Manuela De Gregori

A recurrent 15q13.3 microdeletion syndrome associated with mental retardation and seizures

We report a recurrent microdeletion syndrome causing mental retardation, epilepsy and variable facial and digital dysmorphisms. We describe nine affected individuals, including six probands: two with de novo deletions, two who inherited the deletion from an affected parent and two with unknown inheritance. The proximal breakpoint of the largest deletion is contiguous with breakpoint 3 (BP3) of the Prader-Willi and Angelman syndrome region, extending 3.95 Mb distally to BP5. A smaller 1.5-Mb deletion has a proximal breakpoint within the larger deletion (BP4) and shares the same distal BP5. This recurrent 1.5-Mb deletion contains six genes, including a candidate gene for epilepsy (CHRNA7) that is probably responsible for the observed seizure phenotype. The BP4–BP5 region undergoes frequent inversion, suggesting a possible link between this inversion polymorphism and recurrent deletion. The frequency of these microdeletions in mental retardation cases is ∼0.3% (6/2,082 tested), a prevalence comparable to that of Williams, Angelman and Prader-Willi syndromes.

Cryptic deletions are a common finding in “balanced” reciprocal and complex chromosome rearrangements: a study of 59 patients

Using array comparative genome hybridisation (CGH) 41 de novo reciprocal translocations and 18 de novo complex chromosome rearrangements (CCRs) were screened. All cases had been interpreted as “balanced” by conventional cytogenetics. In all, 27 cases of reciprocal translocations were detected in patients with an abnormal phenotype, and after array CGH analysis, 11 were found to be unbalanced. Thus 40% (11 of 27) of patients with a “chromosomal phenotype” and an apparently balanced translocation were in fact unbalanced, and 18% (5 of 27) of the reciprocal translocations were instead complex rearrangements with >3 breakpoints. Fourteen fetuses with de novo, apparently balanced translocations, all but two with normal ultrasound findings, were also analysed and all were found to be normal using array CGH. Thirteen CCRs were detected in patients with abnormal phenotypes, two in women who had experienced repeated spontaneous abortions and three in fetuses. Sixteen patients were found to have unbalanced mutations, with up to 4 deletions. These results suggest that genome-wide array CGH may be advisable in all carriers of “balanced” CCRs. The parental origin of the deletions was investigated in 5 reciprocal translocations and 11 CCRs; all were found to be paternal. Using customised platforms in seven cases of CCRs, the deletion breakpoints were narrowed down to regions of a few hundred base pairs in length. No susceptibility motifs were associated with the imbalances. These results show that the phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions and that spermatogenesis is more prone to generate multiple chaotic chromosome imbalances and reciprocal translocations than oogenesis.

Morphine metabolism, transport and brain disposition

The chemical structures of morphine and its metabolites are closely related to the clinical effects of drugs (analgesia and side-effects) and to their capability to cross the Blood Brain Barrier (BBB). Morphine-6-glucuronide (M6G) and Morphine-3-glucuronide (M3G) are both highly hydrophilic, but only M6G can penetrate the BBB; accordingly, M6G is considered a more attractive analgesic than the parent drug and the M3G. Several hypotheses have been made to explain these differences. In this review we will discuss recent advances in the field, considering brain disposition of M6G, UDP-glucoronosyltransferases (UGT) involved in morphine metabolism, UGT interindividual variability and transport proteins.

Genetic variability at COMT but not at OPRM1 and UGT2B7 loci modulates morphine analgesic response in acute postoperative pain

PurposeTo investigate interindividual variability in response to pain treatment, we characterized postoperative patients for morphine metabolism and for COMT, OPRM1 and UGT2B7 polymorphisms.MethodsA total of 109 patients treated with morphine were genotyped by DNA sequencing for 12 DNA polymorphisms of the COMT, OPRM1 and UGT2B7 genes. The plasma concentration of morphine and of M3G/M6G metabolites were evaluated by means of reversed phase high-performance liquid chromatography coupled with mass spectrometry.ResultsAn association between average morphine consumption during the first 24 postoperative hours by patient-controlled analgesia (PCA) and COMT haplotypes was found. Specifically, patients with the diplotype for average pain intensity (APS/APS) required the lowest morphine doses compared to the other subjects (p = 0.011). The APS haplotype contains an adenine corresponding to methionine, instead of valine, at position 158 of the COMT protein. Met/Met homozygous patients consumed significantly lower morphine doses than other subjects (p = 0.014); accordingly, Val158Met genotyping alone might be used in the clinical setting to predict PCA morphine need. Considering both COMT Val158Met and OPRM1 A118G polymorphisms, carriers of both the Met/Met and AA genotypes required less morphine than other subjects, although the difference was not significant. The analysis of UGT2B7 revealed the occurrence of two common haplotypes (G_C_C_A_C and A_T_T_G_T) that did not prove to be related with plasma morphine and M3G/M6G concentration.ConclusionsBy considering COMT, OPRM1, and UGT2B7 genotypes, as well as pharmacokinetic results, only COMT polymorphisms appear to be predictive of morphine need in postoperative pain therapy.

