Identification of connexin43 (alpha1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE).

Gap junction channels formed by the connexin43 protein are considered to play crucial roles in development and function because they allow the direct cell-to-cell exchange of molecules that mediate multiple signaling events. Previous results have shown that connexin43 channels are intricately gated by phosphorylation and that disruption of this regulation gives rise to severe heart malformations and defects of laterality in human, chick and frog. Here we report the identification of connexin43 gene mutations that represent a minor population of connexin43 alleles, which could be reliably detected by using denaturing gradient gel electrophoresis (DGGE) to visualize normal and mutant DNAs that were separately sequenced. In contrast, sequencing of total PCR products without DGGE-pre-selection failed to consistently identify these mutations. Forty-six controls and 20 heart transplant recipients were examined in this study. In the latter group, 14 children had hypoplastic left heart syndrome (HLHS) in which connexin43 gene defects were detected in eight. The remaining six transplant patients with HLHS and all controls showed no defects. All eight HLHS children with gene defects had the same four substitutions: two that were silent polymorphisms, and two that were missense, replacing arginine codons at positions 362 and 376 with codons for glutamines. All four of these substitutions are identical to the nucleotide sequence of the connexin43 pseudogene, suggesting the possibility of an illicit recombination. A breakpoint region was identified 5' to the mutation site in a 63bp domain that is 100% identical in the gene and pseudogene. Results from in vitro phosphorylation indicate that the absence of arginines 362 and 376 completely abolishes phosphorylation in the connexin43 channel regulation domain suggesting a possible mechanism for the pathologies associated with HLHS.

Naumoff short-rib polydactyly syndrome compounded with Mohr oral-facial-digital syndrome.

A stillborn baby boy had findings of severe constitutional dwarfism with short limbs, short ribs, and polydactyly that were consistent with Naumoff (type III) short-rib polydactyly syndrome. He also had additional congenital anomalies, including cleft palate, notching of the upper lip, small tongue with accessory sublingual tissue. These oral and pharyngeal anomalies were consistent with Mohr (type II) oral-facial-digital syndrome. We suggest the stillborn infant represented a compound of Naumoff short-rib polydactyly syndrome (SRPS-III) and Mohr oral-facial-digital syndrome (OFDS-II).

Langerhans' cell histiocytosis presenting with an intracranial epidural hematoma.

An 8-year-old boy developed vomiting and severe headache following minor head trauma. A CT scan of the head demonstrated a lytic lesion of the skull and adjacent epidural hematoma. Surgical evacuation and removal of the skull lesion and hematoma were carried out, and pathologic evaluation resulted in a diagnosis of Langerhans' cell histiocytosis (LCH). Epidural involvement of Langerhans' cell histiocytosis is very rare, and we report the first case of LCH presenting as an intracranial epidural hematoma.

Congenital disseminated malignant rhabdoid tumor: a distinct clinicopathologic entity demonstrating abnormalities of chromosome 22q11.

The clinical, pathologic, and immunohistochemical features of a widely disseminated tumor with rhabdoid phenotype are described in nine infants < or = 3 months of age. Five neonates had tumor evident at birth, two of which had placental metastases. The average survival following diagnosis was < 6 weeks. None of the infants had an apparent primary tumor in either the kidney or brain. In four cases, the dominant mass involved the head and neck region, and in two cases, the primary mass was paraspinal. The histologic features were those of a high-grade, round cell neoplasm with abundant cytoplasm and containing cells with cytoplasmic filamentous inclusions. Immunohistochemical studies revealed polyphenotypic antigen expression. Genetic information was available from eight of nine cases. Karyotype analysis revealed abnormalities of chromosome band 22q11-12 in three of six tumors. Fluorescence in situ hybridization studies or molecular studies demonstrated 22q11.2 deletions in all five cases with available frozen tissue, two of which had translocations involving 22q by karyotype analysis. The similar clinical and pathologic findings in these rapidly fatal tumors in infants and the demonstration of abnormalities of chromosome 22q11 in a majority of the cases supports their histogenetic and nosologic relationship to the family of malignant rhabdoid tumors that typically occur in young children in several anatomic sites, including kidney, soft tissues, liver, and brain. Like neuroblastoma and rhabdomyosarcoma, malignant rhabdoid tumor can appear as disseminated disease at birth or shortly thereafter.

