Early Pulmonary Manifestation of Cystic Fibrosis in Children With the ΔF508/R117H-7T Genotype

EXPERIENCE & REASON

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“In Medicine one must pay attention not to plausible theorizing but to experience and reason together.. . .

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Early Pulmonary Manifestation of Cystic Fibrosis in

Children With the

⌬

F508/R117H-7T Genotype

Brian P. O’Sullivan, MDa_{, Robert G. Zwerdling, MD}a_{, Henry L. Dorkin, MD}b,c_{, Anne Marie Comeau, PhD}a,d_{, Richard Parad, MD}c,d,e

a_{Department of Pediatrics, University of Massachusetts Medical School, Worcester, Massachusetts;}b_{Department of Pediatrics, Massachusetts General Hospital, Boston,}

Massachusetts;d_{New England Newborn Screening Program, University of Massachusetts Medical School, Jamaica Plain, Massachusetts;}e_{Department of Newborn}

Medicine, Brigham and Women’s Hospital and Children’s Hospital, Boston, Massachusetts;c_{Department of Pediatrics, Harvard Medical School, Boston, Massachusetts}

The authors have indicated they have no financial relationships relevant to this article to disclose.

ABSTRACT

We report 3 cystic fibrosis newborn screen–positive infants with the ⌬F508/R117H-7T genotype who had Pseudo-monas aeruginosadetected in oropharyngeal cultures early in life and a fourth who had pulmonary symptoms and Gram-negative growth on multiple oropharyngeal cultures. All 4 patients were followed prospectively from the time of genetic diagnosis. As many regions implement newborn screening for cystic fibrosis, there is concern regarding which mutations should be included in genetic panels used to make the cystic fibrosis diagnosis. Some have recommended that mutations not specifically associated with classic cystic fibrosis be excluded. Our cases highlight the importance of considering keeping so-called mild mutations on cystic fibrosis newborn screening panels and the need to follow children with these mutations closely.

T

have indicated that the risk/benefit ratio favors newborn screening for cystic fibrosis (CF),1_{and the US} CF Foundation has recommended nationwide imple-mentation. Massachusetts began CF newborn screening in February 1999 using a 2-tiered process.2_{Initially, the} newborn screen dried blood spot is tested for immuno-reactive trypsinogen (IRT), which is typically elevated in infants with CF. Infants who have IRT values in the top 5% each day are designated as being suspicious for hav-ing CF. The same blood spots from these infants are then analyzed for the presence of a limited number of CF mutations. Using this algorithm, CF screening programs need to determine which mutations will be included on their genetic panels and to determine follow-up proto-cols for all infants who screen positive. Typically, the choice of mutations screened for has taken into account mutation frequency and the phenotypic correlates of the mutation. Whether to include so-called mild CF muta-tions (classes IV, V, and VI)3 _{in such a panel has been} debated because of the implications of making a genetic

diagnosis of CF in newborns who might not become symptomatic for years, if at all.4,5

One mutation that generates debate in the CF new-born screening community is R117H, a substitution of arginine for histidine at the 117th position in the CF transmembrane conductance regulator (CFTR) protein, which is in a membrane-spanning domain.6_{R117H is a} missense mutation in exon 4 that reduces single-channel conductance and reduces chloride-ion transport.6 _The expression of a CFTR gene containing R117H is influ-enced by the polythymidine sequence of intron 8, which

Key Words:cystic fibrosis, pulmonary, newborn screening, gene mutation, R117H

Abbreviations:CF, cystic fibrosis; IRT, immunoreactive trypsinogen; CFTR, cystic fibrosis transmembrane conductance regulator;nT,n-thymidine sequence; CBAVD, congenital bilateral absence of the vas deferens

www.pediatrics.org/cgi/doi/10.1542/peds.2006-0399

doi:10.1542/peds.2006-0399

Accepted for publication May 3, 2006

precedes the exon 9 splicing acceptor site. The CFTR gene occurs with 5, 7, or 9 thymidines in the intron 8 sequence. The more thymidines, the more efficient the splicing. Alone, R117H may not cause sufficient mal-function of the CFTR protein to contribute to overt CF disease; however, when it is found in cis with a short-ened polythymidine sequence in intron 8, the combina-tion can lead to significantly decreased funccombina-tion of the CFTR protein. The clinical characteristics of a person who is a compound heterozygote for a severe CFTR mutation on one chromosome and R117H on the other depends in part on the length of the polythymidine sequence inherited along with R117H. When R117H with a 5-thymidine sequence (R117H-5T) is inherited along with a severe CFTR mutation (the most common of which is a loss of phenylalanine at the 508th position,

