Case study - JPS

1. Case study - JPS Mike Oldridge FRC path 14/01/2011

2. Juvenile Polyposis Syndrome (JPS) • Autosomal dominant hamartoma polyposis syndrome • Incidence of between 1/16,000 and 1/100,000 • Polyps develop from infancy through adulthood – most individuals with JPS have some polyps by age 20 • Some individuals may only have 4-5 polyps over their lifetime, others >100 • Polyps are of juvenile form - occur in the gastrointestinal tract (stomach, lower bowel, small intestine) • Majority are benign, however, a proportion undergoes malignant transformation leading to a lifetime risk of 10-60% of developing bowel cancer, and occasionally pancreatic cancer, in JPS families – Knudson’s 2 hitmodel

3. Testing strategy and patient Bi-directional sequencing of coding regions and intron/exon boundaries of BMPR1A and SMAD4 (11 coding exons each) MLPA to detect large scale deletion / duplications (MRC Holland kit P158-B1 – also tests PTEN) Cascade screening is available to families in which a pathogenic mutation has been identified, which directs clinical management 57 year old male presented with JPS and a family history

4. Results Sequencing results normal MLPA showed deletion in 5’ UTR of BMPR1A

5. Results Two most 5’ of four 5’ UTR probes deleted Sequencing revealed no variants under probe binding sites Does not extend to start codon Exons are in uppercase Introns are in lowercase MLPA probes which are present are highlighted in turquoise MLPA probes which are deleted are highlighted in green The initiating ATG is highlighted in yellow

6. Report 1 Investigations into pathogenicity – No dels reported in literature REPORT ‘it is possible that this deleted region includes functionally important sequences’ Reported as variant of unknown pathogenicity Segregation studies advised Predictive testing NOT available to unaffected family members

7. Further investigation – CpG islands • Regulatory sequences required for transcription? • CpG island analysis • 82,980bp of 5’UTR searched for CpG islands • CpG island was predicted to span nucleotides 1-1760 • includes the deleted probes - nucleotides 799-818 and 874-897

8. Further investigation 2 - segregation AH RH GH SH Samples were received from the proband’s two children (SH and GH) and his brother (RH) for segregation analysis. All were clinically affected with JPS. All 3 other affected family members were found to be heterozygous for the BMPR1A 5’UTR deletion of two probes. Co-segregation of the deletion with disease was established between second-degree relatives.

9. AH C/C RH C/C GH C/A SH C/C Further investigation – RNA analysis • SNP analysis of the 4 affected family members revealed GH to be heterozygous for the coding SNP c.4C>A • Other 3 family members were homozygous for the C allele • Deletion in cis with the C allele

10. Report 2 • Segregation studies reported REPORT • ‘The pathogenicity of this deletion remains uncertain although this result, together with other segregation studies in the family, increases the likelihood of it being pathogenic’ • ‘molecular analysis of the polymorphisms in BMPR1A indicate that RNA studies of GH may be useful in determining the pathogenicity of this deletion.’ • Fresh blood sample requested for RNA studies

11. RNA analysis results • RNA extracted from GH blood sample and cDNA synthesised • Exonic primers flanking c.4C>A SNP designed. Both genomic DNA and cDNA amplified C/A A genomic DNA (rev strand) GH is heterozygous for the c.4C>A SNP cDNA (rev strand) GH is hemizygous for the c.4C>A allele, consistent with loss of expression of the ‘C’ allele which is in cis with the deletion.

12. Report 3 Conclusion RNA studies have shown GH to have PCR product only from the wild-type allele of the BMPR1A gene*. This is consistent with loss of expression from the allele carrying the 5’ UTR deletion. These results further support the interpretation that the 5’ UTR deletion is pathogenic and causitive of juvenile polyposis in this family. Further work This result has implications for other family members who may wish to consider molecular testing. However, complete removal of at risk family members (shown not to have the 5’ UTR deletion) from clinical screening is at the discretion of the referring clinician. *this analysis was undertaken on cDNA synthesised from the RNA extracted from peripheral lymphocytes. We cannot exclude the possibility that the BMPR1A gene expression pattern observed in lymphocytes is different to that seen in tissues relevant to JPS Results

13. References • www.cpgislands.com • Sequence is based on Ensembl release 48, OTTHUMG00000018657