Prolonged discordant xenograft survival and delayed xenograft rejection in a pig-to-baboon orthotopic cardiac xenograft model.

OBJECTIVE: Our objectives were to study delayed xenograft rejection and the effectiveness of pretransplantation total lymphoid irradiation combined with immunosuppression on rejection in a pig-to-baboon cardiac xenograft model. METHODS: Baboons were treated with pretransplantation total lymphoid irradiation, cyclosporine A (INN: ciclosporin), and methotrexate. Orthotopic pig-to-baboon cardiac transplantations were performed after depletion of circulating xenoreactive natural antibody by pretransplantation donor organ hemoperfusion. Tissue samples were collected for immunologic and immunopathologic evaluation. RESULTS: Pig cardiac xenografts survived more than 18 and 19 days without evidence of hyperacute rejection. Immunologic analysis of serum samples demonstrated that circulating xenoreactive natural antibody levels did not return to pretransplantation levels. The production of xenoreactive natural antibodies from the recipient's splenocytes was inhibited completely. Histologic examination of xenografts showed the feature of acute vascular rejection. Immunohistochemical studies demonstrated infiltration of cardiac xenografts by large numbers of macrophages, small numbers of natural killer cells, and a few T cells. The infiltrating macrophages also showed expression of interleukin-1 and tumor necrosis factor. Diffuse deposition of immunoglobulin G, C1Q, C3, and fibrin on xenograft vasculature was observed. Interleukin-2 expression was not found in rejected cardiac xenografts. Xenograft endothelial cells also showed evidence of activation (expression of cytokines interleukin-1 and tumor necrosis factor). CONCLUSIONS: This study demonstrates prolonged discordant cardiac xenograft survival and delayed xenograft rejection in a pig-to-baboon model. The delayed xenograft rejection is mediated by both humoral and cellular mechanisms. Pretransplantation total lymphoid irradiation combined with cyclosporine A and methotrexate can inhibit xenoreactive natural antibody production but not elicited antipig antibody production and the xenoreactivity of macrophages.

Handling and evaluation of pediatric renal tumors.

Optimal pathologic evaluation of pediatric renal tumors, which share many features in common with their adult counterparts, also includes some relatively unique considerations, in large part related to specialized classification, grading or staging systems, and ongoing collaborative clinical and biologic studies. Important factors in the gross evaluation, procurement of tissue for special studies, and selection of tissue blocks for light microscopy are briefly reviewed.

Prolonged discordant cardiac xenograft survival in newborn recipients.

BACKGROUND: We previously demonstrated very low levels of xenoreactive natural antibodies in newborns, suggesting the possibility of prolongation of xenograft survival in newborn recipients. We used a pig-to-newborn goat heterotopic cardiac xenograft model to examine our hypothesis that hyperacute rejection would be absent in newborn recipients and that both humoral and cellular rejection would participate in the late phase of discordant xenograft rejection. METHODS AND RESULTS: The serum of newborn goats was found to have very low titers of natural anti-pig antibodies. Newborn pig hearts were transplanted heterotopically into the neck of four unmanipulated newborn goats: none of these xenografts underwent hyperacute rejection. Dilation of the xenografts and decreased contractility were observed 4 to 6 days after transplantation, and the xenografts eventually ceased functioning between 6 and 8 days after transplantation. Blood samples collected after transplantation demonstrated a dramatic increase in anti-pig xenoantibody titers and correlated with histological studies demonstrating features consistent with delayed humoral rejection, including reactive vascular endothelial and perivascular stromal cells, marked capillary congestion, and interstitial hemorrhages. Scant to diffuse perivascular and interstitial infiltration of activated lymphoid cells occurred. CONCLUSIONS: Our study demonstrates that hyperacute rejection does not occur, allowing limited prolongation of xenograft survival in a pig-to-newborn goat cardiac xenograft model. We propose that this is attributable, at least in part, to the very low titers of natural antibodies in newborn recipients. Delayed xenograft rejection, however, remains an important problem in these newborn recipients. This delayed xenograft rejection is likely the result of both humoral and cellular rejection mechanisms.

