A.2. A.2.1. CF is usually clinically diagnosed with supporting evidence of a CFTR abnormality, either by sweat chloride measurement or identification of mutations in the CFTR gene known to cause CF. An elevated immunoreactive trypsinogen (IRT) level during screening can replace clinical features as a diagnostic criterion in newborns (see page 38). Diagnosis is usually simple following NBS or clinical presentation with an elevated sweat chloride level, but in some situations the combined information makes the diagnosis difficult, for example mild symptoms, a (borderline) positive sweat test, or a new CFTR sequence variation of unknown significance (). As stated previously, diagnostic testing for hereditary mutations in the CFTR gene occurs in three distinct groups/indications: in people with a high clinical suspicion of CF for prenatal CF diagnosis in partners of people with known CFTR mutations for the purpose of reproductive planning. The first group includes individuals presenting with classic or non-classic CF symptoms (including men with CBAVD). Prenatal diagnosis would be indicated for couples who have had a previous child with CF or a CFTR-related disorder, or who have been identified by other means to both be carriers of a CFTR mutation. It could also be used in cases where the fetus is found to have an ‘echogenic gut’. In this scenario the fetus’s parents would undergo CFTR mutation testing to determine if they are carriers, prior to the fetus being tested (if both parents are found to be carriers). The third group includes the testing of a partner of someone with at least one known CFTR mutation, which would influence their reproductive planning to allow an additional option of pre-implantation genetic diagnosis (PGD) if both partners have at least one CFTR mutation. Each person or fetus being tested for hereditary mutations in the CFTR gene would only need to be tested once in their lifetime. Most CF patients would be diagnosed through NBS (see page 38), and the ultrasound examination showing ‘echogenic gut’ leads to approximately 11% of CF diagnoses (). A small percentage of patients are diagnosed when older, as they often have a milder form of the disease (such as men with CBAVD). Different gene tests The common CFTR tests are: F508del mutation single mutation common mutation poly-T (for mild disease and infertility)
Clinical sensitivity of common mutation test Varied in the range 80%–98% $1,898 $1,215 High; higher clinical sensitivity favours intervention Cost of whole gene sequencing Varied in the range $500–$1,000 $1,477 $1,898 Moderate; lowering the cost favours intervention Model 2 – Fetus has FEB (Base-case ICER $23,254/CF birth averted) Incidence of CF in FEB population Varied in the range 2%–13% $53,565 $7,398 High; higher incidence of CF in FEB population favours intervention Uptake of termination of affected pregnancy Varied in the range 30%–100% $52,077 $14,607 High; lower termination uptake rates favour comparator Clinical sensitivity of common mutation test Varied in the range 80%–92% $23,254 $18,800 Low; higher clinical sensitivity favours intervention Uptake of invasive testing when one parent is identified as a carrier Varied in the range 50%–90% $23,254 $20,993 Low; higher values favour intervention CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; FEB = fetal echogenic bowel; ICER = incremental ratio; NBS = newborn screening
Table Study profiles of included studies on diagnostic accuracy Study Country Study design Quality Study population Inclusion criteria / Exclusion criteria Index test(s) Reference standard Axton & Brock () UK Level IV: A study of diagnostic yield (no reference standard) Quality: poor with a high risk of bias N=193 mouthwash samples from CF patients None reported Restriction generation PCR. This technique uses a mismatch primer to introduce a base substitution designed to create or destroy a restriction site when a mutation is present. No reference standard Bareil et al. () France Level III-2: A comparison with reference standard (not blinded or blinding not known) Quality: poor with a high risk of bias N=182 samples from men with a clinical diagnosis of CBAVD Inclusion criteria: Clinical diagnosis of CBAVD Exclusion criteria: Patients with renal abnormalities A complete scan of the 27 coding/flanking sequences of the CFTR gene was performed by either DGGE or DHPLC. In addition, two intronic mutations, 18111.6kbAG in IVS11 and 384910kbCT in IVS19, and variations at locus IVS8-Tn were screened by specific PCR restriction tests. Samples with only 1 or no CFTR mutations were further investigated for large rearrangements by a semi-quantitative fluorescent PCR assay. Samples showing abnormal profiles were directly