Living-related liver transplantation for siblings with progressive familial intrahepatic cholestasis 2, with novel genetic findings.

Progressive familial intrahepatic cholestasis is a syndrome of severe cholestasis progressing to biliary cirrhosis and liver failure that develops in childhood. This report describes two siblings with PFIC-2 who underwent living-related liver transplantation from their genetically proven heterozygous parents. Both patients had normal gamma-glutamyl transpeptidase levels, but showed severe pruritus with sleep disturbance, cholestasis, jaundice and growth failure. Genetic testing of each patient revealed two missense mutations of the bile salt export pump, S901R and C1083Y, which have not previously been associated with PFIC-2. Usual medical treatment failed to improve their clinical symptoms, and the two siblings underwent living-related liver transplantation from their heterozygous parents. The transplants improved their clinical symptoms significantly, and the patients have since shown age-appropriate growth. Electron microscopic findings of the explanted liver of the younger sister revealed dense and amorphous bile, which is characteristic of PFIC-2. In the cases presented here, living-related liver transplantation from a heterozygous donor was associated with better quality of life and improvement of growth, and thus appears to be a feasible option for PFIC-2 patients. Mutation analysis is a useful tool to help decide the course of treatment of PFIC.

Cri-du-Chat Syndrome Cytogenetically Cryptic Recombination Aneusomy of Chromosome 5: Implications in Recurrence Risk Estimation.

Cri-du-chat syndrome is caused by haploinsufficiency of the genes on the distal part of the short arm of chromosome 5, and characteristic features include microcephaly, developmental delays, and a distinctive high-pitched mewing cry. Most cri-du-chat syndrome cases result from a sporadic de novo deletion that is associated with a low recurrence risk. On rare occasions, however, cri-du-chat syndrome with 5p monosomy can be accompanied by 5q trisomy. This combination is virtually always associated with parental large pericentric inversions. Among previously reported cri-du-chat syndrome cases with 5p monosomy accompanied by 5q trisomy, the aneusomy of chromosome 5 in all but one case was cytogenetically visible using G-banding. When an accompanying 5q trisomy is detected, a significant recurrence risk is expected. We here report on a patient with cri-du-chat syndrome phenotype who initially exhibited a normal karyotype on G-banding but in whom molecular analysis using multiplex ligation-dependent probe amplification and array comparative genomic hybridization revealed a 5p deletion accompanied by a 5q duplication. Parental chromosomal testing led to the identification of a very large pericentric inversion, of which breakpoints resided at the terminal regions of 5p15.31 and 5q35.1. This information was vital for counseling the family regarding the significantly high recurrence risk.

Immunohistological study of infiltrated cells and cytokines in murine herpetic keratitis.

PURPOSE: To identify localization and kinetics of infiltrated cells and cytokines in murine herpetic keratitis. METHODS: HSV-1 was inoculated onto the scarified BALB/c corneas. At given times post infection (PI), eyes were removed and studied immunohistochemically using monoclonal antibodies against several infiltrated cells and cytokines. RESULTS: Neutrophils and NK cells infiltrated as early as 1 day PI reaching a maximum number at 2 day PI in initial stage. gamma delta TCR positive cells were observed in the corneal stroma from 1 day PI to 8 day PI. IL-2 and IFN-gamma were positive in the cell-infiltrated areas of the epithelial and stromal lesions, whereas IL-4 was negative throughout the experiment. CONCLUSION: Our results indicated that cytokine profile upon herpes infection on the cornea is Th1 dominant. Together with neutrophils in the early phase of infection, gamma delta positive T cells may play an additional role in protecting the cornea against incoming pathogens.

Mutation analysis of left-right axis determining genes in NOD and ICR, strains susceptible to maternal diabetes.

BACKGROUND: Genetic background of the fetus contributes to the pathogenesis of congenital malformation after teratogen exposure. Such contribution is illustrated in left-right axis malformations observed in the F1 offspring of nonobese diabetic (NOD) mouse dams and sires from different strains. When sires of the NOD, ICR, or C57BL/6J were mated with NOD dams, incidence varied depending on the fetal genotype, with 65% in NOD x NOD, 24% in NOD x ICR, and 7% in NOD x C57BL/6J. METHODS: As a first step in elucidating the molecular basis of the interstrain differences in susceptibility to situs defects, we compared genomic sequences of six genes HNF3beta, Acvr2b, Nodal, ZIC3, Lefty1, and Smad2, which are involved in the normal development of left-right axis among NOD, ICR, and C57BL/6J strains. RESULTS: The outbred strain ICR had 1) a 0.2-kb insertion in the putative promoter region of the isoform E of HNF3beta together with a G to A change that could create a potential splice acceptor in the exon 3 of HNF3beta (gene frequency P = 0.36), 2) five single base substitutions within the 5' controlling element and a proline to serine substitution (P2S) of Lefty1 (P = 0.77), and 3) a tyrosine to histidine substitution within the prodomain of Nodal (P = 0.48). The inbred strain NOD had the same G to A change as ICR and a three-base deletion in the putative promoter of isoform E of HNF3beta. CONCLUSIONS: We suggest that sequence variations in HNF3beta, Lefty1, and Nodal might account, in part, for the interstrain differences in susceptibility to situs abnormalities among the offspring of diabetic dams.

