Comments on CF best Practice Guidelines

Testing Guidelines for molecular diagnosis of Cystic Fibrosis. Prepared by Schwarz M1, Gardner A2, Jenkins L3, Norbury G3, Renwick P4, Robinson D5. 1. NW Molecular Genetics Laboratory, Saint Mary’s Hospital, Hathersage Road, Manchester M13 OJH, United Kingdom 2. Bristol Genetics Department, The Lewis Laboratories, Southmead Hospital, Bristol, BS10 5NB, United Kingdom. 3. North East Thames Regional Molecular Genetics Laboratory, Level 6 York House, 37 Queen Square, London WC1N 3BH, United Kingdom. 4. DNA Laboratory, Genetics Centre, 5th Floor Guy’s Tower, Guy’s Hospital, London, SE1 9RT, United Kingdom. 5. Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, Wiltshire, SP2 8BJ United Kingdom THESE GUIDELINES ARE CURRENTLY PROVISIONAL AND SUBJECT TO CHANGE

Reflex testing. An additional test carried out in response to initial analytical results to confirm or to aid interpretation. 1.Confirmatory reflex test in cases of apparent homozygosity. i) Presence of SNPs e.g. In the region of p.Phe508del there are at least three polymorphisms which can prevent binding of the appropriate primer. Therefore reflex testing required on apparent homozygotes, particularly if the referral reason is ‘soft’. OLA reflex kit or any source of alternative primers will clarify result. Request parental samples.

Reflex testing. ii) Possibility of UPD7 Particularly when rare mutation seen in apparent homozygous state. Particularly if patient has growth retardation (recessive gene for growth on chr7). Particularly if patient has features of RSS (matUPD7). Request parental samples. iii) CFTR deletion Deletion of part or all of CFTR gene could result in apparent homozygosity. Particularly if rare CFTR mutation in a child with no evidence of consanguinity in parents. Request parental samples.

Apparent homozygosity, examples :- Testing the parents to confirm genotypes of CF patients is highly recommended: report of two cases. Stuhrmann M et al EJHG , (Comment by McDevitt and Barton pp ). Case 1. p.Phe508del and c.1545_1546delTA Case 2 p.Phe508del and large deletion Implications for family testing - assignment of carrier status and prenatal diagnosis. N.B. General implications for all recessive diseases. Reflex testing.

c.443T>C; p.Ile148Thre No longer thought to be pathogenic. Linked to c.3067_3072del6 (3199del6) in a small number of cases. May be of use to look for c.3067_3072del6 if patient has a causative mutation and c443C>T.

Nomenclature Particular problem with CF reporting because of established use of traditional nomenclature. Often advisable to use both HGVS and traditional.

Linked markers Useful when:- diagnosis clear and only one or no mutations identified. :- enough family members are available for analysis. :- very useful in PGD

i) Intron 8 polythimidine repeat (5T,7T,9T). a)5T allele associated with CBAVD (also 5% of general pop. have 5T). b) polyT modifies expression of p.Arg117His when in cis e.g. compound heterozygotes for p.Arg117His/5T and a severe CF mutation have classic CF of variable severity. If 7T more variable phenotype and can be benign. Analyse parents to establish phase (N.B p.Phe508del in strong disequilibrium with 9T). 2. Reflex test to aid interpretation. Reflex testing.

c) 5T in trans with severe CFTR mutation, or homozygosity for 5T. Variable phenotype often associated with CFTR related disease e.g. CBAVD, bronchiectasis. d) Thus carry out reflex testing for intron 8polyT in:- Males with infertility with CBAVD. Patients with bronchiectasis/pancreatitis in whom one CFTR mutation has been found. Patients with p.Arg117His. Reflex testing.

ii) Intron 8 poly TG TG repeats immediately upstream of intron 8 polyT T12 or T13 in cis with 5T leads to increased exon 9 skipping. Patients with a severe CFTR mutation in trans with 5T/12 or 13TG are more likely to have CFTR related disease than those with TG11 (the common allele).