Double homozygous Cystic Fibrosis Transmembrane Regulator gene (CFTR) mutation: A case series and review of the literature

Results: A total of 396 patients (312 families) confi rmed CF that were positive for the cystic fi brosis transmembrane conductance regulator (CFTR) variants from January 1998 to December 2018. A total of four families that constitute 4 patients were positive for double homozygous CFTR mutations in Trans position status. All parents were fi rst-degree cousins. Their clinical pictures were of the severe type in relation to chest disease and failure to thrive. Family screening showed that 7 family members were carriers with double heterozygous mutations in Cis position. The Prevalence of double homozygous CFTR mutation in our study is 4/312 families or 1-2:100 families which is the highest that has been reported in the medical literature. Literature review showed only 2 cases of double homozygous were reported in 1995 and 2017.

Citation: Banjar  gene. Mutations in the gene lead to altered protein synthesis, which leads to insuffi cient active CFTR at the cell surface that affect the chloride ion channel function lead to dysregulation of epithelial fl uid transport in the lung, pancreas and other organs, resulting in cystic fi brosis. Mutations consist of replacements, duplications, deletions or shortenings in the CFTR gene [4]. This may result in proteins that may not function, work less effectively, are more quickly degraded, or are present in inadequate numbers [3,4].
Several studies about CF showed that chest infection, steatorrhea, failure to thrive and Pseudo-Barrter's Syndrome are the main clinical presentations [5,6]. In other Gulf countries, CF populations shared common disease traits [7,8].
CF population in Saudi Arabia has poor compliance with chest physiotherapy and medication, poor nutrition and early colonization of bacteria [5,6]. Around 50% of CF patients had colonization with Pseudomonas Aeruginosa at a mean age of 34 months [3], and this is signifi cantly earlier than the age reported in Europe, where the mean age for colonization in these studies is 5 -7 years of age [1,2].
It is reported that there are more widely-varied differences in Cystic Fibrosis transmembrane conductance Regulator gene mutations (CFTR) in the Saudi population than in other populations [6,9]. This can be due to consanguineous marriages that were reported to be 80% compared to the general Saudi population of 50%, which maintained the existence of these mutations [9]. The importance of identifi cation of the CFcausing mutations is to provide genetic counseling, studying phenotype/ genotype correlation, family index and cascade testing of its carriers. Promisingly, identifi cation of mutation type helps in targeting the best treatment for CF patient and to develop specifi c treatment for each case [10].
In the United Kingdom, 56 different types of CFTR mutations that account for 86% of CF genes were reported [11].
Double homozygous CFTR, is a very rare phenomenon with the presence of double mutations in each allele in transposition, and has been only described twice in the medical literature since the description of the CFTR mutation in 1989 [12,13]. One of these cases were describe in 1995 as part of complex CFTR mutation [12], and the other case was described by the current author from the same center [13].
The aim of our study is to fi nd the prevalence of double homozygous in our Cf population and describe our (CFTR) mutations and their clinical picture, their family screening, and literature review of this phenomenon since the discovery of the CFTR mutation in 1989.

Methodology
This study is a retrospective case series study performed through reviewing all referred CF patients to our clinics in a tertiary-care center in Saudi Arabia from 1998 to the end of 2018. The diagnosis of CF was based on a typical clinical picture of cough, sputum production, and two subsequent samples of high sweat chloride levels (>60 mmol\L) by the Wescor quantitative method [5].
Respiratory cultures were taken from nasopharyngeal aspirates for patients younger than 5 year of age; and from induced sputum for patients older than 5 years of age. Cultures of broncho-alveolar lavage specimens were taken from 10% percent of the encountered CF population.
Pancreatic insuffi ciency was diagnosed based on stool elastase measurements of > 100-200 ug/g, or a 72-hour fecal fat estimation using the van de Kamer method [12] of which a positive result corresponded to a fecal fat content >7 g/24 hours. Bronchiectasis was identifi ed as dilated bronchi through radiological studies like chest X-rays or Computed Tomography (CT) scans [12].