Human homolog of the mouse imprinted gene Impact resides at the pericentric region of chromosome 18 within the critical region for bipolar affective disorder.

Several mapping studies of families with multiple individuals who have bipolar affective disorder (BPAD) have demonstrated possible linkage of the trait to the pericentric region of chromosome 18 (18cen). Currently, the large size of the critical interval defined by these studies makes effective selection of candidate genes formidable. However, documentation of 18cen-linked families in which a parent-of-origin effect was observed in the transmission of the BPAD trait provides a clue to the nature of the putative gene; it may be imprinted. In the present study, we cloned IMPACT, the human homolog of the mouse imprinted gene Impact and mapped it to 18cen within the critical interval for BPAD. Human IMPACT encodes a protein with 320 amino acids and is expressed at high levels in the brain. Since only a small number of imprinted genes are estimated to be present in the entire genome, very few imprinted genes would be expected to be present in this particular chromosomal region. Hence, IMPACT represents a candidate gene for BPAD susceptibility. Alternatively, other as yet unknown imprinted gene(s) adjacent to IMPACT could contribute to the BPAD trait, since multiple imprinted genes may occasionally form clusters. Localization of human IMPACT at 18cen in this study defines a promising target region in which to search for putative BPAD genes.

Fluorescence-based DHPLC for allelic quantification by single-nucleotide primer extension.

We have investigated the possibility of determining quantitatively the alleles of binary DNA polymorphisms by single-nucleotide primer extension (SNuPE) and fluorescence-based DHPLC. Using a polymorphism of interest to our group, ROX-labeled dideoxy CTP (ROX-ddCTP) was incorporated at the 3' end of the primer annealed to the template adjacent to the polymorphic site. The primer extension product was then resolved from the unincorporated dye terminator by ion-pair reversed-phase liquid chromatography. The signal intensity of incorporated ROX-ddCTP correlated well over one order of magnitude with the relative amount of the C-allele present in the genomic DNA template. We conclude that SNuPE, when combined with fluorescence-based DHPLC, can accurately determine the relative molar proportion of one allele in total DNA.

Imprinting of human GRB10 and its mutations in two patients with Russell-Silver syndrome.

Documentation of maternal uniparental disomy of chromosome 7 in 10% of patients with Russell-Silver syndrome (RSS), characterized by prenatal and postnatal growth retardation and dysmorphic features, has suggested the presence of an imprinted gene on chromosome 7 whose mutation is responsible for the RSS phenotype. Human GRB10 on chromosome 7, a homologue of the mouse imprinted gene Grb10, is a candidate, because GRB10 has a suppressive effect on growth, through its interaction with either the IGF-I receptor or the GH receptor, and two patients with RSS were shown to have a maternally derived duplication of 7p11-p13, encompassing GRB10. In the present study, we first demonstrated that the GRB10 gene is also monoallelically expressed in human fetal brain tissues and is transcribed from the maternally derived allele in somatic-cell hybrids. Hence, human GRB10 is imprinted. A mutation analysis of GRB10 in 58 unrelated patients with RSS identified, within the N-terminal domain of the protein, a P95S substitution in two patients with RSS. In these two cases, the mutant allele was inherited from the mother. The fact that monoallelic GRB10 expression was observed from the maternal allele in this study suggests but does not prove that these maternally transmitted mutant alleles contribute to the RSS phenotype.

Immunohistochemical evidence for the brevican-tenascin-R interaction: colocalization in perineuronal nets suggests a physiological role for the interaction in the adult rat brain.

Brevican is one of the most abundant chondroitin sulfate proteoglycans in the adult rat brain. We have recently shown that the C-type lectin domain of brevican binds fibronectin type III domains 3-5 of tenascin-R. Here we report strong evidence for a physiological basis for this interaction. Substantial brevican immunoreactivity was detected in a number of nuclei and in the reticular formations throughout the midbrain and hindbrain, including, but not limited to, the deep cerebellar nuclei, the trapezoid body, the red nucleus, the oculomotor nucleus, the vestibular nucleus, the cochlear nucleus, the gigantocellular reticular nucleus, the motor trigeminal nucleus, and the lateral superior olive. Most of the brevican immunoreactivity exhibited pericellular and reticular staining patterns. In almost all of these sites, brevican immunoreactivity colocalized with that of tenascin-R, which was also substantially codistributed with versican, another member of the lectican family. Detailed analysis revealed that the pericellular staining of brevican resembled that in perineuronal nets in which tenascin-R has been localized. Immunoelectron microscopy identified brevican immunoreactivity in the intercellular spaces surrounding presynaptic boutons and on their surfaces, but not in the synaptic clefts or in their immediate vicinity, a distribution pattern consistent with perineuronal nets. Taken together, our results provide strong evidence that the previously reported interactions between brevican and tenascin-R may play a functional role within the perineuronal nets.

