Intra-abdominal extra lobar pulmonary sequestration communicating with gastric duplication -- a case report.

Gastric duplications with associated pulmonary sequestration are very infrequent abnormalities. Although often asymptomatic, they should be considered in the differential diagnosis of intra-abdominal or retro-peritoneal masses. The authors report the case of a ten-year-old boy who had an occasional finding of poorly symptomatic intra-abdominal mass, recognised at intervention as a gastric duplication with associated extralobar pulmonary sequestration. The authors describe the surgical treatment of this case and briefly discuss the current views on the embryological pathogenesis of such malformations.

Influence of smoking habits on Down's syndrome risk evaluation at mid-trimester through biochemical screening.

Expectant mothers who smoke have higher levels of maternal serum alpha-fetoprotein and lower levels of unconjugated estriol and total human chorionic gonadotrophin than non-smoking mothers. This significantly affects performance of screening for Down's syndrome. This study includes 22,169 pregnant women: 18,876 non-smokers, 2,660 smoking < or = 10 cigarettes/day, and 633 smoking > 10 cigarettes/day. Mean maternal age (32.6 years), maternal weight (60.5 kg), and gestational age (114.7 days) were similar or only slightly different between the three groups. To verify the effects of smoking on screening, we studied retrospectively 130 sequential Down's syndrome cases (47 from the screening program, 83 from the prenatal diagnosis program). The proportion of smokers in the Down's syndrome and unaffected pregnancies was similar, whilst the false-positive rate and detection rate, based on fetal outcome, differed: false-positive rates were 5.63% in smokers and 9.42% in non-smokers, and detection rate 55.6% in smokers and 83.0% in non-smokers. Since the prevalence of Down's syndrome pregnancies was the same at mid-trimester in smokers and non-smokers and the proportion of smokers was not related to maternal age, we propose an adjustment of the Down's syndrome risk evaluation algorithm according to smoking habits.

Molecular characterization of immunoglobulin G4 gene isoallotypes.

The molecular bases of classical serological immunoglobulin allotypes are progressively uncovered through detailed characterization of the relevant genes. Here we describe two isoallotypic determinants of the G4 gene. In the first, Leu 309, as in G1 and G3, is changed to Val, as in G2; studies on myeloma proteins have long assigned the immunologically defined nG4 m(a)/(b) to the same position. The two molecular variants, here called IGHG4*L309 and IGHG4*V309, are allelic in IGHC haplotypes with a single G4 gene, but can be found together in cis in G4-duplicated haplotypes. A second isoallotypic variant was found at codon 409, where either Arg, as in G1 and G3, or Lys, as in G2, can be found. Both isoallotypes are associated with several 'silent isoallotypic' substitutions dispersed through the hinge, CH2 and CH3 domains. This suggests segmental gene conversion as the common mechanism of origin.

Congenital bilateral absence of vas deferens with a new missense mutation (P499A) in the CFTR gene.

We describe a congenital bilateral absence of the vas deferens (CBAVD) patient with a compound heterozygosity in the cystic fibrosis transmembrane regulator (CFTR) gene for a stop mutation W1282X and a new missense mutation P499A. The P499A is interpreted as a mild mutation whose phenotypic effects, in this case limited to the development of wolffian duct derivatives, are revealed only in combination with a severe CFTR mutation.

The G4 gene is duplicated in 44% of human immunoglobulin heavy chain constant region haplotypes.

The structure of the human immunoglobulin heavy chain constant region (IGHC), on chromosome 14q32, comprises nine CH genes and two pseudogenes, all originating from multiple duplication events. Continuing evolution of the region is demonstrated by the finding of various types of duplicated and deleted haplotypes, which together add up to 6%. Here we provide molecular and genetic evidence that the G4 gene is duplicated in 44% of IGHC haplotypes in the Italian population. The duplication spans about 20 kb of genomic DNA and probably originated through unequal crossing over. Refined characterisation of the genomic region downstream from the G4 gene improves our knowledge of the evolutionary history of CH genes.

Repeat testing of mothers with high human chorionic gonadotrophin levels in Down's syndrome screening.

