Southern Europe.

We used fluorescence in situ hybridization to localize the human gene for cytokeratin 3 (KJRT3), a member of the type II subfamily of cytokeratins, within the human genome. The results show that KRT3 is located within chromosome region 12q12→q13. All human type II keratin genes mapped to date have been assigned to chromosome 12, where they are likely to be organized into one homotypic cluster.

A YAC clone (211F11) containing approximately 0.5 Mb of human DNA was isolated from a human genomic library by PCR-based screening with cytokeratin (KRT) 13-specific primers. The YAC clone was mapped by FISH to the long arm of chromosome 17 (17q12→q21), a region to which several other type I KRT genes had been mapped previously. We now show by Southern blot hybridization and PFGE analyses that KRT13, 14, 15, and 16 are all contained within YAC clone 211F11. Long-range restriction mapping analysis of clone 211F11 and of two smaller YAC clones that were also isolated with KRT13-specific primers, suggests that KRT13, 14, 15, 16 and their linked type I genes KRT17 and 19, are contained in less than 150 kb of genomic DNA. According to our reconstruction it then appears that at least six type I KRT genes are arranged in a highly compact cluster. The three YACs reported in this study represent a new tool to dissect the molecular structure of the locus of the human type I KRT genes.