Characterization and mutation analysis of human LEFTY A and LEFTY B, homologues of murine genes implicated in left-right axis development.

Members of the transforming growth factor (TGF)-beta family of cell-signaling molecules have been implicated recently in mammalian left-right (LR) axis development, the process by which vertebrates lateralize unpaired organs (e.g., heart, stomach, and spleen). Two family members, Lefty1 and Lefty2, are expressed exclusively on the left side of the mouse embryo by 8.0 days post coitum. This asymmetry is lost or reversed in two murine models of abnormal LR-axis specification, inversus viscerum (iv) and inversion of embryonic turning (inv). Furthermore, mice homozygous for a Lefty1 null allele manifest LR malformations and misexpress Lefty2. We hypothesized that Lefty mutations may be associated with human LR-axis malformations. We now report characterization of two Lefty homologues, LEFTY A and LEFTY B, separated by approximately 50 kb on chromosome 1q42. Each comprises four exons spliced at identical positions. LEFTY A is identical to ebaf, a cDNA previously identified in a search for genes expressed in human endometrium. The deduced amino acid sequences of LEFTY A and LEFTY B are more similar to each other than to Lefty1 or Lefty2. Analysis of 126 human cases of LR-axis malformations showed one nonsense and one missense mutation in LEFTY A. Both mutations lie in the cysteine-knot region of the protein LEFTY A, and the phenotype of affected individuals is very similar to that typically seen in Lefty1-/- mice with LR-axis malformations.

Overproduction of hyaluronan by expression of the hyaluronan synthase Has2 enhances anchorage-independent growth and tumorigenicity.

Hyaluronan (HA) has long been implicated in malignant transformation and tumor progression. However, due to the lack of molecular tools to directly manipulate production of HA, which does not require a core protein for its synthesis, our understanding of the role of HA in tumor cells has been largely circumstantial. In this study, we genetically manipulated the production of HA by transfection of a mammalian HA synthase Has2 into human HT1080 cells and examined the malignant phenotype of transfected cells. We found that increased production of HA promotes anchorage-independent growth and tumorigenicity of the cells. Has2-transfected cells formed greater numbers of colonies in semisolid medium. Tumors in nude mice derived from Has2-transfected cells grew more rapidly and were 2-4 times larger than those derived from control cells at termination of experiments. Histological and biochemical analyses of tumors revealed no significant differences in cell density and tissue structures between them, indicating that the larger size of the tumors was due to enhanced cell proliferation, not to increased accumulation of tumor stroma or increased angiogenesis. These results demonstrate that HA production by tumor cells per se promotes proliferation of these cells in tissues and provides direct evidence for the role of HA in tumorigenicity.

Left-right axis malformations associated with mutations in ACVR2B, the gene for human activin receptor type IIB.

Targeted disruption of the mouse activin receptor type IIB gene (Acvr2b) results in abnormal left-right (LR) axis development among Acvr2b-/- homozygotes [Oh and Li, 1997: Genes Dev 11:1812-1826]. The resulting malformations include atrial and ventricular septal defects, right-sided morphology of the left atrium and left lung, and spleen hypoplasia. Based on these results, we hypothesized that mutations in the type IIB activin receptor gene are associated with some cases of LR axis malformations in humans. We report here characterization of the ACVR2B genomic structure, analysis of ACVR2B splice variants, and screening for ACVR2B mutations among 112 sporadic and 14 familial cases of LR axis malformations. Two missense substitutions have been identified, one of which appears in two unrelated individuals. Neither of these nucleotide changes has been found in 200 control chromosomes. We conclude that ACVR2B mutations are present only rarely among human LR axis malformation cases.

Corneal infiltration and CMV retinitis in a patient with AIDS.

PURPOSE: To describe the clinical features of stromal keratitis in a patient with cytomegalovirus (CMV) retinitis and acquired immunodeficiency syndrome (AIDS). METHODS: Case report. RESULTS: Human CMV genome was detected in plasma, urine, and aqueous humor by polymerase chain reaction. CMV retinitis subsided and the corneal infiltrate was scarred within 4 weeks of systemic ganciclovir treatment. CONCLUSION: This is the first report of the corneal infiltration seen in an AIDS patient with CMV retinitis. Etiological significance of the finding is discussed.