Multiples of medians of serum markers are assumed to be independent of gestational age: every algorithm used for Down's syndrome risk evaluation is based on this hypothesis. However, our former observations suggested that multiples of medians of human chorionic gonadotrophin in Down's syndrome are dependent on gestatational age. Furthermore, observations on 84 Down's syndrome cases confirmed that human chorionic gonadotrophin multiples of medians in samples drawn at 15-17 weeks are approximately 10% lower than in samples drawn at 18-21 weeks, thus showing that the human chorionic gonadotrophin concentration decreases about 10% less than expected. The control group comprised 554 women with two blood samples and normal human chorionic gonadotrophin at first sampling. A further group of 532 women with multiples of medians at first sampling > 1.8 was examined with the aim of excluding an association between the human chorionic gonadotrophin trend in Down's syndrome and high starting values. The trend is peculiar to human chorionic gonadotrophin in Down's syndrome pregnancies and may help to explain the increase in detection rate with gestational age. Based on these findings, screening can be optimized, thus improving performance.

Autosomal dominant polycystic kidney disease (ADPKD) in an Italian family carrying a novel nonsense mutation and two missense changes in exons 44 and 45 of the PKD1 Gene.

Sixty-seven Italian patients with autosomal dominant polycystic kidney disease (ADPKD) were screened for mutations in the 3' unique region of the PKD1 gene, using heteroduplex DNA analysis. Novel aberrant bands were detected in 3 patients from the same family. DNA sequencing showed a C to T transition in exon 44 (C12269T), resulting in a premature stop codon (R4020X), predicted to impair the synthesis of the putative intracytoplasmic C-terminus tail of the PKD1 protein, polycystin. The mutation also generates a novel DdeI restriction site, and the abnormal restriction pattern was observed both on genomic DNA and on cDNA from the affected relatives, indicating that this is indeed the pathogenetic molecular lesion. Reverse transcriptase-polymerase chain reaction (RT-PCR) performed on lymphocyte mRNA showed that the mutant transcript is normally present and stable. No aberrantly spliced mRNAs were detected. Interestingly, the mutant PKD1 chromosome in this family also bears two missense mutations downstream (A12341G and C12384T), not found in the other ADPKD families studied.

Salivary IgG subclasses in individuals with and without homozygous IGHG gene deletions.

In this study, the levels of salivary IgG1, IgG2, IgG3 and IgG4 from individuals with and without homozygous immunoglobulin heavy chain constant gene deletions were quantified by enzyme-linked immunosorbent assay (ELISA). To analyse the restriction of salivary IgG subclasses, we used unstimulated whole saliva and sera collected at the same time from individuals with homozygous gene deletions, two with G1 deletion, one with G4 deletion, six with both G2 and G4 deletions and from eight individuals without IGHG gene deletions and expressing all four IgG subclasses. The median values of salivary IgG from individuals with homozygous G1, or G4, or both G2 and G4 deletions, and from individuals expressing all four subclasses were 24.2 mg/l and 23.4 mg/l, respectively. The median values of serum IgG were 13.7 g/l and 15.9 g/l, respectively. Our results show that the salivary and serum IgG levels were both within the normal range in individuals with homozygous gene deletions of either G1, or G4, or both G2 and G4.

[Molecular genetics of X-linked primary immunodeficiencies: advances in diagnosis and prevention]. / La genetica molecolare delle immunodeficienze primitive legate al cromosoma X: nuove prospettive nella diagnosi e nella prevenzione.

