ABCMdb

PMID: 15371908

Buyse IM, McCarthy SE, Lurix P, Pace RP, Vo D, Bartlett GA, Schmitt ES, Ward PA, Oermann C, Eng CM, Roa BB
Use of MALDI-TOF mass spectrometry in a 51-mutation test for cystic fibrosis: evidence that 3199del6 is a disease-causing mutation.
Genet Med. 2004 Sep-Oct;6(5):426-30., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Ile148Thr Use of MALDI-TOF mass spectrometry in a 51-mutation test for cystic fibrosis: Evidence that 3199del6 is a disease-causing mutation Inge M. Buyse, PhD1 , Sarah E. McCarthy1 , Paul Lurix, PhD1 , Robert P. Pace, MS1 , David Vo1 , George A. Bartlett1 , Eric S. Schmitt, PhD, MS1 , Patricia A. Ward, MS1 , Christopher Oermann, MD2 , Christine M. Eng, MD1 , and Benjamin B. Roa, PhD1 Purpose: We developed a 51-mutation extended cystic fibrosis (CF) panel that incorporates the 25 previously recommended CFTR mutations, plus 26 additional mutations including 3199del6, which was associated with I148T. Login to comment 2 ABCC7 p.Gly542* Results: CF testing was performed on over 5,000 individuals, including a 3-year-old Hispanic-American patient with a compound heterozygous G542X/3199del6 genotype. Login to comment 3 ABCC7 p.Ile148Thr He is negative for I148T, or other mutations assessed by CFTR gene sequencing. Login to comment 7 ABCC7 p.Ile148Thr The disease-causing CFTR gene lies on chromosome 7q31.2 and was cloned in 1989.1-3 The clinical spectrum ranges from a severe CF phenotype characterized by progressive lung disease, pancreatic insufficiency, male infertility, and elevated sweat chloride, to a milder presentation that includes congenital bilateral absence of the vas deferens (CBAVD) in males.4 Over 1300 CFTR mutations have been identified to date, wherein certain mutations are correlated with a mild or severe clinical phenotype.5 CF carrier screening for couples of reproductive age is recommended in the U.S. using a core panel of 25 common CF mutations.6 Although laboratories vary in the number of mutations and methodologies used for CF testing, many labs offer a CF testing panel that is applied to both carrier screening and diagnostic cases. One controversial component of the recommended panel is I148T, which was initially classified as a severe CF mutation. Login to comment 8 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr However, in vitro studies indicated that the major CFTR functions are retained by the I148T protein.7 Population screening studies revealed that the frequency of I148T is approximately 100-fold greater in asymptomatic carriers versus CF patients, which is more consistent with a benign variant than a disease-causing mutation.8,9 An in-frame deletion in exon 17a, 3199del6, was previously identified in association with I148T; moreover, I148T was found to be associated with a severe CF phenotype only when present in a complex haplotype of I148T/3199del6/9T, paired with a severe CF mutation on the opposite chromosome.4,8 The clinical significance of the 3199del6/I148 haplotype is unclear from the limited data available, because the few laboratories that test for 3199del6 mostly do reflex testing limited to patients who are positive for I148T. Login to comment 12 ABCC7 p.Ile148Thr Moreover, our testing strategy evaluates each patient`s 3199del6 status independently of I148T, which is useful for refining genotype-phenotype correlations. Login to comment 18 ABCC7 p.Ile148Thr An additional 11 patients who were previously identified as I148T carriers by allele-specific oligonucleotide (ASO) anal- From the 1 Medical Genetics Laboratories, Department of Molecular and Human Genetics, and the 2 Department of Pediatrics, Baylor College of Medicine, Houston, Texas. Login to comment 28 ABCC7 p.Arg117His In compliance with ACMG recommendations, this assay provides reflex testing on R117H positive samples for the 5T, 7T, and 9T polymorphic alleles at the intron 8 polythymidine tract.6 Robotic automation was applied at the pre-PCR (Multiprobe II HT, Perkin Elmer and Biomek FX, Beckman) and post-PCR processes (Multimek, Beckman). Login to comment 39 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Statistical analysis The observed CF carrier frequency in our screening population, and the frequency of I148T in particular, were statistically compared to previously reported CF and I148T carrier frequencies8 using the Fisher exact test, with a two-sided P-value reported. Login to comment 40 ABCC7 p.Glu60* RESULTS CFTR mutation panel analysis Our expanded CF assay using a MALDI-TOF mass spectrometry system (Sequenom) was initially validated against the ASO hybridization assay that was