sequenced with BigDye Terminator sequencing. Clinical diagnosis of CBAVD was based on clinical examination with impalpable vas deferens, transrectal ultrasonography, semen analysis and sperm count in accordance with World Health Organization guidelines. Bernardino, Lima & Zatz () Brazil Level III-2: A comparison with reference standard (not blinded or blinding not known) Quality: poor with a high risk of bias N=17 patients with CBAVD Inclusion criteria: 20 unrelated men (ages years) with obstructive azoospermia; 17 were diagnosed with CBAVD; none had been diagnosed with CF Mutation detection studies were carried out on all 27 exons and exon–intron boundaries of the CFTR gene by SSCP and heteroduplex analysis. CFTR gene variants of the T tract length of intron 8 were also investigated. Abnormally migrating fragments were subsequently sequenced to confirm the Diagnosis of CBAVD was based on scrotal examination, ultrasound and semen analysis. 49
presence of mutations. Bickmann et al. (2009) Germany Level III-2: A comparison with reference standard (not blinded or blinding not known) Quality: good with a low risk of bias N=92 CF patients Not reported Pyrosequencing: single-stranded DNA was prepared and used for pyrosequencing, and the quantitative signal was detected with the PSQ 96MA instrument. CF patients had typical symptoms and positive sweat test results. Bonizzato et al. (1995) Gasparini et al. (1993) Italy Level III-2: A comparison with reference standard (not blinded or blinding not known) Quality: intermediate with some risk of bias N=225 chromosomes from 133 CF patients N=24 chromosomes (12 patients) from whom DNA was not available N=17 chromosomes shared by 8 siblings and 2 cousins were excluded from analysis Inclusion criteria: All CF patients born between 10 January 1973 and 12 December 1981 in two northern Italian regions (Veneto and Exclusion criteria: For mutation frequency analysis, 8 brothers or sisters and 1 common chromosome shared by cousins were discarded RFLP, RNA-SSCP and DGGE analysis of the CFTR gene The presence of a given mutation in electrophoretically altered fragments was always determined by DNA sequencing. Diagnosis was confirmed by at least two positive sweat tests. Bonizzato et al. () Italy Level III-2: A comparison with reference standard (not blinded or blinding not known) Quality: good with a low risk of bias N=806 chromosomes from 403 CF patients Not reported Reverse dot-blot hybridisation assay designed to detect the 15 most common CFTR mutations in Italy Diagnosis of CF was by sweat test.
Castaldo et al. (2000) Italy Level IV: A study of diagnostic yield (no reference standard) Quality: good with a low risk of bias N=32 fetal samples collected through CVS from high-risk couples (1 dizygotic twin pregnancy) Inclusion criteria: Pregnant couples were identified during a CF-carrier screening study and offered prenatal screening; 32/33 couples gave informed consent for prenatal testing. An ASOH dot-blot semi-automated procedure using a panel of 13 CF mutations was used on all the CF families. When no mutations were detected, STR genotyping of intragenic polymorphisms by PCR and gel electrophoresis was followed by extragenic polymorphisms by PCR and restriction enzyme analysis. No reference standard Collazo et al. () Cuba Level III-2: A comparison with reference standard (not blinded or blinding not known) Quality: good with a low risk of bias N=61 fetal samples collected through amniocentesis from couples with some risk of having children affected by CF Inclusion criteria: Couples with some risk of having children affected by CF, referred from all parts of Cuba during 1988—2011 ARMS to detect F508del, G542X and R1162X PCR-based restriction enzyme analysis to detect R334W, R553X and 3120+1G>A Clinical diagnosis at birth Danziger et al. () USA Level III-2: A comparison with reference standard (not blinded or blinding not known) Quality: good with a low risk of bias N=16 male patients with CAVD (n=13 with CBAVD and n=1 CUAVD) or with idiopathic epididymal obstruction (n=2) Prospectively selected infertile men attending the male infertility clinic over a period who were diagnosed with obstructive azoospermia Inclusion criteria: (i) new diagnosis with no prior genotyping (ii) members of an ethnic group with an assumed low detection rate by common mutation panel mTTGE and DNA sequencing The Ambry CF Test includes a full mutation scan of all CFTR exons as well as relevant intronic regions by mTTGE analysis followed by DNA sequencing of suspect regions. A detailed history and physical examination was performed on all patients by an experienced urologist. The diagnosis of CAVD or idiopathic obstruction was based on physical examination findings.