Over the past 2 years, progress in medical genetics has brought about major advances in the field of primary immunodeficiencies. The genes underlying four X-linked defects in humans (X-linked agammaglobulinemia-XLA, X-linked severe combined immunodeficiency-XSCID, hyper IgM syndrome-HIGM1, and Wiskott-Aldrich syndrome- WAS), have recently been identified. These syndromes are all associated with increased susceptibility to infections due to defects of cell mediated and/or humoral immunity. The X-linked disorders described here are due to mutations in genes whose products are involved in fundamental steps in the development and maturation of lymphoid cells. XLA results from a defect in a non-receptor tyrosine kinase that is likely to be involved in a lineage-specific pathway of growth signal transduction. XSCID is due to a defect of the subunit gamma, common to a family of multichain lymphokine receptors (i.e., IL-2R; IL-4R). HIGM1 results from a partial failure of the interaction between T helper cells and B cells owing to mutation of the ligand (CD40L) expressed on T cells which normally interacts with B cell receptor CD40. The WAS protein has recently been identified as the mutated protein in the WAS, although it has not yet been fully characterized. Analysis of X-inactivation in different hematopoietic cell lineages of carrier females has been done to identify the lineage affected by the genetic defect: non random X-inactivation is observed in cell lines where the mutated protein plays a fundamental role. Moreover, X-inactivation analysis serves as a test to identify obligate carriers and to perform prenatal diagnosis. These results have enabled better understanding of the pathogenetic mechanisms underlying some immunodeficiencies and have laid the foundation for future therapeutic possibilities.

Prenatal diagnosis of limb-girdle muscular dystrophy type 2A.

A branch of a highly inbred family was referred for prenatal counseling with an initial misdiagnosis of Becker Muscular Dystrophy (BMD) due to the limited clinical and laboratory data obtained in pre-dystrophin era and hidden family information. In a second branch of the family with a diagnosis of limb-girdle muscular dystrophy type 2A (LGMD2A) molecular studies revealed a homozygous 550 delta A mutation in the calcium-activated neutral protease 3 (calpain 3, CANP3) gene in the affected members. Finally, in the third branch of the family, it turned out that both parents were heterozygous for the 550 delta A mutation and the 13-week-old fetus was homozygous. The same mutation subsequently also was found in the first branch of the family. The parents were informed that the risk of their child of developing the disease would be very high given that he was carrying the same homozygous mutation of the other affected members. They were informed also that in another population (in Reunion Island) the same disease does not necessarily follow such a simple pattern of inheritance. After counseling the parents decided to terminate the pregnancy.

Analysis of the rhodopsin and peripherin/RDS gene in two families with pattern dystrophy of the retinal pigment epithelium.

Mutations of the peripherin/retinal degeneration slow (RDS) gene have been reported in autosomal dominant retinitis pigmentosa and variable forms of pattern dystrophy of the retinal pigment epithelium. We screened the rhodopsin and the peripherin/RDS gene in the members of two families who presented the clinical features of pattern dystrophy of the retinal pigment epithelium transmitted as an autosomal dominant trait. No migration patterns were detected in single strand conformation polymorphism or hydrolink gels. Both the rhodopsin and the peripherin/RDS gene were normal in one family. In the second, the proband had a normal rhodopsin gene and, although he passed a different haplotype to each of his affected daughters, there was no linkage with the peripherin/RDS gene. The origin of the retinal disturbance in our two pedigrees must therefore be sought, if indeed DNA is involved, elsewhere in the genome. Our findings provide additional evidence that pattern dystrophies of the retinal pigment epithelium may be pathogenically related in spite of different etiological origins. The genetic polymorphism can probably account for the wide range of phenotypes.

Pattern dystrophies of the retinal pigment epithelium.

Pattern dystrophies of the retinal pigment epithelium are infrequent fundus abnormalities arranged in various patterns of dots, lines and branches. The basic lesion appears to be yellow deposits of abnormal lipofuscin accumulated within degenerated retinal pigment epithelium cells. Examinations were carried out on two families who had developed different patterned alterations in the retinal pigment epithelium. The proband of family 1 had diffuse changes associated with equatorial folds. One sister had a macular alteration. A daughter was normal; a son had bilateral atrophy of the temporal retinal pigment epithelium. The proband of family 2 had bilateral, symmetrical retinal pigment epithelium lesions that simulated fundus flavimaculatus. His first daughter had a central lesion in her right eye. The second daughter, a peripapillary crescent of hyperpigmentation in her right eye, and circumpapillary chorioretinal atrophy associated with foveolar abnormalities in the left. This report provides further evidence that variable types of pattern dystrophy can occur within a single family pedigree and support the current opinion that all forms of pattern dystrophies are variants of a single pathogenic mechanism.

Detection of two different nonsense mutations in exon 44 of the PKD1 gene in two unrelated Italian families with severe autosomal dominant polycystic kidney disease.