How and why to screen for CYP2D6 interindividual variability in patients under pharmacological treatments

Cytochromes P450 are members of a superfamily of hemoproteins that catalyze a variety of oxidative reactions in the metabolism of endogenous and exogenous hydrophobic substrates. Fifty-eight cytochrome P450 (CYP) isoenzymes belonging to 18 families have been identified in human cells; the corresponding genes are highly polymorphic, and genetic variability underlies interindividual differences in drug response. The polymorphisms of CYP2D6 significantly affect the pharmacokinetics of about 50% of the drugs in clinical use, which are CYP2D6 substrates. The number of functional CYP2D6 alleles per genome determines the existence of four different phenotypes, i.e. poor, intermediate, extensive, and ultrarapid metabolizers. CYP2D6 genetic variants include copy number variations, single nucleotide substitutions, frameshift and insertion/deletion mutations. This review reports some of the different methodological approaches used to screen for CYP2D6 variants and focuses on methods that have improved variation detection, from conventional techniques to more recent microarray technology and high throughput DNA sequencing. In addition, this review reports some results on clinical relevance of CYP2D6 polymorphisms and provides examples of variability in drug response associated with interindividual phenotypic differences.

Inverted duplications: how many of them are mosaic?

The best-known situation indissolubly linked to mosaicism is the uniparental disomy where a trisomic or monosomic zygote develops at least one cell line with 46 chromosomes. The mosaicism normal/abnormal cell lines may remain confined to placenta or persist in the embryo. Here, we describe a second situation that might also be indissolubly linked to a mosaic condition or at least to a confined placental mosaicism. We analysed the case of a mosaicism del(8p)/inv dup(8p) found in prenatal diagnosis. We had already demonstrated that the first product of the abnormal meiotic recombination at the basis of the inv dup rearrangements is a dicentric chromosome. Its breakage leads to the formation of a deleted and an inv dup chromosome. Although we had previously assumed that the dicentric underwent a breakage at meiosis II so that the zygote inherited the inv dup chromosome, our findings and those of others indeed indicate that the dicentric may be inherited in the zygote and that it might persist as such in early postzygotic stages, then undergoing different breakages in different cells leading to different abnormal chromosomes, either deleted or duplicated. Selection versus the most viable cell line(s) results either in a confined placental mosaicism with the inv dup cell line as the only one present in the embryo or in children with both the deleted and the inv dup cell lines. Phenotype/karyotype relationships in inv dup rearrangements must also take into account the influence of the other abnormal cell line during embryogenesis.

A 7 Mb Duplication at 22q13 in a Girl With Bipolar Disorder and Hippocampal Malformation

We identified a duplication of 22q13.1‐q13.2 in a 10‐year‐old girl and demonstrated that this duplication was the recombinant product of a maternal intrachromosomal insertion. Phenotypic characteristics included prominent forehead, small low‐set ears, hypertelorism, epicanthal folds, small palpebral fissures, short philtrum, and syndactyly. MRI of the brain revealed high signal abnormalities in the periventricular white matter, a hypoplastic corpus callosum, under‐rotated hippocampus on the left and atrophic hippocampus on the right. Since age 5, the childs behavior has shown cyclic maniacal episodes with severely disorganized mood and behavior. Psychiatric and cognitive assessment led to a diagnosis of bipolar disorder not otherwise specified, manic episodes, attention deficit hyperactivity disorder and moderate mental retardation. Array‐CGH revealed an interstitial duplication of 6.9 Mb at chromosome 22q: dup(22)(q13.1q13.2). FISH using BAC clones confirmed the array‐CGH results and demonstrated that the duplication was inverted. G‐banding analysis in the propositas mother revealed a banding pattern suggestive of an intrachromosomal insertion, as demonstrated by dual‐color FISH with BACs that were duplicated in the proposita and multicolor‐banding (MCB) based on microdissection derived region‐specific libraries for chromosome 22. Our findings suggest that in both seemingly de novo deletions and duplications, the parent transmitting the imbalance should be investigated for possible balanced rearrangements. This report reinforces previous evidence that chromosome imbalances, and thus gene dosage effects, may be at the basis of some psychiatric disorders. Stringent correlations between submicroscopic imbalances, specific behavioral phenotypes and brain imaging will possibly help in dissecting complex behavioral traits.

