A large deletion in the LDL receptor gene--the cause of familial hypercholesterolemia in three Italian families: a study that dates back to the 17th century (FH-Pavia).

In the LDL-receptor gene, a large rearrangement causing hypercholesterolemia was detected in three apparently unrelated families living in northern Italy. In all probands, binding, internalization, and degradation of 125I-LDL measured in skin fibroblasts were found to be 40%-50% of control values, indicative of heterozygous familial hypercholesterolemia (FH). Southern blot analysis revealed that the probands were heterozygous for a large (25-kb) deletion of the LDL-receptor gene eliminating exons 2-12. The affected subjects possessed two LDL-receptor mRNA species: one of normal size (5.3 kb) and one of smaller size (3.5 kb). In the latter mRNA, the coding sequence of exon 1 is joined to the coding sequence of exon 13, causing a change in the reading frame and thereby giving rise to a premature stop codon. The receptor protein deduced from the sequence of the defective mRNA is a short polypeptide of 29 amino acids, devoid of any function. Tracing these three families back to the 17th century, we found both their common ancestor and the possible origin of the mutation, in a region which is called "Lomellina" and which is located in southwest Lombardy, near the old city of Pavia. Therefore we named the mutation "FH-Pavia.

Four novel partial deletions of LDL-receptor gene in Italian patients with familial hypercholesterolemia.

In this study, we report four new partial deletions of the LDL-receptor (LDL-R) gene discovered during a survey of 326 Italian patients with familial hypercholesterolemia (FH). All deletions were found in FH heterozygotes whose LDL-R activity in skin fibroblasts ranged from 52% to 43% of the values found in control cells. The size and boundaries of the deletions were defined by Southern blotting and, in some cases, by polymerase chain reaction (PCR) amplification of genomic DNA. The sequence of the deletion joint was performed after the reverse transcription and PCR amplification of the appropriate regions of LDL-R mRNA. FHMassa is a 12-kilobase deletion spanning from intron 2 to intron 10. RT-PCR showed that the mutant allele is transcribed into one major and two minor mRNAs. In the most abundant mRNA species, exon 2 joins exon 11, as expected from DNA analysis. In one minor mRNA, which was sequenced, exon 2 joins exon 13, with exons 11 and 12 skipped as a result of an alternative splicing. FHGenova is a 4-kb deletion spanning from intron 10 to intron 12 and eliminating exons 11 and 12. FHRoma is a 4.7-kb deletion spanning from the 5′ end of intron 12 to the middle of intron 14 and eliminating exons 13 and 14. This deletion differs in size from the previously described deletion (FHChieti/Macerata), which is located in the same region of the LDL-R gene but is smaller (3.7 kb). In both FHRoma and FHChieti/Macerata, the mutant LDL-R mRNA is present in a minute amount, suggesting that the deletion of exons 13 and 14 may increase mRNA degradation. FHPadova-2 is a 2-kb deletion spanning from intron 15 to intron 16 and eliminating the sole exon 16. All deletions except FHPadova-2 produce a shift in the reading frame, leading to either a very short peptide or a truncated protein. In FHPadova-2, elimination of exon 16 does not change the reading frame but is predicted to produce a receptor protein of 813 amino acids, lacking 18 amino acids of the O-linked sugar and 8 amino acids of the transmembrane domain. Ligand blot experiments with rabbit 125I-β VLDL indicate that half the amount of LDL-receptor is present in FHPadova-2 fibroblasts, suggesting that the in-frame deletion of 26 amino acids may disrupt the intracellular transport and/or the insertion of the receptor in the plasma membrane or may increase its degradation

Assessment of prenatal exposure to ethanol by meconium analysis: results of an Italian multicenter study.

Background: This study estimated in 7 Italian cities the prevalence of prenatal exposure to ethanol by determining fatty acid ethyl esters (FAEEs; palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic esters) and ethyl glucuronide (EtG) in neonatal meconium samples. Methods: A total of 607 meconium samples were obtained from neonatal wards of 7 public hospitals: Verona and San Daniele del Friuli in the northeast of the country, Reggio Emilia in the middle east, Florence and Rome in the center, and Naples and Crotone in the southwest of the peninsula. Meconium biomarkers were assessed by a validated methodology using liquid chromatography- tandem mass spectrometry and the results categorized using the accepted cutoff of 2 nmol / g total amount of 7 FAEEs and 2 nmol / g EtG, to differentiate between heavy maternal ethanol use during pregnancy and occasional or no use at all. Results: On the basis of the above- reported cutoffs, the overall prevalence of newborns prenatally exposed to maternal ethanol was 7.9%: 0% in Verona, 4.0% in San Daniele del Friuli, 4.9% in Naples, 5.0% in Florence, 6.2% in Crotone, up to 10.6% in Reggio Emilia, and 29.4% in Rome. Low maternal education level and younger maternal age were associated with biomarker scores over the cutoff. There was also a significant correlation between the highest percentage of prenatal exposure in the capital and certain maternal sociodemographic characteristics. Conclusions: These results indicate considerable variability in the prevalence of fetal exposure to ethanol in different Italian cities, as determined by the objective measurement of biomarkers in meconium. These data, together with previous ones obtained in Barcelona, Spain, indicate that gestational ethanol exposure is widespread, at least in parts of Europe

Testing ethylglucuronide in maternal hair and nails for the assessment of fetal exposure to alcohol: comparison with meconium testing

