Genome Technology and Genetic Disease
(Human and Clinical Genetics - LUMC)

Student research training

(modified last June 29, 2001)

Wolf-Hirschhorn Syndrome
(Pitt-Rogers-Danks Syndrome)

Children with Wolf-Hirschhorn syndrome (WHS, OMIM# 194190) are born with specific facial malformations including a prominent forehead and widely spaced eyes (also known as “Greek helmet”). They suffer from severe mental retardation, show growth retardation and often have defects of the heart and midline of the body (e.g. cleft palate). WHS is allelic with the milder Pitt-Rogers-Danks Syndrome (PRDS, OMIM # 262350). The phenotype of PRDS patients largely overlaps part with that of WHS patients.

WHS and PRDS patients have large genomic deletions, measuring several megabase pairs, of the short arm of chromosome 4. At the department of Human and Clinical Genetics, we have recently identified a gene, designated the Wolf-Hirschhorn Syndrome Candidate 1 gene (WHSC1, Stec et al.). This gene falls for two-thirds of its length in the 165 kb WHS critical region, as determined by Wright et al.

The WHSC1 gene is very complex in regard to alternative splicing and polyA-addition. Several mRNA’s are transcribed from this gene, probably resulting in proteins of different lengths and containing different sets of domains. Whether these proteins are all functionally active has to be determined. WHSC1 is a good candidate gene as it encodes a protein containing domains that are found in proteins which are usually involved in development and which function as a transcription factor. As the only gene described thus far, WHSC1 contains two so called PWWP-domains (Stec et al).

In patients with multiple myeloma (MM), suffering from tumours which destroy bone marrow structures, translocations are present between the IgH locus on chromosome 14 and the WHSC1 gene on chromosome 4. Possibly, gene fusion products play a causative role in the etiology of multiple myelomas.

Recently, two highly homologous genes were discovered in the human genome (Stec et al., Genomics, in press). WHSC1L1, located on chromosome 8p11.2, shows a similar overall structure as WHSC1 with the same complex transcription. WHSC1L1P is an intron-less pseudogene covering only the 5' half of WHSC1L1. It is located on chromosome 17.

Research

Currently, we pursue several lines of research, all with excellent possibilities for student research training periods. The exact subject will be dependent on how far the research has progressed at the start of the training period.

1. Homologous genes in other species
   By hybridisation of part of the human WHSC1 gene on a Zoo blot containing DNA of different organisms signals were seen in cow, dog, mouse, rat and primate, indicating that the gene has been conserved during evolution. To get an idea about the importance of the different domains that are present in the human gene, we will try to isolate the gene from other species. We have started to isolate the homologous mouse and puffer fish (Fugu rubripes) genes. Since function is conserved during evolution, the analysis should reveal whether the extensive differential splicing and use of different polyA-addition sites are conserved and thus of functional significance.
   Techniques: cDNA synthesis, PCR, screening of clone libraries, DNA isolation, sequence analysis, etc
2. Antibodies
   Antibodies will be needed to study WHSC1-protein expression and localization. Expected is the presence of several proteins because of alternative splicing of the WHSC1 gene. We have made several synthetic peptides according to the predicted protein sequence. One is unique for the largest predicted protein, another should detect the smaller proteins encoded. Antibodies are raised in rabbits, tested for specificity and used to detect the size, localization and expression of the WHSC1 protein.
   Techniques: Western blot analysis, ELISA, immunohistochemistry, in vitro translation, immunoprecipitation, immunohistochemistry, etc.

Information

Johan den Dunnen, +31 - 71 - 527 6105, ddunnen @ HumGen.nl

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