Compound Heterozygosity for CFTR Phe508del/Pro750Leu in Two Siblings with Normal Sweat Chloride, Lung Function, Growth, and Fecal Elastase

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INTRODUCTION
As genetic testing is increasingly utilized to aid in making a diagnosis, accurate interpretation of sequence variants is critically important.This is particularly relevant for the disorders included in the newborn screen, in which a diagnosis is often suggested in an otherwise asymptomatic individual.CF is one of the most common autosomal recessive disorders, especially in the Northern European (Caucasian) population.Although the clinical spectrum of CF is highly variable, it is mainly characterized by recurrent respiratory infections, disruption of the exocrine function of the pancreas, Congenital Bilateral Absence of the Vas Deferens (CBAVD) and other clinical problems.CF mortality and morbidity are mainly due to pulmonary complications [1].
In the United States, all states screen for CF by measuring the level of IRT [2].For patients who screen positive, additional diagnostic testing includes sweat chloride testing and genetic testing of CFTR (panels of common mutations or sequencing of the entire coding region).Although one pathogenic variant, p.Phe508del (legacy name is known as deltaF508), accounts for at least one mutation in over half of CF patients, the disorder exhibits significant allelic heterogeneity, with over 2,000 pathogenic variants identified to date.For many of these rare variants, phenotypic data is not readily available or is limited due to low frequency, making interpretation difficult.Of note, when two variants are on the same parental chromosome, they are called in cis, and when they are on different parental chromosomes, they are called in trans.However, two variants on the same parental chromosome (in cis) is called a complex allele [3][4].CF is inherited in an autosomal recessive pattern, where the disease will manifest when patients have two pathogenic variants in trans.This report describes two siblings with one such rare variant, Pro750Leu, in trans with Phe508del (p.[(Phe508del)]; [(Pro750Leu)]) who have normal phenotypes.

Sanger sequencing
The entire coding regions of all 27 exons of the CFTR gene (exons plus 50 bp upstream and 50 bp downstream of exon/intron boundaries) were amplified using specific forward and reverse primers, and bidirectionally sequenced using Sanger methodology.

Multiplex Ligation-dependent Probe Amplification (MLPA)
CFTR MLPA was performed using the SALSA MLPA P091 CFTR probe mix (MRC Holland, Netherlands), and each

Clinical findings
Patients A and B are full sisters.Patient A, who was 36 months old at the time of presentation to the University of Michigan C.S. Mott Children's Hospital, was diagnosed with cystic fibrosis in California by newborn screening.Genetic testing of CFTR revealed the presence of two variants, Phe508del and Pro750Leu.CFTR MLPA was negative for intragenic deletions or duplications.Patient A had two normal sweat chloride tests in early infancy, with levels of 21 and 12 mmol/L (normal <40).She had a third Sweat chloride test at 21 months of age that was 27 mmol/l.Patient A, was born at term with a birth weight of 3.8 kg.
Her fecal elastase test was normal.During early childhood, her growth was normal, and her stools were non-greasy.She was occasionally treated for respiratory infections with antibiotics, prednisone, albuterol, and chest physiotherapy.Respiratory infections were characterized primarily by wet cough.She contracted RSV infection at 25 months of age.Her Throat cultures usually grew normal flora, but the culture after RSV infection grew beta-lactamase-negative Hemophilus influenzae.Her chest X-rays showed peribronchial cuffing.Patient A also had a history of recurrent ear infections.Laboratory studies were normal except for a 25-hydroxy vitamin D level three months prior to presentation of 12 ng/mL (normal 25-100 ng/mL).Her medications were vitamin D (2,000 IU cholecalciferol/day) and albuterol nebulizer treatments.She also performed vest physiotherapy 2-3 times per day as needed.Patient A has been followed at the University of Michigan for four years.At 7 years of age, her weight was 46.9 kg (>99 percentile) and height was 130 cm (87 percentile).Her physical exam has remained normal.Her laboratory tests have included seven throat cultures with normal flora and three chest radiographs, each of which were read as normal except for a few  Testing of the patients' parents revealed that the mother carries the Phe508del pathogenic variant and the father carries the Pro750Leu variant, confirming that both siblings inherited the variants in trans.CF is the most common autosomal recessive genetic disease in the Northern European (Caucasian) population.Allelic heterogeneity within the CFTR gene with more than 2,000 pathogenic variants identified, can present challenges for genotype-phenotype predictions.This report describes two siblings with two CFTR variants, Phe508del and Pro750Leu, with normal sweat chloride testing, minimal respiratory symptoms, normal growth parameters and laboratory testing.

DISCUSSION
There are three previous reports in the literature, with conflicting interpretations of pathogenicity of the Pro750Leu variant.The first is of a male Mexican patient with Pro750Leu in trans with a Phe508del allele [5].This patient had early onset of symptoms at 2 months of age and had pancreatic insufficiency and chronic respiratory disease, leading the authors to conclude that Pro750Leu is a pathogenic variant.In contrast to our patients, the reported patient presented with a severe CF course.Of note, CFTR testing in that study was performed by Single-Strand Conformation Polymorphism (SSCP) and multiplex Heteroduplex (mHET) analyses, followed by sequencing of affected fragments.Thus, there is a possibility that another pathogenic variant in cis with Pro750Leu was not detected.
A second report is of a patient identified by newborn screening to have an elevated IRT with a borderline sweat chloride test (44 mmol/L) [6].Sequencing identified a complex allele p.
[(Arg352Trp; Pro750Leu)] in combination with a Phe508del allele in trans.This patient had negative stool elastase testing and is being treated prophylactically in a CF clinic [6].The phenotype described in this report is similar to our patients.
A third report described a Chinese patient with congenital bilateral absence of the vas deferens.Sequencing revealed two variants in trans, Pro750Leu and Gly970Asp [7].CBAVD is not an outcome that we can assess in our patients.Pro750Leu has also been reported, based on personal correspondence, in the Cystic Fibrosis Mutation Database (CFTR1) in 2 more patients with incomplete clinical and molecular data [8].However, Pro750Leu is not listed in the CFTR2 mutation database [9].