previously implemented in our laboratory.11 A total of 1080 patient samples that were analyzed by ASO were tested in parallel by MALDI-TOF mass spectrometry, and concordant results were obtained with the exception of two cases. One sample was heterozygous for a rare mutation, E60X, which was not included in the smaller (37 mutation) ASO panel. Login to comment 41 ABCC7 p.Gly314Glu ABCC7 p.Gly314Ala In the second case, an ASO result of a heterozygous G314E mutation was further characterized by MALDI-TOF mass spectrometry as a different allelic variant, G314A (data not shown). Login to comment 42 ABCC7 p.Gly314Ala DNA sequence analysis confirmed the G314A allele, which is a novel missense variant of inconclusive clinical significance. Login to comment 45 ABCC7 p.Ile148Thr positives (Freq) I148T 3199del6 4839 Carrier screening 173 (3.5% Ϸ1/28) 16 1 155 Diagnostic 30 (19.4% Ϸ1/5) 0 1 34 Parental/FEBa 3 (8.8%) 1 0 10 Prenatal study 5 (50%)b 0 0 53 Family history 20 (37.7%) 0 0 277 Not specified 10 (3.6%) 0 0 a Fetal echogenic bowel b Either heterozygous or homozygous positive for parental mutations. Login to comment 46 ABCC7 p.Ile148Thr Table 2 Description of 28 identified I148T heterozygous individualsa No. Login to comment 47 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Genotype Indication EthnicityI148T 3199del6 Others 1 ϩ - - Diagnostic Not specified 1 ϩ - ⌬F508 Parental FEBb Caucasian 18 ϩ - - Carrier screen Caucasian 1 ϩ - - Carrier screen Asian Indian 4 ϩ - - Carrier screen Not specified 2 ϩ - - Not specified Not specified 1 ϩ ϩ - Carrier screen Caucasian a Includes 17 I148T carriers found in a consecutive dataset by MALDI-TOF Mass Spectrometry (also contributed to ref 14), and 11 I148T carriers previously identified by ASO analysis. Login to comment 55 ABCC7 p.Ile148Thr We observed a disproportionately high frequency of the I148T allele among heterozygous carriers with no family history (16/173 CF carriers), compared to patients referred with a definite or possible diagnosis of CF (0/155 CF patients). Login to comment 56 ABCC7 p.Ile148Thr Our findings are consistent with significantly elevated I148T allele frequencies in asymptomatic carriers versus CF patients by previous reports.8,9 In addition, two patients out of 5368 consecutive cases were found to be positive for 3199del6 (Table 1), and details on these two cases are provided in the following sections. Login to comment 57 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Description of I148T-positive individuals A composite group of 28 individuals who tested positive for the I148T allele is listed in Table 2. Login to comment 58 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr This group included 17 individuals in our consecutive dataset who were simultaneously tested for I148T and 3199del6, plus 11 additional I148T carriers who were previously identified by ASO and subsequently tested by MALDI-TOF. Login to comment 59 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Population screening was the indication for testing among the majority of the I148T carriers (24 out of 28); among these, 23 I148T carriers tested negative for 3199del6. Login to comment 60 ABCC7 p.Ile148Thr One individual of European Caucasian descent referred to our lab for carrier screening was genotyped positive for both I148T and 3199del6. Login to comment 61 ABCC7 p.Ile148Thr However, the configuration of the I148T and 3199del6 alleles could not be determined on this asymptomatic individual because parental samples were not submitted for analysis. Login to comment 62 ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr Of the four remaining I148T carriers who were referred for reasons other than carrier screening, one was a male patient with obstructive azoospermia who was heterozygous positive for I148T and negative for the 3199del6 and 5T alleles. Login to comment 64 ABCC7 p.Ile148Thr She tested heterozygous positive for the I148T and ⌬F508 mutations, and negative for the 3199del6 and the 5T alleles (9T/9T). Login to comment 66 ABCC7 p.Ile148Thr These findings are consistent with reports of apparently healthy individuals with a ⌬F508-positive, I148T-positive, and 3199del6-negative genotype.8,9 This individual`s spouse was also referred for testing and found to be negative for 51 CF mutations and the 5T allele (7T/7T). Login to comment 67 ABCC7 p.Gly542* Identification of a CF patient with a G542X/3199del6 genotype In the course of clinical testing with the extended CF panel, we studied a 3-year old Hispanic-American male referred by the Baylor Cystic Fibrosis Care Center at Texas Children`s Hospital. Login