Use of electron microscopy and other special techniques in the investigation of suspected specimen contamination.

Contamination of a biopsy or surgical specimen with spurious tissue is an uncommon but potentially disastrous event. In this regard, the case of a 5-year-old boy referred for treatment of an abdominal tumor is presented. Sections made from paraffin blocks brought by the family showed both neuroblastoma and a spindle cell sarcoma, initially suggesting the possibility of divergent or mixed differentiation. However, the resemblance of the spindle cell component to well-differentiated leiomyosarcoma rather than rhabdomyosarcoma raised the suspicion that a specimen contamination had occurred. Electron microscopy was instrumental in confirming the smooth muscle nature of the sarcomatous component, leading to a fluorescence in situ hybridization study, which established that this component was incompatible with the patient's gender. This case illustrates that even when the light microscopic differential has been compromised by specimen mishandling, electron microscopy can at times provide useful information regarding specimen identity, as well as assist in sorting out the correct diagnosis.

Primitive neuroectodermal tumor in a child with tuberous sclerosis.

The case of a 13-year-old boy with tuberous sclerosis and primitive neuroectodermal tumor of bone is presented. A pathogenetic association between these two entities is postulated, the unifying underlying mechanism being a maldevelopment of the neural crest, or neurocristopathy.

Limitations to survival for infants with hypoplastic left heart syndrome before and after transplant: the Loma Linda experience.

Untreated, hypoplastic left heart syndrome is a lethal cardiac defect. Heart transplant has become an accepted therapeutic option for this condition. However, significant limitations to survival remain for infants with this condition who are referred for heart transplantation. Attention to the prevention, early detection, and management of common problems occurring at each stage of the transplantation process is important for improving survival rates. This study retrospectively reviewed the cases of 195 infants with hypoplastic left heart syndrome registered for heart transplantation at Loma Linda University Medical Center between November 1985 and July 1996 to determine causes of death. During the waiting period, progressive cardiac failure and complications from interventional procedures were the leading causes. In the early postoperative period, technical issues and acute graft failure were most important, whereas late deaths (more than 30 days after transplant) were most often related to rejection and posttransplant coronary artery disease.

Neonatal osteofibrous dysplasia: report of two cases.

Skeletal neoplasms of the newborn and neonatal period are infrequently encountered and often misdiagnosed. Osteofibrous dysplasia is a tumor of childhood that may be seen in the newborn. Because of its unique natural history, with and without surgical treatment, and the similarity in radiographic appearance of this tumor to other lesions, a high index of suspicion must be maintained when a tibial lesion is encountered in the newborn. We present two additional cases of neonatal osteofibrous dysplasia and discuss the differential diagnostic considerations.

Orthotopic transplantation for total anomalous pulmonary venous connection associated with complex congenital heart disease.

BACKGROUND: When total anomalous pulmonary venous connection is associated with other complex cardiac malformations, early and late postsurgical morbidity and mortality are excessive. METHODS: In an attempt to modify this outcome, twelve children (4 days to 6.8 years of age) with total anomalous pulmonary venous connection and various congenital cardiac defects were treated with orthotopic heart transplantation. Associated cardiac diagnoses included the following: hypoplastic left heart syndrome (n = 2), unbalanced atrioventricular canal with pulmonary atresia (n = 2), and single ventricle with severe pulmonary stenosis (n = 3) or atresia (n = 5). Two patients had situs inversus, and two had dextrocardia with situs ambiguous. Eight patients had asplenia and one had polysplenia. Palliative pretransplantation procedures in five patients included the following: systemic to pulmonary artery shunt (n = 5), atrioventricular valve annuloplasty (n = 1) and classical Glenn shunt (n = 1). The donor left atrium was anastomosed directly to a common pulmonary venous pool in nine patients; whereas three children required complex reconstruction to baffle the pulmonary venous flow to the donor left atrium. RESULTS: There was one operative death related to an oversized heart and vena caval thrombosis. Follow-up ranged from 16 months to 4.5 years (average 3 years). In two patients (18%) pulmonary venous obstruction developed 3 and 4 months after transplantation. Reoperation to relieve the obstruction was successful in one patient. The second patient underwent three such reoperations and died of sepsis 10 months after orthotopic heart transplantation. CONCLUSION: Orthotopic transplantation is a viable option for children with complex total anomalous pulmonary venous connection that precludes a biventricular repair. Transplantation may improve the dismal prognosis of those children, but it does not eliminate the potential for late pulmonary venous obstruction.

