Histological demonstration of BSEP/ABCB11 inhibition in transient neonatal cholestasis: a case report.

BACKGROUND: Idiopathic or transient neonatal cholestasis (TNC) represents a group of cholestatic disorders with unidentified origin and remains a diagnosis of exclusion. Dysfunction of hepatobiliary transporters mediating excretion of biliary constituents from hepatocytes may play a central role in the pathogenesis of cholestasis. Despite variants of bile salt (BS) export pump (BSEP/ABCB11) have already been described in TNC, the pathogenic role of BSEP dysfunction in TNC remained so far elusive. CASE PRESENTATION: We report on a newly-identified heterozygous ABCB11 missense variant (c.1345G > A, p.Glu449Lys) which was associated with prolonged cholestasis in a term infant after a complicated neonatal period. Moreover, we show for the first time almost completely abolished BSEP expression on the hepatocellular membrane in TNC. CONCLUSION: This report demonstrates for the first time a close association between the prolonged cholestasis in infancy and impaired BSEP expression on the hepatocyte canalicular membrane in a heterozygous carrier of newly-identified ABCB11 variant.

NOD2 gene variants confer risk for secondary sclerosing cholangitis in critically ill patients.

Sclerosing cholangitis in critically ill patients (SC-CIP) is a progressive cholestatic disease of unknown aetiology characterized by chronic biliary infections. Hence we hypothesized that common NOD2 (nucleotide-binding oligomerisation domain containing 2) gene variants, known risk factors for Crohn's disease and bacterial translocation in liver cirrhosis, increase the odds of developing SC-CIP. Screening of 4,641 endoscopic retrograde cholangiography procedures identified 17 patients with SC-CIP, who were then genotyped for the three common NOD2 mutations (Cohort 1, discovery cohort). To validate the association, we subsequently tested these NOD2 variants in 29 patients from SC-CIP cohorts of three additional medical centers (Cohort 2, replication cohort). In Cohort 1, the NOD2 variants were present in 5 of 17 SC-CIP patients (29.4%), which is twice the frequency of the general population. These results were replicated in Cohort 2 with 8 patients (27.6%) showing NOD2 mutations. In contrast, polymorphisms of hepatocanalicular transporter genes did not have major impact on SC-CIP risk. This first study on genetic susceptibility in SC-CIP patients shows an extraordinary high frequency of NOD2 variation, pointing to a critical role of inherited impaired anti-bacterial defense in the development of this devastating biliary disease.

An X-linked myopathy with postural muscle atrophy and generalized hypertrophy, termed XMPMA, is caused by mutations in FHL1.

We have identified a large multigenerational Austrian family displaying a novel form of X-linked recessive myopathy. Affected individuals develop an adult-onset scapulo-axio-peroneal myopathy with bent-spine syndrome characterized by specific atrophy of postural muscles along with pseudoathleticism or hypertrophy and cardiac involvement. Known X-linked myopathies were excluded by simple-tandem-repeat polymorphism (STRP) and single-nucleotide polymorphism (SNP) analysis, direct gene sequencing, and immunohistochemical analysis. STRP analysis revealed significant linkage at Xq25-q27.1. Haplotype analysis based on SNP microarray data from selected family members confirmed this linkage region on the distal arm of the X chromosome, thereby narrowing down the critical interval to 12 Mb. Sequencing of functional candidate genes led to the identification of a missense mutation within the four and a half LIM domain 1 gene (FHL1), which putatively disrupts the fourth LIM domain of the protein. Mutation screening of FHL1 in a myopathy family from the UK exhibiting an almost identical phenotype revealed a 3 bp insertion mutation within the second LIM domain. FHL1 on Xq26.3 is highly expressed in skeletal and cardiac muscles. Western-blot analysis of muscle biopsies showed a marked decrease in protein expression of FHL1 in patients, in concordance with the genetic data. In summary, we have to our knowledge characterized a new disorder, X-linked myopathy with postural muscle atrophy (XMPMA), and identified FHL1 as the causative gene. This is the first FHL protein to be identified in conjunction with a human genetic disorder and further supports the role of FHL proteins in the development and maintenance of muscle tissue. Mutation screening of FHL1 should be considered for patients with uncharacterized myopathies and cardiomyopathies.

Genomic analysis of five chromosome 7p deletion patients with Greig cephalopolysyndactyly syndrome (GCPS).