to comment 77 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Gly314Glu ABCC7 p.Arg347His ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Arg352Gln ABCC7 p.Ile148Thr ABCC7 p.Gln493* ABCC7 p.Val520Phe ABCC7 p.Ser549Arg ABCC7 p.Gly85Glu ABCC7 p.Ser549Asn ABCC7 p.Tyr122* ABCC7 p.Arg560Thr ABCC7 p.Asp1152His ABCC7 p.Lys710* ABCC7 p.Phe508Cys ABCC7 p.Ile506Val ABCC7 p.Arg117Cys ABCC7 p.Gly480Cys ABCC7 p.Glu60* ABCC7 p.Ile507Val ABCC7 p.Gly178Arg ABCC7 p.Ile506Met ABCC7 p.Gly330* ABCC7 p.Ala559Thr ABCC7 p.Phe316Leu This assay also demonstrated heterozygosity for the G542X mutation, and reflex testing for the 5T variant at CFTR intron 8 showed a genotype of 7T/9T in this patient (data not Table 3 Description of the 16 multiplex assays designed to analyze 51 CFTR mutations Multiplex Mutations Exon 1 1078delT, G314E, R352Q, G330X 7 2 R347H, R347P, R334W, 1717-1A 7, 11 3 R553X, S549N, R1162X 11, 19 4 A559T, R560T, G551D 11 5 G542X, S549R, 621ϩ1T, Y122X 4, 11 6 W1282X, 3876delA, 3905insT, D1152H 18, 20 7 3849ϩ4G, 3659delC, 1898ϩ1A 12, 19 8 405ϩ1A, 405ϩ3C, 3120A, 3120ϩ1A 3, 16 9 394delTT, E60X, G85E 3 10 A455E, ⌬F508a 9, 10 11 G480C, Q493X, V520F 10 12 711ϩ1T, G178R, 3199del6 5, 17a 13 2143delT, 2184delA, K710X, F316L 7, 13 14 I148T, R117H, R117C 4 15 N1303K, 2789ϩ5A, 3849ϩ10kbT 14b, intron19, 21 16 ⌬I507a 10 17 5Tb intron 8 a F508C and I507V, I506V, I506M variants are tested for concurrently with the ⌬F508 and ⌬I507 assays respectively. Login to comment 78 ABCC7 p.Arg117His ABCC7 p.Arg117Cys b 5T reflex testing is performed for R117H- and R117C-positive individuals. Login to comment 80 ABCC7 p.Gly542* ABCC7 p.Gly542* Compound heterozygosity for the 3199del6 and G542X mutations was confirmed by parental studies, which identified 3199del6 in the patient`s mother and G542X in the father. Login to comment 84 ABCC7 p.Gly542* No definitive mutations were identified in addition to G542X and 3199del6. Login to comment 93 ABCC7 p.Ala455Glu ABCC7 p.Gly314Glu ABCC7 p.Ala455Val ABCC7 p.Gly314Ala Two previously unreported missense alleles were identified: G314A (allelic to G314E) and A455V (allelic to A455E). Login to comment 94 ABCC7 p.Arg553* ABCC7 p.Arg553Gly In addition, the R553G mutation which is allelic to the R553X, was identified in one patient (data not shown). Login to comment 103 ABCC7 p.Ile148Thr Among unaffected individuals who tested positive on carrier screening, the I148T allele was found in approximately 9.2% of carriers (16/173), compared to Ϸ6.4% from the previously cited study8 (P ϭ 0.21). Login to comment 104 ABCC7 p.Ile148Thr In contrast to carrier screening results, none of the 155 diagnostic cases tested by MALDI-TOF mass spectrometry were positive for I148T. Login to comment 105 ABCC7 p.Ile148Thr This finding is consistent with the significantly elevated I148T frequency in carrier screens versus diagnostic cases,8,9 and the relatively small number of diagnostic tests in our study. Login to comment 106 ABCC7 p.Ile148Thr This observed difference in frequency suggests that I148T is a benign variant, and that 3199del6 could instead represent the actual mutation. Login to comment 107 ABCC7 p.Ile148Thr Up until now, limited data had been available for 3199del6 because systematic analysis was largely based on reflex testing of I148T-positive samples. Login to comment 109 ABCC7 p.Ile148Thr In contrast, our assay tests for 3199del6 simultaneously with I148T in an extended panel for CF. Login to comment 111 ABCC7 p.Gly178Arg The mass spectrometry chromatogram indicates the expected wild-type and mutant peaks for each mutation in the multiplex CF assay shown (31999del6, G178R, 711ϩ1T). Login to comment 113 ABCC7 p.Gly542* ABCC7 p.Ile148Thr The identification of a CF patient with a compound heterozygous 3199del6/G542X genotype represents the first report of 3199del6 that is not associated with I148T on a CF chromosome. Login to comment 114 ABCC7 p.Ile148Thr This indicates that 3199del6 is a disease-causing mutation that can occur on a haplotype without I148T. Login to comment 116 ABCC7 p.Ile148Thr In addition to our Hispanic CF patient, we identified an asymptomatic Caucasian individual who tested positive on carrier screening for 3199del6 and I148T (phase unknown). Login to comment 119 ABCC7 p.Ile148Thr Our results provide additional support to the interpretation of I148T as a benign variant, although its interaction with the 3199del6 mutation is not fully delineated. Login to comment 120 ABCC7 p.Ile148Thr These findings provide additional support for removing I148T from the ACMG recommended carrier screening panel for CF. Login to comment