Sixty-seven Italian patients with autosomal dominant polycystic kidney disease (ADPKD) were screened for mutations in the PKD1 gene. We used PCR, heteroduplex and single-strand conformation polymorphism DNA analysis, and automated DNA sequencing for exons 35, 36, 38, 44 and 45. We detected abnormal heteroduplexes in affected individuals from two unrelated families with clinically severe ADPKD phenotype. These changes were absent in other, unaffected members, as well as in the probands of the other families studied. DNA sequencing revealed in both cases different C to T transitions in exon 44, which created premature stop codons. Both mutations altered restriction sites, and the abnormal patterns were observed in all the affected family members. RT-PCR performed on lymphocyte mRNA showed that both the mutant and the normal transcript are represented. To our knowledge these are the first nonsense mutations described in the PKD1 gene.

Carrier detection of Duchenne muscular dystrophy through analysis of DNA from deciduous teeth of a dead affected child.

The sister of a child affected by Duchenne muscular dystrophy (DMD) was referred for genetic counselling to assess the risk of her being a carrier. Her brother had died 15 years previously at the age of 8. There were no other affected males in the family. There were no methods for DNA investigation at the time of the child's death and the family had never been studied for linkage with polymorphic probes on the chromosomal region Xp21. The only tissue from which an assessment of the risk could be made by DNA linkage analysis was two of the child's deciduous teeth that the parents had kept. DNA was extracted using a protocol described for the recovery of ancient DNA from museum specimens and archaeological finds. Multiplex amplification did not reveal deletions in 19 exons spanning the hot-spot regions for deletions within the dystrophin gene in Xp21. Linkage analysis using three highly polymorphic microsatellites demonstrated that the sister had not received the X chromosome borne by her brother. These results show that DNA extracted from teeth is a reliable source for molecular diagnosis.

Variability of the immunoglobulin heavy chain constant region locus: a population study.

The Immunoglobulin Heavy chain Constant region (IGHC) locus is a multigene family composed of highly homologous segments often involved in unequal crossings over that lead to deleted and duplicated haplotypes. The frequencies of these haplotypes in 558 individuals from Lombardy, Veneto, Puglia and Sardinia were determined by Pulsed Field Gel Electrophoresis (PFGE), followed by Southern blotting with four IGHC probes, and compared with those observed in 110 subjects from Piedmont. Twenty deletions and 60 duplications were characterized, all in heterozygous individuals except for 2 homozygous deletions. The differences in frequency between the five populations were not significant. The deletions/duplications involved one or more genes: GP-A2, A1-E and G4 duplications, and A1-E and GP-A2 deletions were the most common. Four new duplications are described: three, involving the genes from GP to A2, from G2 to G4, and G4, are counterparts of known deletions. The fourth duplication spans from GP to G2. A G1 deleted heterozygous individual never previously described in Italy is reported. All the rearranged haplotypes seem to be the result of unequal crossing over. The difference between the number of duplications and deletions was significant in Sardinia, Lombardy, Puglia and in the total of 668 subjects (P < 0.001). This may be due to selection or genetic drift.

Molecular characterization of Gm(n+) and G2m(n-) allotypes.

Immunoglobulin (Ig) allotype typing is usually performed with serological methods based on hemagglutination inhibition. The recent development of molecular techniques has allowed the molecular typing of several Ig markers. The hinge, CH2, and CH3 domains of the G2 gene from six unrelated individuals (three G2m(n+) and three G2m(n-)) were amplified and cloned to establish the molecular basis of the G2mn+ and G2mn- . Comparison of the allele sequences revealed three changes: two (codons 308 and 437) are silent exonic substitutions, one is a G to A transition corresponding to an amino acid difference in position 282: Val (GTG) in G2mn- , Met (ATG) in G2mn+ . These substitutions were identified via two approaches: 282 polymorphism, after digestion of a specific polymerase chain reaction product with Nla III followed by acrylamide electrophoresis; 308 and 437, by a dot-blot technique using allele-specific oligonucleotides. These molecular typing results correspond exactly to those obtained serologically; moreover, the three substitutions defining the G2mn+ and G2mn- alleles are always associated in a strict linkage disequilibrium.

