Familial juvenile hyperuricaemic nephropathy (FJHN): linkage analysis in 15 families, physical and transcriptional characterisation of the FJHN critical region on chromosome 16p11.2 and the analysis of seven candidate genes

Background: Bicuspid aortic valve disease (BAV), the most common congenital cardiovascular malformation, has an incidence of 0.5–1.0% of live births. While most cases of BAV appear to be sporadic, familial inheritance patterns have been observed consistent with autosomal dominant inheritance with variable penetrance. However, little is known about specific genetic loci responsible for familial BAV. Here, we performed linkage analysis on a large multi-generational pedigree affected with BAV. Methods: We identified a large, five-generation pedigree (136 family members) with 10 individuals having BAV. Two-dimensional echocardiography was used to assign aortic valve phenotype. Genome-wide linkage analysis using 430 microsatellite markers (Marshfield Clinic) and fine mapping using 100 single nucleotide polymorphisms (Affymetrix) on chromosome 9 was performed on genomic DNA from all available family members. Logarithm of odds (LOD) scores of >2.0 were considered suggestive of linkage. Comprehensive splice site/open reading frame mutational analysis of candidate genes residing in the putative locus is underway using PCR, DHPLC, and DNA sequencing. A candidate gene, KLF9, Krüppel-like factor 9 was analyzed for mutations because of its role in cardiogenesis. Results: Multi-point genome-wide linkage analysis demonstrated a 7 cM region on chromosome 9q21 that was suggestive of linkage for familial BAV with a maximum multipoint LOD score of 2.8 flanked by the microsatellite markers GATA7D12 and D9S1834. This region contains several candidate genes with biological plausibility for BAV phenotype. KLF9- encoded Krüppel-like factor 9, localized to chromosome 9q21, was targeted as a prime candidate gene for familial BAV. However, no mutations involving the translated exons of KLF9 were detected. Further fine mapping studies and candidate gene analysis are currently underway. Conclusions: We report a novel susceptibility locus on chromosome 9q21 for BAV in a large multi-generational family. Although coding region mutations in KLF9 are not responsible for BAV in this pedigree, several candidate genes with biological plausibility for the development of congenital BAV lie within this region and warrant further scrutiny.

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Combined QTL-Seq and Traditional Linkage Analysis to Identify Candidate Genes for Purple Skin of Radish Fleshy Taproots

Frontiers in Genetics ◽

10.3389/fgene.2019.00808 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 1

Author(s):

Tongjin Liu ◽

Jinglei Wang ◽

Chunhui Wu ◽

Youjun Zhang ◽

Xiaohui Zhang ◽

...

Keyword(s):

Linkage Analysis ◽

Candidate Genes

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Linkage analysis of candidate genes and the small, dense low-density lipoprotein phenotype

Atherosclerosis ◽

10.1016/s0021-9150(98)00193-2 ◽

1999 ◽

Vol 142 (1) ◽

pp. 79-87 ◽

Cited By ~ 21

Author(s):

Melissa A. Austin ◽

Karen Stephens ◽

Carolyn E. Walden ◽

Ellen Wijsman

Keyword(s):

Linkage Analysis ◽

Candidate Genes ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Low Density ◽

Lipoprotein Phenotype

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Uncovering the Epigenetic Pathomechanism in 13q14.

Blood ◽

10.1182/blood.v110.11.487.487 ◽

2007 ◽

Vol 110 (11) ◽

pp. 487-487

Author(s):

Daniel Mertens ◽

Melanie Ruppel ◽

Angela Philippen ◽

Verena Fleig ◽

Bianca Brakel ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Tumor Suppressor ◽

Candidate Genes ◽

Critical Region ◽

Replication Timing ◽

Complete Loss ◽

Gene Copy ◽

Active Copy ◽

Suppressor Mechanism

Abstract INTRODUCTION: Deletions in chromosomal band 13q14.3 distal to RB1 occur in a variety of human neoplasms like B-cell chronic lymphocytic leukaemia (CLL), indicating a tumor suppressor mechanism in this region. Intriguingly, several characteristics of the region of interest point to an epigenetic pathomechanism: candidate genes lack point mutations, yet these genes are downregulated in tumors, the presence of large non-coding RNA genes in 13q14.3 is reminiscent of imprinted regions where only one gene copy is active. The data we show here led us to propose a novel oncogenic mechanism where already in healthy tissue only one gene copy is active while one gene copy is randomly chosen for silencing. Loss of the single active copy is then sufficient for complete loss of gene function in tumor cells. Currently we are trying to identify the (epi-)genetic element that controls the whole locus. AIM: Identification of the epigenetic regulatory mechanism localized in 13q14.3. METHODS and RESULTS: We performed FISH analyses of hematopoietic and non-hematopoietic cell lines to assess replication timing and chromatin packaging of the critical region. In line with an imprinting mechanism, we find that the two copies of the critical region replicate asynchronously and/or show delayed chromatid segregation, suggesting differential chromatin packaging of the two copies of 13q14.3. Next, we found by sequencing of SNPs that 13q14.3 candidate genes are expressed from one copy only in healthy probands. However, expression originated from either the maternal or paternal copy, excluding an imprinting mechanism. We could also show a functional interconnection of DNA methylation and gene expression, as demethylating agents and histone hyperacetylation induced biallelic expression. However, replication timing was not affected. Currently we are employing array- and capillary electrophoresis-based analysis of DNA-methylation (aPRIMES and bioCOBRA) and chromatin-immunoprecipitation on arrayed CpG-libraries (chIP on chip) with antibodies specific for histone modifications in order to identify the epigenetic element regulating the critical region. CONCLUSIONS: We propose that differential replication timing represents an early epigenetic mark that distinguishes the two copies of 13q14.3, resulting in differential chromatin packaging and monoallelic expression. This has profound effects for the tumor suppressor mechanism localized in 13q14.3: Deletion of the single active copy of the region at 13q14.3, which is detected in more than 50% of CLL tumors, will suffice for complete loss of tumor suppressor function, as the remaining gene copies are epigentically silenced. In addition, we are currently identifying the locus control region that orchestrates gene expression in the critical region. Thus, we provide a model for the pathomechanism of 13q14.3 in CLL by the interaction of genetic lesions and epigenetic silencing.

