Identification of a mutation in CNNM4 by whole exome sequencing in an Amish family and functional link between CNNM4 and IQCB1.

We investigated an Amish family in which three siblings presented with an early-onset childhood retinal dystrophy inherited in an autosomal recessive fashion. Genome-wide linkage analysis identified significant linkage to marker D2S2216 on 2q11 with a two-point LOD score of 1.95 and a multi-point LOD score of 3.76. Whole exome sequencing was then performed for the three affected individuals and identified a homozygous nonsense mutation (c.C1813T, p.R605X) in the cyclin and CBS domain divalent metal cation transport mediator 4 (CNNM4) gene located within the 2p14-2q14 Jalili syndrome locus. The initial assessment and collection of the family were performed before the clinical delineation of Jalili syndrome. Another assessment was made after the discovery of the responsible gene and the dental abnormalities characteristic of Jalili syndrome were retrospectively identified. The p.R605X mutation represents the first probable founder mutation of Jalili syndrome identified in the Amish community. The molecular mechanism underlying Jalili syndrome is unknown. Here we show that CNNM4 interacts with IQCB1, which causes Leber congenital amaurosis (LCA) when mutated. A truncated CNNM4 protein starting at R605 significantly increased the rate of apoptosis, and significantly increased the interaction between CNNM4 and IQCB1. Mutation p.R605X may cause Jalili syndrome by a nonsense-mediated decay mechanism, affecting the function of IQCB1 and apoptosis, or both. Our data, for the first time, functionally link Jalili syndrome gene CNNM4 to LCA gene IQCB1, providing important insights into the molecular pathogenic mechanism of retinal dystrophy in Jalili syndrome.

Four novel Loci (19q13, 6q24, 12q24, and 5q14) influence the microcirculation in vivo.

There is increasing evidence that the microcirculation plays an important role in the pathogenesis of cardiovascular diseases. Changes in retinal vascular caliber reflect early microvascular disease and predict incident cardiovascular events. We performed a genome-wide association study to identify genetic variants associated with retinal vascular caliber. We analyzed data from four population-based discovery cohorts with 15,358 unrelated Caucasian individuals, who are members of the Cohort for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, and replicated findings in four independent Caucasian cohorts (n  =  6,652). All participants had retinal photography and retinal arteriolar and venular caliber measured from computer software. In the discovery cohorts, 179 single nucleotide polymorphisms (SNP) spread across five loci were significantly associated (p<5.0×10(-8)) with retinal venular caliber, but none showed association with arteriolar caliber. Collectively, these five loci explain 1.0%-3.2% of the variation in retinal venular caliber. Four out of these five loci were confirmed in independent replication samples. In the combined analyses, the top SNPs at each locus were: rs2287921 (19q13; p  =  1.61×10(-25), within the RASIP1 locus), rs225717 (6q24; p = 1.25×10(-16), adjacent to the VTA1 and NMBR loci), rs10774625 (12q24; p  =  2.15×10(-13), in the region of ATXN2,SH2B3 and PTPN11 loci), and rs17421627 (5q14; p = 7.32×10(-16), adjacent to the MEF2C locus). In two independent samples, locus 12q24 was also associated with coronary heart disease and hypertension. Our population-based genome-wide association study demonstrates four novel loci associated with retinal venular caliber, an endophenotype of the microcirculation associated with clinical cardiovascular disease. These data provide further insights into the contribution and biological mechanisms of microcirculatory changes that underlie cardiovascular disease.

Novel ABCA4 compound heterozygous mutations cause severe progressive autosomal recessive cone-rod dystrophy presenting as Stargardt disease.