⌬F508) a child may have an elevated or borderline sweat test, moderate lung disease, pancreatic exocrine suffi-ciency, and male infertility.7 _{In contrast, it has been} thought that when a person is a compound heterozygote for ⌬F508 and R117H with the 7T sequence (⌬F508/ R117H-7T), the individual may have a negative or bor-derline sweat test, late-onset CF, congenital bilateral absence of the vas deferens (CBAVD) alone, or no dis-ease at all.4,5,8,9

Several groups have found no evidence of CF-related lung disease in men with CBAVD when R117H-7T is found on one chromosome and the⌬F508 mutation on the other,10–12 _{and one report strongly urged clinicians} not to diagnose CF on the basis of this genotype alone.5 Furthermore, the US CF Foundation consensus state-ment on the diagnosis of CF states that “diagnosis of CF in patients carrying R117H-7T or 5T will require dem-onstration of a CFTR abnormality by sweat testing or nasal potential difference testing.”13_{Therefore, when an} asymptomatic infant is found to have an elevated IRT and the⌬F508/R117H-7T genotype, it is difficult for the treating physician to know what to tell the parents; in fact, at present, the literature concludes that physicians should refrain from calling such a child a CF pa-tient.4,5,11,14

Here we report 4 infants with the⌬F508/R117H-7T genotype who were identified very early in life through newborn screening or prenatal testing and then followed prospectively at accredited CF centers. All 4 patients have clinical signs and symptoms of pulmonary disease and have grownPseudomonas aeruginosaor other Gram-negative organisms from oropharyngeal cultures. Knowledge of the surprisingly early onset of overt dis-ease in at least some children with the⌬F508/R117H-7T genotype is crucial for physicians in regions that are starting CF newborn screening programs who might otherwise consider children with this genotype to be unaffected.

CASE REPORTS

CASE 1.A.B. was born at term without complications. Her newborn dried blood spot demonstrated an elevated IRT, which led to mutation analysis. This proved the child to be a compound heterozygote for ⌬F508 and R117H. Determination of her intron 8 thymidine sequences re-vealed 7T and 9T variants. Because ⌬F508 is generally (but not always) seen in conjunction with the 9T vari-ant,15,16_{the child was assumed to have both R117H and} 7T on the same chromosome. Sweat samples were col-lected in duplicate at 6 weeks of age, and her chloride concentrations were normal (Table 1). The family was apprised of the normal sweat test results and the uncer-tainty of the significance of the⌬F508/R117H-7T geno-type. The child was monitored in a CF clinic as previ-ously determined by the Massachusetts CF Newborn Screening Workgroup protocol. A repeat sweat test per-formed at 21 months of age showed sweat chloride concentrations of 52 and 54 mmol/L. An oropharyngeal swab culture at the same time showed growth of 3 strains of nonmucoid P aeruginosa and Escherichia coli. Therapy with inhaled tobramycin was given for 28 days, and (at the time of this writing) Pseudomonas has not been isolated from subsequent oropharyngeal cultures, although a later culture did grow Serratia marcescens. A chest radiograph obtained at 24 months of age showed hyperinflation and peribronchial cuffing consistent with small-airways inflammation.

CASE 2.J.B., the younger brother of A.B., also had an elevated IRT at birth. He was found to have the same genotype as his sister,⌬F508/R117H-7T. A sweat chlo-ride test was performed at 4 weeks of age and was normal. Because of his sister’s course of early coloniza-tion withP aeruginosa, oropharyngeal cultures were ob-tained at 2, 7, and 10 months of age. At all 3 times the cultures were positive for 2 strains of E coli. A chest radiograph at 5 months of age was normal, but when repeated at 18 months of age, the chest radiograph showed peribronchial thickening and mild hyperinfla-tion.