Tomaiuolo et al. (2013) Southern Italy Retrospective case series Level: IV Quality: 13/18 149 couples who underwent pre-test counselling for CF underwent 181 prenatal diagnoses. 11 couples did not have PND due to miscarriage, disagreeing with PND, too difficult to make a decision about termination, and mild mutations in 1 of the parents (risk of a child with a disorder) Indications: Affected child: 148 Cascade screening: 28 Consanguinity: 1 Preconceptional screen: 2 Echogenic bowel: 1 Screening for IVF: 1 Inclusion: Couples referred for molecular diagnostics for CF because of being at high risk of giving birth to a child affected by CF between January 1993 and December 2012 Fetal DNA was only tested for parental mutations, as the CFTR genotype of all couples was known. 1/181: PND not possible due to insufficient DNA in sample: CF –ve: 38 Carriers: 98 CF +ve: 42, 41 TOP Twins (1 CF +ve, 1 carrier): 1 (selective TOP) Twins (1 carrier, 1 CF –ve): 1 CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; CFTR-RD = CFTR related disorder CVS = chorionic villus sampling; DNA = deoxyribonucleic acid; FEB = fetal echogenic bowel; GP = general practitioner; PND = prenatal diagnosis; IVF = in-vitro fertilisation; TOP = termination of pregnancy
Italy bias (1 dizygotic twin pregnancy) normal fetuses reported Saker et al. (2006) France Level IV Some risk of bias N=12 fetus samples from carrier couples PCR and STR genotyping F508del and STRs 1 (8%) 7 (58%) 4 (33%) Unknown No clinical outcomes reported ARMS = amplification refractory mutation system; ASOH = allele-specific oligonucleotide hybridisation; CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; DGGE = denaturing gradient gel electrophoresis; DNA = deoxyribonucleic acid; FEB = fetal echogenic bowel; PCR = polymerase chain reaction; STR = short tandem repeats Table CFTR mutation test limitations, mutation identification errors and failure rates Study Country Evidence level and risk of bias Population Test(s) Test limitations and resolutions Mutation identification errors Failure rate Tomaiuolo, Spina & Castaldo (2003) Italy Level III-2 Low risk of bias N=129 chromosomes from 85 DNA samples from CF subjects with known mutations CF(12) ARMS kit ARMS technology cannot distinguish between heterozygous and homozygous point mutations. 5 samples with homozygous point mutations were correctly identified but the CF(12) ARMS assay could not distinguish between the heterozygous and homozygous state. Not reported Strom et al. (2003) USA Level III-2 Low risk of bias N=7 confirmed CF patients with positive sweat tests 4 patients had 1 CF mutation and 3 had no mutations identified by either the ACMG or extended panel screening. DNA sequencing reactions were performed with an ABI Prism Big Dye™ Terminator v3.0 cycle sequencing reaction kit according to the manufacturer’s protocol. This sequencing assay detects 991 of the 1,004 (98.7%) described mutations as of August 2002. All but 2 of these mutations involve large deletions of CFTR and would not be detectable by any sequencing assay. None reported Not reported