PURPOSE: To identify the gene causing a severe form of progressive autosomal recessive cone-rod dystrophy presenting as Stargardt disease and to characterize clinical features in a large American family. METHODS: We characterized an American family who had an unusual retinal dystrophy with clinical features of Stargardt disease and severe progressive cone-rod dystrophy. Family members underwent complete ocular examinations with evaluation of visual acuity, visual fields, fundus examination, fluorescein angiography, and electroretinography. Genome-wide linkage analysis of the family was performed using 408 microsatellite markers spanning the entire human genome. Direct DNA sequence analysis was used for mutational analysis of the ABCA4 gene in all exons and exon-intron boundary regions and for testing cosegregation of the mutations with the disease in the family. DNA sequence analysis was used to determine the presence of the mutations in 200 unrelated controls. RESULTS: The proband presented with a clinical phenotype that was initially compatible with Stargardt disease, only to progress to a severe cone-rod dystrophy over the course of a few years. The disease-causing gene in the family was linked to the ABCA4 locus on chromosomal 1p22. One novel mutation, c.655A>T, was identified in exon 6 and another novel splicing mutation, c.5312+3A>T, was identified in intron 37 of ABCA4. The mutations were not present in 200 controls. The two affected sisters in this pedigree were compound heterozygotes for the mutations. Unaffected family members either did not carry either or had only one of the two mutations. CONCLUSIONS: We have identified two novel ABCA4 mutations, c.655A>T and c.5312+3A>T. When present as a compound heterozygous state, the mutations cause a phenotype of retinal dystrophy that initially manifests as Stargardt disease and slowly progresses to a severe cone-rod dystrophy. These results expand the wide range of clinical manifestations of ABCA4 mutations.

Interaction between the photoreceptor-specific tubby-like protein 1 and the neuronal-specific GTPase dynamin-1.

PURPOSE: Tubby-like proteins (TULPs) are a family of four proteins, two of which have been linked to neurosensory disease phenotypes. TULP1 is a photoreceptor-specific protein that is mutated in retinitis pigmentosa, an inherited retinal disease characterized by the degeneration of rod and cone photoreceptor cells. To investigate the function of TULP1 in maintaining the health of photoreceptors, the authors sought the identification of interacting proteins. METHODS: Immunoprecipitation from retinal lysates, followed by liquid chromatography tandem mass spectrometry and in vitro binding assays, were used to identify TULP1 binding partners. RT-PCR was performed on total RNA from wild-type mouse retina to identify the Dynamin-1 isoform expressed in the retina. Immunocytochemistry was used to determine the localization of TULP1 and Dynamin-1 in photoreceptor cells. Electroretinography (ERG) and light microscopy were used to phenotype tulp1-/- mice at a young age. RESULTS: Immunoprecipitation from retinal lysate identified Dynamin-1 as a possible TULP1 binding partner. GST pull-down assays further supported an interaction between TULP1 and Dynamin-1. In photoreceptor cells, Dynamin-1 and TULP1 colocalized primarily to the outer plexiform layer, where photoreceptor terminals synapse on second-order neurons and, to a lesser extent, to the inner segments, where polarized protein translocation occurs. ERG analyses in young tulp1-/- mice indicated a decreased b-wave at ages when the retina retained a full complement of photoreceptor cells. CONCLUSIONS: These data indicated that TULP1 interacts with Dynamin-1 and suggested that TULP1 is involved in the vesicular trafficking of photoreceptor proteins, both at the nerve terminal during synaptic transmission and at the inner segment during protein translocation to the outer segment. These results also raised the possibility that normal synaptic function requires TULP1, and they motivate a closer look at synaptic architecture in the developing tulp1-/- retina.

Tubby-like protein 1 (TULP1) interacts with F-actin in photoreceptor cells.