Defi nition of CFTR genotype
A detailed family history was taken and screening to identify other family members affected with CF was performed. A homozygous genotype was identifi ed as identical homozygous pathogenic CFTR variants. Compound heterozygous mutations were identifi ed as two different pathogenic CFTR variants occurred in a trans confi gurations at two different alleles for the same chromosome. In a heterozygous genotype, one variant was identifi ed in one allele [9].
In double homozygous genotype, two different CFTR pathogenic variants in one chromosome with a carbon copy of the same variant in trans position location in the other allele [9].

CFTR allele counts for patients and families
Multiple siblings with a homozygous CFTR variant within the same family are counted as having two alleles. In a compound heterozygous CFTR variant, each variant is counted as one mutant allele separately (i.e., multiple CF-affected siblings within the same family will be counted with each CFTR variant separately). The percentage of alleles for the patients is calculated as the number of affected alleles for a certain CFTR variant divided by the total number of alleles for the whole CF population (396 patients/792 alleles). The percentage of alleles for the families is calculated as the number of families with the affected alleles for a certain CFTR variant divided by the total number of alleles for whole CF population (312 families) [9].

CFTR identifi cation
The CFTR gene screening methodology involves DNA isolation, Polymerase Chain Reaction (PCR) amplifi cation of genomic DNA, mutational analysis, and sequencing methods [14]. Genomic DNA from the patient's lymphocytes was This method is used in some of our patients recently. information was kept strictly confi dential. Each patient was given a study number, and all patient data were entered into the designed data sheet without patient identifi cation. Family screening found 7 family members were carriers with double heterozygous state in cis position (in the same allele) with normal sweat chloride test and normal growth. The range age of diagnosis is between 3 to 12 months of age [ Table  1] [18][19][20][21].

Resu lts
The P revalence of double homozygous CFTR mutation in our study is 4/312 families or 1-2:100 families which is the highest that has been reported in the medical literature.
Only one study described the double homozygous mutation as part of a complex CFTR description of their CF population [13], and the other 4 cases are from our CF population ( Table 2).
Out of 312 screened families, four cases in four different families had both mutations in the two alleles in an identical way and were diagnosed as double homozygous mutations. Therefore, the prevalence of this genotype is accounted as 1.3% out of all reported cases. Such conditions may be due to the consanguinity between relatives in Gulf Arab communities that can be between two or more consecutive generations. Repetition of consanguinity will pool the present mutations in the affected family. More regulations and precautions should be taken to prevent such extremely rare phenomena. Family counseling and educational campaigns to increase the awareness about the risk of these marriages would help to decrease the inheritance of these mutations. For this reason, the presence of four cases indicates that it is relatively common in the Gulf Arab communities and we suggest more research to be done on the prevalence and distribution of this phenomena.
The clinical pictures were of the severe type in relation to chest disease, failure to thrive, progressive bronchiectasis, worse Pulmonary function test parameters, persistent vitamins ADEK defi ciencies compared to the rest of the CF population, more frequent (MRSA) colonization compared to the general CF population.
Reporting the phenotype of this newly discovered genetic phenomenon will help in comparing it to other common and rare CF conditions and for studying the contributions of these mutations to the course of the disease. Moreover, it would help in individualized therapy and planning of the management [10] Family counseling and screening before marriage, and educational public campaigns might reduce or eliminate this extremely rare conditions that combine double identical mutations in one individual.
We be lieve identifi cation of double homozygous mutations could help in personalized medicine and fi nding pharmacological treatment in the future such as Orkambi ® (lumacaftor/ivacaftor) and Symdeko (Tezacaftor/Ivacaftor) from vertex for F508del mutation [36,37] and Ivacaftor (Kalydeco) for patients with G551D mutations and some other mutations [38].
Citation: Banjar  Our study has limitation due to small number 4 cases.
Further study is needed to do extensive family screening of the relatives of such families to understand the effect of such phenomenon.

Double homozygous CF is relatively common in Saudi
Arabia and might be present in the other Arab Gulf countries  due to having similar societies and consanguinity among their families. More public awareness is needed in these countries and similar communities to prevent such phenomena. It is important to report these newly discovered genotypes for personalized medicine of CF patients.