Changes in total plasma and serum N-glycome composition and patient-controlled analgesia after major abdominal surgery

Systemic inflammation participates to the complex healing process occurring after major surgery, thus directly affecting the surgical outcome and patient recovery. Total plasma N-glycome might be an indicator of inflammation after major surgery, as well as an anti-inflammatory therapy response marker, since protein glycosylation plays an essential role in the inflammatory cascade. Therefore, we assessed the effects of surgery on the total plasma N-glycome and the association with self-administration of postoperative morphine in two cohorts of patients that underwent major abdominal surgery. We found that plasma N-glycome undergoes significant changes one day after surgery and intensifies one day later, thus indicating a systemic physiological response. In particular, we observed the increase of bisialylated biantennary glycan, A2G2S[3,6]2, 12 hours after surgery, which progressively increased until 48 postoperative hours. Most changes occurred 24 hours after surgery with the decrease of most core-fucosylated biantennary structures, as well as the increase in sialylated tetraantennary and FA3G3S[3,3,3]3 structures. Moreover, we observed a progressive increase of sialylated triantennary and tetraantennary structures two days after surgery, with a concomitant decrease of the structures containing bisecting N-acetylglucosamine along with bi- and trisialylated triantennary glycans. We did not find any statistically significant association between morphine consumption and plasma N-glycome.

Clinical pharmacokinetics of morphine and its metabolites during morphine dose titration for chronic cancer pain.

Background: Pain is one of the most prevalent and distressing symptoms in patients with cancer. There is evidence from observational studies that many patients do not get adequate relief. Although data in the literature confirm the effectiveness of most opioid drugs for the treatment of chronic pain, there is limited information about opioid titration. Methods: The aim of this study was to evaluate the clinical pharmacokinetics of morphine (M) and their correlation with pharmacodynamic results (effective daily dose of M and side effects) during the M titration phase, in the management of chronic cancer pain. Fifty-two consecutive patients were administered Oramorph (Molteni Farmaceutici, Scandicci, Florence, Italy; beginning with 5 mg every 6 hours), to maintain pain intensity at low levels (visual analog scale <4). M, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) plasma concentrations were determined by a mass spectrometric assay. Results: Expected pharmacokinetic parameters were based on a pharmacokinetic profile extrapolated from 39 patients: M total clearance varied between 1.5 and 6.42 L·h−1·kg−1; the median apparent volume of M distribution was 25.0 L/kg, and the elimination half-life was 4.4 hours. Over the entire period of treatment, a weak correlation between M and M3G or M6G concentrations was found, but the metabolite ratio (M3G/M6G) remained quite stable for each patient and at different sampling times. At the end of titration, the M6G/M ratio was significantly higher in the patients whose effective M concentration was below the median (5.2 ng/mL), than in patients in whom the concentration was above the median (M6G/M: 13.0 and 9.0, respectively). Conclusions: This article presents the pharmacokinetic profiles of M and its metabolites: their concentration ratio could help clinicians to optimize individual therapies and tailor the dose to individual needs. Our results indicate that the relationship between M6G and M could represent a potentially useful parameter to personalize M dosing.

A case of autism with an interstitial 1q deletion (1q23.3-24.2) and a de novo translocation of chromosomes 1q and 5q†

Chromosomal abnormalities may cause autism by disrupting a gene or by providing a permissive genetic environment for mutations elsewhere in the genome to become expressed as autism. We report here on a patient with an apparently balanced de novo translocation of chromosomes 1q and 5q. He presented with minor dysmorphic features and renal malformations, mental retardation, and autism. Further characterization of the chromosomal rearrangement by FISH revealed a deletion in chromosome 1 from q23.3 to q24.2 corresponding to a region of rising interest in the research of autism susceptibility genes. The array‐CGH technique gave better resolution of the breakpoints and the size of the deletion was calculated to be 4.97 Mb.