Identification of a complex congenital heart defect susceptibility locus by using DNA pooling and shared segment analysis

The identification of genetic loci involved in most forms of congenital heart disease has been hampered by the complex inheritance patterns of these disorders. Atrioventricular canal defects (AVCDs) are most commonly associated with Down syndrome, although non-syndromic cases also occur. Non-syndromic AVCDs have been attributed to multifactorial inheritance. However, the occurrence of a few kindreds with multiple affected individuals has suggested that a major genetic locus can account for the disorder in some families. We have used a combination of DNA pooling and shared segment analysis to perform a high density screen of the entire autosomal human genome in an extended kindred. In so doing, we have identified a genetic locus on chromosome 1 shared by all affected individuals. Our data demonstrate the existence of a congenital heart defect susceptibility gene, inherited as an autosomal dominant with incomplete penetrance, involved in AVCD. Furthermore, our data demonstrate the power of using key isolated kindreds in combination with high density genomic screens to identify loci involved in complex disorders such as congenital heart defects.      

Internet databases for clinical geneticists - an overview1

In this paper, we provide an overview of databases that are of importance to clinical geneticists. Some suggestions for the fruitful use for both research and diagnosis are given. For beginning ‘web-surfers’ we also list some well-known search engines and give a short overview of how to use these and other services. In addition, the URLs of some of the most important databases, gateways and tutorials are listed.     

Maternal Na+ intake induces renal function injury in rats prevented by a short‐term angiotensin converting enzyme inhibitor

The Na⁺‐ATPase, a secondary pump in the proximal tubule, is only weakly responsive to angiotensin II in adult offspring exposed perinatally to high Na⁺ intake. We have investigated whether the offspring from mothers given 0.3 mol/L NaCl show an ineffective angiotensin II action to increase in blood pressure. It was hypothesized that functional alterations at adult life are associated with the number of angiotensin II‐positive cells in the developing kidney, with increased oxidative stress in maternal/foetal organs, or with morphometrical changes in placentas. Wistar female rats were maintained on 0.3 mol/L NaCl in their drinking water from 20 days before conception until weaning. After weaning, some of the male offspring were treated with enalapril for 21 days. Glomerular filtration rate was recorded up to 210 days of age, when mean arterial pressure was measured after infusion of angiotensin II. To investigate the placenta and foetal kidneys, mothers on tap water or NaCl were also treated with alpha‐tocopherol, pregnancy being interrupted on the 20th day. There were no changes in the number of cells positive for angiotensin II in the foetal kidney and unchanged lipid peroxidation in the placenta of offspring exposed to NaCl, but the intermediate trophoblast area in the junctional zone was increased, possibly reducing maternal–foetal exchange. Glomerular filtration rate was reduced and there was an attenuated effect of angiotensin II on elevation of blood pressure, which could be mediated by an elevated angiotensin II during early life, once these disturbances had been prevented by early and short‐term treatment with enalapril.     

Disparity of amniotic fluid volume and fetal size: problem of the stuck twin--US studies

The "stuck twin" phenomenon in monochorionic diamniotic (MCDA) pregnancies is characterized by marked disparity in both fluid volume and fetal size between the twin gestations. To determine the prevalence, sonographic characteristics, and clinical outcome of this phenomenon, discharge summaries, placental pathologic reports, and prenatal sonograms from 307 twin pregnancies were reviewed. Of 52 cases of MCDA pregnancies, 18 (35%) demonstrated marked disparity in amniotic fluid volume. In 16 of these 18 cases there was discordant twin growth, further suggesting the diagnosis of twin transfusion syndrome. All 16 cases and an additional nine cases supplied by another center demonstrated a small, morphologically normal fetus in an oligohydramniotic sac suspended anteriorly (72%) or laterally (28%) in the uterus. The amniotic membrane separating this twin from the larger twin in the polyhydramniotic sac was thin, closely applied to the smaller fetus, and difficult to detect. Perinatal morbidity was 100% for all twin pairs, and premature labor occurred in all cases. Perinatal mortality ranged from 88% for the larger/poly twin to 96% for the small/oligo twin.     

Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region ("tethered ligand") into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin- induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34-52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34-52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83-94). Anti-TR34-52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34-52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83-94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34-52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34-52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34-52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34-52 prevented cleavage of the TR34-52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.