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High Levels of Protein C Are Determinated by PROCR H3 in a Dutch Family.

Blood ◽

10.1182/blood.v114.22.2139.2139 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2139-2139

Author(s):

Maria Carolina Pintao ◽

Sara Roshani ◽

Marieke C.H. de Visser ◽

Cris Tieken ◽

Michael W.T. Tanck ◽

...

Keyword(s):

Linkage Analysis ◽

Candidate Genes ◽

Protein C ◽

Transmembrane Domain ◽

Family Members ◽

Endothelial Protein C Receptor ◽

Lod Score ◽

Chromosome 20 ◽

The Family ◽

The Mean

Abstract Abstract 2139 Poster Board II-116 The natural anticoagulant protein C (PC) circulates in blood at a concentration of about 60 nM. Inter-individual variations in the levels of PC are in part genetically determined, but which loci in the genome are involved is only partially known. In a recent study we identified a locus on chromosome 20 which was associated with high PC levels in a large pedigree from the GENES study (LOD score >5 at 55 cMorgan). Candidate genes related to the PC pathway under the LOD-1 region encoded FOXA2 (previously known as HNF3 beta, a nuclear factor regulating protein C gene transcription), thrombomodulin (THBD,which is key to activation of PC), and the endothelial protein C receptor (PROCR). Here we present data that pinpoint a SNP in PROCRas being responsible for the observed segregation of high PC levels. The pedigree has 218 members and was ascertained through a proband with a family history of venous thrombosis (VT). Classical genetic risk factors for thrombosis (i.e. PC-, PS-, antithrombin deficiency, factor V Leiden and prothrombin G20201A) were not present. Complete medical data, plasma measurements and DNA was available for 161 family members. The mean age was 47±15 (range 15-87) years. The mean PC plasma level was 116±25% (range 72-212). Four family members had experienced VT and 2 had had recurrence. These symptomatic members had normal to high PC levels (66, 82, 114 and 178%).Haplotypes (and genotypes) for PROCR were determined in the family members by TaqMan assay using tag SNPs (single nucleotide polymorphisms) and PROCR H3 was associated with the levels of PC in the family. Furthermore, the promoter, exons, and 3`UTR of the 3 candidate genes were sequenced in 13 individuals, 9 with high and 4 with normal plasma PC levels. Critical SNPs that were encountered during sequencing were genotyped in all family members, namely FOXA2 rs1055080 (3`UTR) and rs2277764 (promoter region). As those 2 SNPs were inherited together in the set of 13 patients and also in the LETS (data not shown), our further analysis used only rs1055080. Plasma soluble endothelial protein C receptor (sEPCR) and soluble thrombomodulin (sTM) levels were measured with an ELISA assay. PC and sEPCR and levels were compared between PROCR H3 and FOXA2 rs1055080 carriers and non carriers by Student's t-test. sTM was analyzed by Mann-Whitney test. Association between PC levels and sEPCR/sTM levels were evaluated using linear regression analysis. Afterwards associations were adjusted for the PROCR H3 and FOXA2 rs1055080 SNP separately to detect their possible confounding effect. DNA sequencing only yielded previously reported SNPs in FOXA2, THBD and PROCR. Only the above mentioned SNPs were associated with PC plasma levels. Linkage analysis for PC levels using the original markers (from Marshifield) and adding the new PROCR and FOXA2 SNPs did not change the LOD score. When the analysis was adjusted for the mentioned markers, the LOD score dropped below 2. sEPCR has a bimodal distribution; mean ± SD was 103±27 ng/ml for the first mode and 262±70 ng/ml for the second mode. Median (range) sTM was 1.2 ng/ml (0.1-4). Linkage analysis for sEPCR levels yielded a high LOD score (above 6) that was accentuated to above 8 when PROCR H3 was included as a marker. For sTM, the LOD score was low with every combination of markers. PC, sEPCR and sTM levels were compared between PROCR H3 carriers and non-carriers and both PC levels and sEPCR levels were influenced by this PROCR haplotype, but not sTM. In conclusion, chromosome 20 harbors a locus which influences PC levels and also the levels of sEPCR, but not the levels of sTM. A detailed analysis with SNPs in PROCR, THBD and FOXA2suggests that the so-called PROCR H3 is directly responsible for the increased PC and sEPCR levels in this family. PROCR H3 is known to represent a g.A6936G substitution leading to a p.Ser219Gly replacement in the transmembrane domain of EPCR. The Gly219 isoform is more sensitive to sheddases (such as the ADAM17 metalloprotease) and is associated with generation of truncated mRNA lacking the transmembrane domain. However, the exact mechanism by which EPCR and sEPCR levels influence the level of PC remains to be determined Disclosures: No relevant conflicts of interest to declare.

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