CASE 3.A.Z. was born at term. Her parents had had pre-natal testing and were aware that the mother carried the

developed a cough, increased work at breathing, and fever. A chest radiograph was remarkable for bilateral lower lobe infiltrates. An oropharyngeal culture ob-tained at the time of this illness was positive for nonmu-coidP aeruginosa. She was treated with oral ciprofloxacin for 2 weeks and inhaled tobramycin for 28 days and has been Pseudomonas-negative since that time; however, she has hadStaphylococcus aureusrecovered from several throat swabs.

CASE 4.J.E. was born at term. He had been diagnosed as having ⌬F508/R117H-7T via amniocentesis prenatally. The family denied permission for CF newborn screening because they already knew the child’s genotype. A sweat chloride test was in the borderline range. At 30 months of age, pan-sensitive, nonmucoidP aeruginosawas recov-ered from an oropharyngeal culture. The child was treated with inhaled tobramycin in 28-day cycles for 1 year. Repeat cultures have not grown any Pseudomonas

species, and the child is asymptomatic. Of note, a chest radiograph at 2 months of age showed signs of mild small-airways disease, with the changes persisting on subsequent films. By 12 months of age there were per-sistent increases in interstitial markings and hyperinfla-tion.

DISCUSSION

In Massachusetts, the panel of mutations used for CF newborn screening includes R117H with automatic test-ing for the 5T, 7T, and 9T variants in intron 8 for any child found to have R117H.2_{The degree of CFTR}

dys-function resulting from the R117H mutation depends, in part, on the number of thymidine repeats in intron 8. Prognosis for an asymptomatic infant whose genotype consists of a severe mutation on one chromosome and R117H on the other (eg, ⌬F508/R117H) is not always clear. When the R117H chromosome also has the 5T sequence, 90% of its CFTR messenger RNA transcripts will not include exon 9; thus, individuals with the ge-notype⌬F508/R117H-5T may have pancreatic-sufficient CF with mild-to-moderate lung disease.7,8,14_{The majority} of diagnostic and prognostic dilemmas occur when a 7T sequence is on the R117H chromosome. Infants with the genotype⌬F508/R117H-7T may have mild lung disease, CBAVD without lung disease, or no disease at all.4,5,8,9 Complicating the issue for both ⌬F508/R117H-5T and 7T, other factors may further influence splicing effi-ciency of exon 9. Just upstream from the polythymidine track in intron 8 are a series of thymidine-guanine re-peats. A smaller number of repeats is associated with better splicing efficiency and, thus, more messenger RNA transcripts including exon 9.17,18_{Thus, the splicing} efficiency of exon 9 depends on several factors, which in part explains the variable penetrance of intron 8 se-quence variants.

Current newborn screening data do not provide the incidence of⌬F508/R117H in our total population. We are aware of 2 infants who have⌬F508/R117H and who were CF screen–negative because their IRT was below the designated cutoff value. One of these 2 infants (who has the 5T sequence, genotype ⌬F508/R117H-5T) pre-sented clinically at 3 months of age. The other (with the

TABLE 1 Data Regarding Infants With the⌬F508/R117H-7T Genotype Patient IRT, ng/

mL

Initial Sweat Chloride Concentration, mmol/L (Age)a

Repeat Sweat Chloride Concentration, mmol/L (Age)a

Oropharyngeal Culture Results, Age,

mo (Organism)

Clinical Signs and Symptoms Pancreatic Status, hFE-1

␮g/gm

Weight for Height, %

A.B. 176 24 and 24 (6 wk) 52 and 54 (21 mo) 21 (P aeruginosa,E coli) Hyperinflation and peribronchial cuffing on chest radiograph; cough

PS (ND) ⬍3

27 (H influenzae) 29 (S marcescens) J.B. 227 23 and 26 (1 mo) 27 and 29 (22 mo) 2 (E coli;Moraxella

catarrhalis)

Hyperinflation and peribronchial cuffing on chest radiograph; no pulmonary symptoms