Chromosomal deletions on chromosome 7p are associated with Greig cephalopolysyndactyly syndrome (GCPS, OMIM 175700) a syndrome affecting the development of the skull, face, and limbs. We have compared data from molecular cytogenetic and genetic analyses with clinical symptoms from five previously published GCPS deletion patients, including a pair of monozygotic twins. The genomic DNA of the probands and their parents, as well as the DNA from monoallelic cell lines of two patients, was analyzed using microsatellite markers. In some cases (e.g. where the microsatellite studies were uninformative) we also used fluorescence in situ hybridization (FISH) with bacterial artificial chromosomes (BAC) probes. The fine mapping results of the deletions and genomic data from chromosome 7, were compared to the clinical symptoms. Common breakpoint sequences or mutation hotspots were not observed. Mutation screening for PGAM2, which is responsible for a form of myopathy with recessive inheritance, was performed in all patients. Loss of heterozygosity for known genes with dominant inheritance, such as the glucokinase gene (GCK), which, when mutated or haploinsufficient, is responsible for maturity-onset diabetes of the young, type II (MODY2, OMIM 125851), was identified and included in a genetic counseling of the patients' families.

Phenotypic and molecular characterisation of a de novo 5q deletion that includes the APC gene.

We report on a 12-year-old female patient with mild dysmorphic signs, including bilateral epicanthal folds, low-set dysplastic ears, a short nose with anteverted nostrils, conically shaped fingers, generalised increase of subcutaneous fat, multiple fine venous teleangiectasia on her back, mild pectus carinatum, and a general muscular hypotonia. Cytogenetic analysis and fluorescence in situ hybridisation (FISH) studies using region-specific BAC and YAC clones indicated a de novo interstitial deletion of the long arm of chromosome 5, resulting in monosomy 5q21.1-q23.1. Molecular analysis of polymorphic markers helped to narrow down the breakpoints and demonstrated that the derivative chromosome 5 is of paternal origin. By using the same panel of polymorphic markers, a reinvestigation of a similar, already published, 5q deletion case [Raedle et al. (2001) Am J Gastroenterol 96:3016-3020] was performed, allowing a more detailed genotype-phenotype correlation. Phenotypic classification was also carried out. Several known genes, including APC and MCC, were found to map to the common deleted genomic segment. Genetic counselling based on the molecular analysis data was performed for the index family.

Cloning, genomic structure, and expression profiles of TULIP1 (GARNL1), a brain-expressed candidate gene for 14q13-linked neurological phenotypes, and its murine homologue.

Previously, we have described the clinical and molecular characterization of a de novo 14q13.1-q21.1 microdeletion, less than 3.5 Mb in size, in a patient with severe microcephaly, psychomotor retardation, and other clinical anomalies. Here we report the characterization of the genomic structure of the human tuberin-like protein gene 1 (TULIP1; approved gene symbol GARNL1), a CpGisland-associated, brain-expressed candidate gene for the neurological findings in our patient, and its murine homologue. The human TULIP1 gene was mapped to chromosome band 14q13.2 by fluorescence in situ hybridization of BAC clone RP11-355C3 (GenBank Accession No. AL160231), containing the 3' region of the gene. TULIP1 spans about 271 kb of human genomic DNA and is divided into 41 exons. An untranscribed, processed pseudogene of TULIP1 was found on human chromosome band 9q31.1. The active locus TULIP1, encoding a predicted protein of 2036 amino acids, is expressed ubiquitously in pre- and postnatal human tissues. The murine homologue Tulip1 spans about 220 kb of mouse genomic DNA and is also divided into 41 exons, encoding a predicted protein of 2035 amino acids. No pseudogene could be found in the available mouse sequence data. Several splicing variants were found. Considering the location, expression profile, and predicted function, TULIP1 is a strong candidate for several neurological features seen in 14q deletion patients. Additionally we searched for mutations in the coding region of TULIP1 in subjects from a family with idiopathic basal ganglia calcification (IBGC; Fahr disease), previously linked to chromosome 14q. We identified two novel SNPs in the intron-exon boundaries; however, they did not segregate only with affected subjects in the predicted model of an autosomal dominant disease such as IBGC.

The C-terminal domain, but not the interchain disulphide, is required for the activity and intracellular trafficking of aminopeptidase A.

Mammalian aminopeptidase A (APA; glutamyl aminopeptidase; EC 3.4.11.7) is a type II membrane-spanning protein consisting of a short N-terminal cytosolic domain, a single transmembrane domain and a large extracellular C-terminal domain containing the active site. The extracellular domain consists of a 107 kDa domain, containing the zinc-binding motif and all the residues involved in catalysis, separated by a protease-susceptible hinge region from the 45 kDA C-terminal domain of unknown function. To investigate the role of the 45 kDa domain, a construct of murine APA (G594Delta) lacking this C-terminal domain was expressed in COS-1 cells. This truncated form of APA, although expressed, lacked enzymic activity and failed to reach the cell surface. Confocal immunofluorescence microscopy revealed that G594Delta co-localized with the lectin concanavalin A and had a similar staining pattern as protein disulphide-isomerase, indicating that it was retained in the endoplasmic reticulum. Thus the C-terminal 45 kDa domain appears to be acting like a pro-domain and seems to be required for the correct folding and trafficking of APA. In contrast, mutation of cysteine-43 to serine, which is involved in the disulphide-linkage of the APA homodimer, did not affect the enzymic activity, cellular location or rate of trafficking through the secretory pathway of APA.