PURPOSE: TULP1 is a photoreceptor-specific protein of unknown function that, when mutated, can cause retinitis pigmentosa in humans and photoreceptor degeneration in mice. Toward a better understanding of the role of TULP1 in retinal disease, its subcellular localization was sought and the TULP1 protein binding partners identified. METHODS: Immunocytochemistry and subcellular fractionation were used to determine the localization of TULP1 and actin in COS7 cells and photoreceptor cells. Immunoprecipitation from retinal lysates followed by liquid chromatography tandem mass spectrometry and in vitro binding assays was used to identify TULP1-binding partners. Phospholipid binding assays were performed with a commercially available kit. RESULTS: TULP1 localizes at or near the plasma membrane and associates with the membranous fraction of COS7 cells, probably through binding phosphorylated phospholipids. In addition, TULP1 partitions to the aqueous phase during Triton X-114 extraction. Immunoprecipitation from retinal lysate identified F-actin as a possible TULP1-binding partner. Co-sedimentation assays further support an interaction between TULP1 and actin. In photoreceptor cells, actin and TULP1 colocalize at the inner segment, connecting cilium, and outer limiting membrane. CONCLUSIONS: TULP1 is a cytoplasmic protein that associates with cellular membranes and the cytoskeleton. TULP1 and actin appear to interact and colocalize in photoreceptor cells of the retina. TULP1 may be involved in actin cytoskeletal functions such as protein trafficking that takes place at or near the plasma membrane from the inner segment through the connecting cilium into the outer segment of photoreceptor cells.

Mutation screen of the membrane-type frizzled-related protein (MFRP) gene in patients with inherited retinal degenerations.

MFRP is a member of the frizzled-related protein family and contains a cysteine-rich domain essential for Wnt binding and signaling. MFRP is highly expressed in the retinal pigment epithelial cells of the eye. A splice donor mutation in the mouse ortholog of Mfrp is responsible for photoreceptor degeneration in the rd6 mouse. For these reasons, we investigated MFRP as a candidate gene for a phenotype associated with mutations. We screened 152 patients with inherited retinal degenerations including retinitis pigmentosa, Leber congenital amaurosis and Stargardt macular dystrophy. We identified five polymorphisms in the 5' untranslated region, four missense changes, six isocoding variants and four intronic changes. None of the sequence variants were interpreted as pathogenic.

On the Mechanism of Growth Inhibition of Epiregulin in A431 Epidermal Carcinoma Cells.

Preliminary investigation on the mechanism of the growth inhibition by recombinant epiregulin(EPI)of epidermal carcinoma cell A431 is reported. Northern blotting indicated that the mRNA level of cyclin dependent kinase(CDK)inhibitor, p21(WAF1/CIP1), was increased significantly after stimulation of the recombinant epiregulin protein. Luc reporter revealed that STAT1 could bind the promoter region of p21 in response to the EPI signal. Flow cytometry assay showed that the EPI-induced growth inhibition was not related to the apoptosis. The above results indicate that the EPI-induced cell growth inhibition might result from the STAT1-stimulated expression of p21, leading to the G1 arrest.

Expression of Human Epiregulin in E.coli.

Human epiregulin cDNA was amplified from the lung cancer cell line A549 using RT-PCR. After adding 6 His codon to its 3' end, it was cloned into a high efficient secretive Escherichia coli system with alkaline phosphatase promoter(phoA promoter)constructed in our lab and induced for expression. The product was purified one-step by Ni-NTA column. Amino acid sequence analysis revealed the identity of our product with that previously reported. The product showed strong proliferative effect on fibroblast cell line Balb/c3T3 and growth inhibitory effect on epithelial carcinoma cell line A431.

Cloning and Characterization of fup1, A Gene Highly Expressed in Hepatocellular Carcinoma.

Primary hepatocellular carcinoma(HCC) is one of the common malignant tumors in China. In our previous work, a gene named fup1(function-unknown protein 1) was isolated that was expressed differently in HCC and in normal liver. We assumed that it might be a candidate oncogene for the HCC. The fup1 gene had a ORF of 1 233 bp, encoding a protein with M(r) of 46 kD and isoelectric point of 5.48. The sequence characteristics showed its possible localization in nuclei. Northern blots showed that this gene was weakly expressed in many types of human tissues, except in the heart, implying its tissue-specific expression pattern. MTT assay of the NIH 3T3 cells transfected with this gene in the form of recombinant eukaryotic expression plasmid showed its enhancing role to cellular proliferation.