PS (⬎500) 5

7 (E coli) 10 (E coli)

A.Z. 64.6 41 and 44 (1 mo) 32 and 39 (5 mo) 3 (E coli) Increased work of breathing, cough, fever at 10 mo of age

PS (⬎500) 75

10 (P aeruginosa)

14 (S aureus) Bilateral lower lobe infiltrates on chest radiograph film

J.E. ND 31 and 32 (1 mo) ND 30 (P aeruginosa) Hyperinflation and peribronchial cuffing on chest radiograph; no pulmonary symptoms

PS (ND) 50

PS indicates pancreatic sufficient (pancreatic enzyme supplements not required); hFE-1, human fecal elastase 1 (values⬎200 indicate normal pancreatic function); ND, not done.

7T sequence, genotype⌬F508/R117H-7T) was identified at 3 years of age after a younger sibling’s screen prompted parental testing that showed that both parents were carriers. We do not know how many other such nonhypertrypsinogenemic infants there are in Massa-chusetts who have yet to come to our attention. In addition, we do not know how many of the infants with R117H who were detected by the newborn screen might yet develop pulmonary symptoms. Thus, any statement about the percentage of children with⌬F508/R117H-7T who develop CF lung disease is unsupportable at this time. A more complete, prospective study of a larger population is needed to answer this important question. Evidence for a lack of lung disease with the ⌬F508/ R117H-7T genotype comes from reviews of men with CBAVD10–12 _{and retrospective cross-sectional studies of} limited populations.8,14 _{The report by Dumur et al}12 in-cludes 3 men evaluated for CBAVD who had the⌬F508/ R117H-7T genotype. All 3 of these men had elevated sweat chloride concentrations (90, 78, and 71 mmol/L), but none had respiratory symptoms; they were well except for their infertility issues. Another 3 men with

⌬F508/R117H-7T were among the cohort with CBAVD reported by Colin et al.11_{Again, these men had elevated} sweat chloride concentrations (106, 80, 79 mmol/L); however, all 3 had normal pulmonary-function testing (forced expiratory volume in 1 second ⬎100% of pre-dicted), with evidence only of mild asthma in one of them. Conclusions drawn from these reports are con-founded by patient-selection bias. Had the population screened been attendees at a clinic for sinus or pancreas disease rather than an infertility clinic, the degree of lung disease discovered might have been quite different. Conversely, women with this genotype may not come to clinical attention at all because they do not suffer from CBAVD.

Massie et al4 _{studied a more general sample. They} examined 57 infants identified by newborn screening, all of whom had elevated IRT levels, one⌬F508 mutation, and sweat chloride concentrations ⬍60 mmol/L. Of these 57 infants, 5 had the⌬F508/R117H-7T genotype, and none had clinical symptoms of CF. The authors concluded that newborn screen genetic test panels should be limited to severe mutations associated with classic clinical CF.4 _{In a subsequent publication, this} group looked at all individuals with the R117H mutation known to CF clinics in Australia and New Zealand.14 They found 20 individuals with the ⌬F508/R117H-7T genotype, 16 of whom were diagnosed via newborn screening. One patient who presented in adulthood with pulmonary symptoms grew S aureus and Haemophilus influenzaefrom her sputum. Two children identified by newborn screening had pulmonary symptoms; a 6-year-old was reported to have recurrent bronchitis, and a 1-year-old was reported to have persistent cough. Un-fortunately, most of these patients did not produce

spu-tum, and the authors did not report the results of oro-pharyngeal swab cultures. The authors concluded that, on the basis of their experience, most individuals with the⌬F508/R117H-7T genotype do not have clinical CF but should be observed over time for development of symptoms.14

Reports such as these have led physicians who care for asymptomatic newborns found to have the ⌬F508/ R117H-7T genotype to hesitate before telling parents that their children have true clinical CF. Our report emphasizes that the genotype⌬F508/R117H-7T may not be benign. In fact, 3 of the 4 children reported here have grown the typical CF pathogen, P aeruginosa, which is found in only approximately one third of infants with CF

