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, 71 (6), 1443-9

A Missense Mutation (R565W) in Cirhin (FLJ14728) in North American Indian Childhood Cirrhosis

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A Missense Mutation (R565W) in Cirhin (FLJ14728) in North American Indian Childhood Cirrhosis

Pierre Chagnon et al. Am J Hum Genet.

Abstract

North American Indian childhood cirrhosis (CIRH1A, or NAIC), a severe autosomal recessive intrahepatic cholestasis described in Ojibway-Cree children from northwestern Quebec, is one of several familial cholestases with unknown molecular etiology. It typically presents with transient neonatal jaundice, in a child who is otherwise healthy, and progresses to biliary cirrhosis and portal hypertension. Clinical and physiological investigations have not revealed the underlying cause of the disease. Currently, liver transplantation is the only effective therapy for patients with advanced disease. We previously identified the NAIC locus by homozygosity mapping to chromosome 16q22. Here we report that an exon 15 mutation in gene FLJ14728 (alias Cirhin) causes NAIC: c.1741C-->T in GenBank cDNA sequence NM_032830, found in all NAIC chromosomes, changes the conserved arginine 565 codon to a tryptophan, altering the predicted secondary structure of the protein. Cirhin is preferentially expressed in embryonic liver, is predicted to localize to mitochondria, and contains WD repeats, which are structural motifs frequently associated with molecular scaffolds.

Figures

Figure  1
Figure 1
Mutation detection. A, Electropherogram of patient (top), parent (middle), and control subject (bottom). At the bottom, the amino acid sequence corresponding to this DNA region is shown. The NAIC mutation changes an arginine codon to a tryptophan codon. B, Detection of the NAIC mutation by a PCR-RFLP test. The NAIC mutation creates an AluI restriction site. Exon 15 of the FLJ14728 gene was amplified by PCR using forward primer 5′-AAT GCC CTT CTG GTC TTT CTG and reverse primer 5′-CGC AAA GTA GAC AAA TAG TCA. PCR was performed under the following conditions: 95°C for 5 min, followed by 35 cycles of 95°C for 45 s, 58°C for 30s, and 72°C for 45s. The amplicon was digested with AluI, and the digestion products were separated on 3.0% agarose gels. The PCR fragment also contains a constant internal AluI site that serves as a positive control for digestion and that yields a 150-bp fragment. Lanes 1–3, patients with NAIC; lanes 4–6, heterozygous parents; lanes 7–10, homozygous normal individuals.
Figure  2
Figure 2
Partial amino acid sequences of Cirhin and its homologues in vertebrates. The underlined arginine (R) is the residue mutated in patients with NAIC. Multiple sequence alignment was performed using the ClustalW alignment tool with Blosum weight matrix at the Baylor College of Medicine Search Launcher Web site. To generate the output, we used BoxShade 3.21 (BoxShade Server Web site). All homologous protein sequences (GenBank ID numbers shown) were originally identified by tblastn analysis at the NCBI BLAST Home Page, using the FLJ14728 protein sequence (GenBank accession number NP_116219 ) as a query against EST databases. In the residue numbering, 1 is the first amino acid in each of the predicted proteins.
Figure  3
Figure 3
Embryonic expression of Cirhin. In situ hybridization on a sagittal section of a day-11.5 mouse embryo with a 3′ probe showed prominent expression of Cirhin in the liver (arrow) and much weaker levels in the somites, brain, and craniofacial structures. Probe 1 specific to the 5′ portion of Cirhin gave identical results, whereas the corresponding sense probes showed no detectable signals. The mouse primer sequences for amplification by RT-PCR of probe 1 (5′ fragment of the gene) are 5′-CAG ATT GGC TGT TTC ACG AAC and TGT TGC CTA TCT AAA ACC ATC, and those for probe 2 (3′ fragment) are 5′-CTT TCC AGC CGC AGT CAG GTA and 5′-CCG CCT CAA GAA GAC ATC TGA T. Total adult mouse liver RNA was used as a template for the RT-PCRs.
Figure  A
Figure A
A, Physical map of the NAIC critical region. All markers genotyped and all genes sequenced in the study are indicated (see the Electronic-Database Information section for a list of GenBank accession numbers). The region of homozygosity for all patients with NAIC is located between D16S3085 and D16S3095. The physical distance was based on the December 2001 browser of the Human Genome Working Draft (UCSC Genome Bioinformatics Web site). B, Structure and genomic organization of the Cirhin gene. The relative position of the Cirhin gene between HAS3 and SNTB2 is shown on BAC RP11-502K10 (GenBank accession number AC009131). Note that the arrows indicate the continuation of the BAC DNA and not the transcriptional polarity of the genes.

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