Serum immunoglobulin levels in heterozygous subjects with immunoglobulin heavy chain constant region gene deletions.

The immunoglobulin heavy chain constant region locus is a multigene family composed of nine genes and two pseudogenes, whose high homology is often responsible for meiotic mispairings leading to deleted and duplicated haplotypes. These rearrangements have a population frequency of about 1.5% and 4.5% respectively, with a significant difference between deletions and duplications (P < 0.001). Both positive selection of duplications or negative selection against deletions can account for this imbalance. Serum levels of IgG and IgA subclasses, of IgE, of isohemagglutinins and of IgG antibodies to tetanus toxoid and pneumococcal antigens were evaluated in 11 heterozygous carriers of constant region deletions. There was no gross abnormality in serum IgG and IgA subclass levels, with the possible exception of G1-deleted individuals; furthermore, isohemagglutinins and anti-tetanus toxoid and pneumococcal IgG antibodies are in the normal range, suggesting that the humoral immune response is normal in these carriers. The influence of single and multiple immunoglobulin heavy chain constant region gene deletions on the humoral response is discussed.

[Cystic fibrosis: molecular genetics and new perspectives of prevention and therapy]. / Fibrosi cistica: genetica molecolare e nuove prospettive di prevenzione e terapia.

Cystic fibrosis (CF) is the most common lethal autosomal recessive disorder among Caucasians, occurring with a frequency of 1/2000 newborn babies. This editorial will consider the clinical features of CF as well as the genomic structure of the CFTR gene and the functional properties of its product, mutations of the gene, correlations between genotypes and phenotypes, strategies for carrier screening, and recent advances in gene therapy. After isolation and cloning of the CFTR gene, different laboratories have characterized over 350 mutations responsible for CF, the most frequent being the delta F508 which accounts for 70% of all CF chromosomes. Studies on correlations between genotypes and phenotypes have confirmed that patients with the homozygous delta F508/delta F508 genotype are severely affected, with major involvement of pancreatic function. Patients with the delta F508/R117H genotype, on the other hand, evidence a mild phenotype with pancreatic sufficiency. Several pilot studies for carrier detection are now in progress. As recent experiments with somatic gene therapy, performed on knock-out mice for the CFTR gene have given satisfactory results, it is hoped that the same approach can soon be used for humans.

Validation of IgA1 and IgA2 measurements by a solid-phase immunoradiometric assay in serum and secretions.

We describe specific, sensitive and reproducible immunoradiometric assays to measure total IgA and IgA subclass levels in biological fluids, which take into account the problem that polymeric forms are differently recognized in immunoassays. Sera from subjects totally deficient in one of the IgA subclasses allowed us to ensure the specificity of the subclass assays and to define the proportions of IgA1 (84%) and IgA2 (16%) in the normal pooled serum (from 30 blood donors) used as standard. With purified milk 11-S secretory IgA1 and 11-S secretory IgA2, we determined a correction factor for the corresponding polymeric forms using, respectively, monomeric IgA1 and monomeric IgA2 from pooled serum as standards. With the monoclonal antibodies used, purified 11-S secretory IgA1 was similarly recognized by both the total IgA assay and the IgA1 assay; both total IgA and IgA1 concentrations were underestimated compared with monomeric IgA or monomeric IgA1. In contrast, 11-S secretory IgA2 was better recognized by the IgA2 assay than by the total IgA assay and the values were thus overestimates. Considering this problem of recognition, we fractionated saliva and lung secretions by sucrose density gradient ultracentrifugation before measuring their IgA1 and IgA2 levels.

Small frameshift deletions within the COL4A5 gene in juvenile-onset Alport syndrome.

Small frameshift deletions within the COL4A5 gene were identified in three Alport syndrome Italian families by non-isotopic single-strand conformation polymorphism (SSCP) screening: in family RMA, a 7-bp deletion (GGGTGAA) in exon 39; in family DGR, a 4-bp deletion (TGGA) in exon 41; in family MIB, deletion of a G in exon 50. The phenotype was characterized by juvenile-onset renal failure with sensorineural hearing loss in males, and a milder clinical pattern in heterozygous females.