Genome-wide linkage scan identifies two novel genetic loci for coronary artery disease: in GeneQuest families.

Coronary artery disease (CAD) is the leading cause of death worldwide. Recent genome-wide association studies (GWAS) identified >50 common variants associated with CAD or its complication myocardial infarction (MI), but collectively they account for <20% of heritability, generating a phenomena of "missing heritability". Rare variants with large effects may account for a large portion of missing heritability. Genome-wide linkage studies of large families and follow-up fine mapping and deep sequencing are particularly effective in identifying rare variants with large effects. Here we show results from a genome-wide linkage scan for CAD in multiplex GeneQuest families with early onset CAD and MI. Whole genome genotyping was carried out with 408 markers that span the human genome by every 10 cM and linkage analyses were performed using the affected relative pair analysis implemented in GENEHUNTER. Affected only nonparametric linkage (NPL) analysis identified two novel CAD loci with highly significant evidence of linkage on chromosome 3p25.1 (peak NPL  = 5.49) and 3q29 (NPL  = 6.84). We also identified four loci with suggestive linkage on 9q22.33, 9q34.11, 17p12, and 21q22.3 (NPL  = 3.18-4.07). These results identify novel loci for CAD and provide a framework for fine mapping and deep sequencing to identify new susceptibility genes and novel variants associated with risk of CAD.

An LRP8 variant is associated with familial and premature coronary artery disease and myocardial infarction.

Our previous genomewide linkage scan of 428 nuclear families (GeneQuest) identified a significant genetic susceptibility locus for premature myocardial infarction (MI) on chromosome 1p34-36. We analyzed candidate genes in the locus with a population-based association study involving probands with premature coronary artery disease (CAD) and/or MI from the GeneQuest families (381 cases) and 560 controls without stenosis detectable by coronary angiography. A nonconservative substitution, R952Q, in LRP8 was significantly associated with susceptibility to premature CAD and/or MI by use of both population-based and family-based designs. Three additional white populations were used for follow-up replication studies: another independent cohort of CAD- and/or MI-affected families (GeneQuest II: 441 individuals from 22 pedigrees), an Italian cohort with familial MI (248 cases) and 308 Italian controls, and a separate Cleveland GeneBank cohort with sporadic MI (1,231 cases) and 560 controls. The association was significantly replicated in two independent populations with a family history of CAD and/or MI, the GeneQuest II family-based replication cohort and the Italian cohort, but not in the population with sporadic disease. The R952Q variant of LRP8 increased activation of p38 mitogen-activated protein kinase by oxidized low-density lipoprotein. This extensive study, involving multiple independent populations, provides the first evidence that genetic variants in LRP8 may contribute to the development of premature and familial CAD and MI.

Mutation screen of the TUB gene in patients with retinitis pigmentosa and Leber congenital amaurosis.

TUB is the first identified member of the TULP family of four proteins with unknown function. A spontaneous mutation in murine tub causes retinal degeneration, obesity, and deafness. Mutations in another member of the TULP family, TULP1, are a cause of autosomal recessive retinitis pigmentosa (RP). These findings prompted us to investigate TUB as a candidate gene for RP and Leber congenital amaurosis (LCA). A mutation screen of the entire coding region of the TUB gene in 159 unrelated patients with autosomal recessive RP, 114 unrelated patients with simplex RP, and 21 unrelated patients with LCA uncovered 18 sequence variations. Of these, seven were missense mutations, six were isocoding changes, and five were intronic polymorphisms. All seven missense mutations were identified as heterozygous changes and no defect could be found in the other allele. None of the isocoding variants or intronic polymorphisms are predicted to create or destroy splice donor or acceptor sites based on splice-site prediction software. Although variant alleles of the TUB gene were found, none could be definitively associated with a specific retinal disease.