⬍2 years of age but in 80% of adults with CF.19 _The fourth child has had multiple cultures positive forE coli. Two of the infants have symptoms referable to small-airways disease, consistent with CF, and all 4 have had abnormal chest radiograph results. Thus, although many people with the genotype ⌬F508/R117H-7T may not have significant pulmonary disease, our experience in-dicates that children identified as having this genotype should be considered to be at increased risk for develop-ing infection or colonization of the oropharynx with CF-related organisms. Positive oropharyngeal culture re-sults do not necessarily reflect lower-airway infection20_; however, Ramsey et al21_{found a strong positive} predic-tive value for the presence ofP aeruginosain the lower airways when oropharyngeal swabs from nonexpecto-rating patients with CF were positive for this organism. It is possible that the presence ofP aeruginosain the upper airway predisposes these children to lower-airway infec-tion even if the lungs are not infected at the time of the first positive oropharyngeal culture.

cepacia. When they had bronchoalveolar lavage per-formed, both men grew strains ofB cepacia(genomovars 4 and 11) from their lower airways.24

Population-based newborn screening for CF is not a comprehensive diagnostic program. By definition, any newborn screen will miss some infants with the disease in question. Which children it is permissible to miss is open for debate. Most CF centers now treat initial P aeruginosa infections aggressively, because early infec-tion seems to be associated with worsening disease.25,26 Eradication ofPseudomonasinfection is easier before mu-coid, biofilm-producing colonies are established. The 4 infants we report and the 10 reported by Lording et al22 illustrate that although CF genotypes with mild muta-tions may not cause classic CF, they can make children susceptible to serious infection with CF-related organ-isms. Failure to follow these children even after a neg-ative sweat test might put them at increased risk of early, sustained Pseudomonas infection, which would be con-trary to the goal of newborn screening—to detect disease before it become irreversible.

The conundrum is that the act of observing these children closely may, in actuality, increase the odds of their becoming infected with a virulent organism. It is possible that some of the positive airway cultures re-ported from children and adults with⌬F508/R117H-7T reflect acquisition of these organisms at the CF center.27 Great care must be taken to isolate newborn infants with CF from older patients with established infection. New-born screen–positive infants should be seen at a separate time and place from other patients with CF to minimize patient-to-patient and caregiver-to-patient transmission of CF-related bacteria. However, the marked nutritional benefits and improved survival for those seen at CF care centers mitigate in favor of these children being seen at such a center. With these caveats, we feel that relatively common mild mutations (specifically, R117H) should be considered for inclusion in newborn screening mutation panels. Furthermore, children with these mutations should have frequent oropharyngeal cultures to detect the onset of serious infections, even if their sweat tests are negative.

The complexity of these issues highlights that physi-cians embarking on CF newborn screening should have a plan in place for counseling parents of children with the⌬F508/R117H-7T genotype.28_{Clearly, much work is} yet to be done to fully understand the reason why some children with⌬F508/R117H-7T become symptomatic at an early age and others may have an extremely late onset of symptoms or no symptoms at all. Long-term follow-up of all those identified with this genotype will be necessary to delineate the true clinical relevance of the⌬F508/R117H-7T mutation. Patients with this geno-type should have clinical assessments, biochemical tests of CFTR dysfunction, and airway cultures performed prospectively. Finally, as more information regarding

the role of thymidine-guanine repeats and non-CFTR genetic modifiers of CF becomes available, it may be possible to better identify which children with the

⌬F508/R117H-7T genotype will develop CF lung dis-ease.

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DOI: 10.1542/peds.2006-0399

2006;118;1260

Pediatrics

Richard Parad

Brian P. O'Sullivan, Robert G. Zwerdling, Henry L. Dorkin, Anne Marie Comeau and

F508/R117H-7T Genotype

∆

Early Pulmonary Manifestation of Cystic Fibrosis in Children With the

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DOI: 10.1542/peds.2006-0399

2006;118;1260

Pediatrics

Richard Parad

Brian P. O'Sullivan, Robert G. Zwerdling, Henry L. Dorkin, Anne Marie Comeau and

F508/R117H-7T Genotype

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Early Pulmonary Manifestation of Cystic Fibrosis in Children With the

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