Background: The deleterious effects exerted by prenatal ethanol exposure include physical, mental, behavioral, and/or learning disabilities that are included in the term fetal alcohol spectrum disorder. The measurement of ethylglucuronide (EtG) in alternative biological matrices, including neonatal and maternal hair, neonatal meconium, and maternal nails, is receiving increasing interest for the accurate evaluation of the in utero exposure to alcohol. Objective: To evaluate the correlation between EtG in maternal hair and nails with EtG in neonatal meconium to further explore the suitability of these biomarkers in disclosing prenatal exposure to ethanol. Methods: A total of 151 maternal hair strands (0-6 cm), nail clips (2-6 mm), and corresponding neonatal meconium and nails samples were obtained from neonatal wards of 4 Mediterranean public hospitals: Rome, Florence, and Belluno in Italy and Barcelona in Spain. Hair, nails, and meconium were analyzed for the presence of EtG by validated liquid chromatography mass spectrometry assay. Meconium was also analyzed for the presence of fatty acid ethyl esters (FAEEs) as a complementary biomarker of potential in utero exposure to alcohol. Results: Eighteen newborns resulted in utero exposed to maternal alcohol consumption by FAEE testing in meconium with EtG values between 0.5 and 1.5 nmol/g. Unfortunately, none of these cases were confirmed by the presence of EtG in maternal hair and nails samples, which resulted all negative to this biomarker. Discussion and Conclusions: The results confirm that FAEEs and EtG in meconium are the best biomarkers to assess in utero exposure to maternal alcohol. EtG in hair and nails are not good biomarkers to disclose alcohol consumption lower than on daily basis and lower than 1-2 alcoholic units per da

Four novel partial deletions of LDL-receptor gene in Italian patients with familial hypercholesterolemia.

In this study, we report four new partial deletions of the LDL-receptor (LDL-R) gene discovered during a survey of 326 Italian patients with familial hypercholesterolemia (FH). All deletions were found in FH heterozygotes whose LDL-R activity in skin fibroblasts ranged from 52% to 43% of the values found in control cells. The size and boundaries of the deletions were defined by Southern blotting and, in some cases, by polymerase chain reaction (PCR) amplification of genomic DNA. The sequence of the deletion joint was performed after the reverse transcription and PCR amplification of the appropriate regions of LDL-R mRNA. FHMassa is a 12-kilobase deletion spanning from intron 2 to intron 10. RT-PCR showed that the mutant allele is transcribed into one major and two minor mRNAs. In the most abundant mRNA species, exon 2 joins exon 11, as expected from DNA analysis. In one minor mRNA, which was sequenced, exon 2 joins exon 13, with exons 11 and 12 skipped as a result of an alternative splicing. FHGenova is a 4-kb deletion spanning from intron 10 to intron 12 and eliminating exons 11 and 12. FHRoma is a 4.7-kb deletion spanning from the 5' end of intron 12 to the middle of intron 14 and eliminating exons 13 and 14. This deletion differs in size from the previously described deletion (FHChieti/Macerata), which is located in the same region of the LDL-R gene but is smaller (3.7 kb). In both FHRoma and FHChieti/Macerata, the mutant LDL-R mRNA is present in a minute amount, suggesting that the deletion of exons 13 and 14 may increase mRNA degradation. FHPadova-2 is a 2-kb deletion spanning from intron 15 to intron 16 and eliminating the sole exon 16. All deletions except FHPadova-2 produce a shift in the reading frame, leading to either a very short peptide or a truncated protein. In FHPadova-2, elimination of exon 16 does not change the reading frame but is predicted to produce a receptor protein of 513 amino acids, lacking 18 amino acids of the O-linked sugar and 8 amino acids of the transmembrane domain. Ligand blot experiments with rabbit I-125-beta VLDL indicate that half the amount of LDL-receptor is present in FHPadova-2 fibroblasts, suggesting that the in-frame deletion of 26 amino acids may disrupt the intracellular transport and/or the insertion of the receptor in the plasma membrane or may increase its degradation

Diversification of the rainfrog <i>Pristimantis ornatissimus</i> in the lowlands and Andean foothills of Ecuador

<div><p>Geographic barriers and elevational gradients have long been recognized as important in species diversification. Here, we illustrate an example where both mechanisms have shaped the genetic structure of the Neotropical rainfrog, <i>Pristimantis ornatissimus</i>, which has also resulted in speciation. This species was thought to be a single evolutionary lineage distributed throughout the Ecuadorian Chocó and the adjacent foothills of the Andes. Based on recent sampling of <i>P</i>. <i>ornatissimus</i> sensu lato, we provide molecular and morphological evidence that support the validity of a new species, which we name <i>Pristimantis ecuadorensis</i> sp. nov. The sister species are elevational replacements of each other; the distribution of <i>Pristimantis ornatissimus</i> sensu stricto is limited to the Ecuadorian Chocó ecoregion (< 1100 m), whereas the new species has only been found at Andean localities between 1450–1480 m. Given the results of the Multiple Matrix Regression with Randomization analysis, the genetic difference between <i>P</i>. <i>ecuadorensis</i> and <i>P</i>. <i>ornatissimus</i> is not explained by geographic distance nor environment, although environmental variables at a finer scale need to be tested. Therefore this speciation event might be the byproduct of stochastic historic extinction of connected populations or biogeographic events caused by barriers to dispersal such as rivers. Within <i>P</i>. <i>ornatissimus</i> sensu stricto, morphological patterns and genetic structure seem to be related to geographic isolation (e.g., rivers). Finally, we provide an updated phylogeny for the genus, including the new species, as well as other Ecuadorian <i>Pristimantis</i>.</p></div