A novel method for treatment of abdominal aortic aneurysms using percutaneous implantation of a newly designed endovascular device.

BACKGROUND: Percutaneous implantation of a stent to bridge abdominal aortic aneurysm (AAA) may provide an alternative to surgical reconstruction in patients with this serious disorder. We developed a self-expandable, stainless steel, woven mesh endovascular device with a delivery catheter and studied its efficacy in a canine model of AAA. METHODS AND RESULTS: Infrarenal AAAs were created surgically in eight adult dogs using autologous tissue. Two types of endovascular stents were used in this study; a plain or uncovered stent, about 14 mm in diameter in the unconstrained configuration, and a covered stent, coated by porous polyurethane, about 16 mm in diameter. All stents were successfully placed on the first attempt. Aortograms revealed a mean aneurysm diameter of 1.86 +/- 0.47 cm, an average of 70% larger than the reference aortic lumen before stent placement. After stent placement, aortograms showed that the aneurysmal cavity disappeared completely in three dogs treated with a covered stent and that the aortic blood flow into the cavity markedly reduced, with faint contrast filling the cavity in the remaining five dogs treated with an uncovered stent. The uncovered stent was intentionally placed across the major arterial branches in two dogs. No acute complications were encountered at the time of stent placement. Two dogs were killed shortly after the procedure for immediate evaluation of the device, which was found to be in place and patent. One dog in which a covered stent was placed was euthanized 2 1/2 weeks later because of paraplegia secondary to a spinal cord infarction noted 48 hours after stent placement. Postmortem study revealed thrombus occluding the stent lumen. The remaining five dogs tolerated the devices well and completed 4 weeks of follow-up. Premortem aortograms showed no residual aneurysmal cavity in four dogs and only a small cavity in one dog that had received an uncovered stent. All stents were fully patent with no thrombus and were either completely or partially surfaced by neointima. Importantly, the major arterial branches over which the uncovered stents were placed were widely patent without obstruction by neointima. CONCLUSIONS: This study demonstrates the feasibility of percutaneous implantation of this new device and its effectiveness in the treatment of surgically created AAA in our canine model. The covered stent was able to exclude AAA immediately upon deployment and is of potential value in the emergency treatment of leaking AAAs. The uncovered stent appears to safely bridge branch arteries as well as significantly reduce the angiographic size of the aneurysm and may be useful in the elective therapy of AAAs. These results are promising, and future clinical trials to investigate the safety and efficacy of this device in humans are warranted.

Mutations of the Connexin43 gap-junction gene in patients with heart malformations and defects of laterality.

BACKGROUND: Gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. Connexin43, the major protein of gap junctions in the heart, is targeted by several protein kinases that regulate myocardial cell-cell coupling. We hypothesized that mutations altering sites critical to this regulation would lead to functional or developmental abnormalities of the heart. METHODS: Connexin43 DNA from 25 normal subjects and 30 children with a variety of congenital heart diseases was amplified by the polymerase chain reaction and sequenced. Mutant DNA was expressed in cell culture and examined for its effect on the regulation of cell-cell communication. RESULTS: The 25 normal subjects and 23 of the 30 children with heart disease had no amino acid substitutions in connexin43. All six children with syndromes that included complex heart malformations had substitutions of one or more phosphorylatable serine or threonine residues. Four of these children had two independent mutations, suggesting an autosomal recessive disorder. Five of these children had substitutions of proline for serine at position 364. A seventh child, with a different heart condition, also had a point mutation in connexin43. Transfected cells expressing the Ser364Pro mutant connexin43 sequence showed abnormalities in the regulation of cell-cell communication, as compared with cells expressing normal connexin43. CONCLUSIONS: Mutations in the connexin43 gap-junction gene, which lead to abnormally regulated cell-cell communication, are associated with visceroatrial heterotaxia.