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Analysis of cystic fibrosis gene mutations in children with cystic fibrosis and in 964 infertile couples within the region of Basilicata, Italy: a research study

  • Domenico Dell’Edera1Email author,
  • Michele Benedetto1,
  • Gemma Gadaleta2,
  • Domenico Carone3,
  • Donatello Salvatore4,
  • Antonella Angione5,
  • Massimiliano Gallo5,
  • Michele Milo1,
  • Maria Laura Pisaturo6,
  • Giuseppe Di Pierro6,
  • Eleonora Mazzone7 and
  • Annunziata Anna Epifania8
Journal of Medical Case Reports20148:339

https://doi.org/10.1186/1752-1947-8-339

Received: 2 April 2014

Accepted: 20 August 2014

Published: 10 October 2014

Abstract

Introduction

Cystic fibrosis is the most common autosomal recessive genetic disease in the Caucasian population. Extending knowledge about the molecular pathology on the one hand allows better delineation of the mutations in the CFTR gene and the other to dramatically increase the predictive power of molecular testing.

Methods

This study reports the results of a molecular screening of cystic fibrosis using DNA samples of patients enrolled from January 2009 to December 2013. Patients were referred to our laboratory for cystic fibrosis screening for infertile couples. In addition, we identified the gene mutations present in 76 patients affected by cystic fibrosis in the pediatric population of Basilicata.

Results

In the 964 infertile couples examined, 132 subjects (69 women and 63 men) resulted heterozygous for one of the CFTR mutations, with a recurrence of carriers of 6.85%. The recurrence of carriers in infertile couples is significantly higher from the hypothetical value of the general population (4%).

Conclusions

This study shows that in the Basilicata region of Italy the CFTR phenotype is caused by a small number of mutations.

Our aim is to develop a kit able to detect not less than 96% of CTFR gene mutations so that the relative risk for screened couples is superimposable with respect to the general population.

Keywords

Cystic fibrosis Cystic fibrosis conductance transmembrane regulator Screening in infertile couples Mutation analysis Polymerase Chain Reaction

Introduction

Cystic fibrosis (CF) is the most common autosomal recessive genetic disease for the Caucasian (white) population. In Italy, the disease occurs in 1/2500 to 1/3000 Caucasian newborns, with a carrier incidence ranging from 1/26 to 1/30 in the general population [1, 2].

CF is a complex multisystem disease related to the buildup of thick, sticky mucus that can damage many of the body's organs (epithelia of the respiratory tract, exocrine pancreas, intestine, male genital tract, hepatobiliary system, and exocrine sweat glands). The pulmonary disease is present in 90% of patients and it is the major cause of morbidity and mortality in CF.

The CFTR gene provides instructions for making a glycoprotein called cystic fibrosis transmembrane conductance regulator (1,480 amino acid residues). This protein works as a channel across the membrane of cells producing mucus, sweat, saliva, tears, and digestive enzymes. The channel negatively transports charged particles called chloride ions into and out of the cells. The transport of chloride ions helps control the movement of water in tissues, which is necessary for the production of thin, freely flowing mucus. Mucus is a slippery substance that lubricates and protects the lining of the airways, digestive system, reproductive system and other organs and tissues [3]. In particular, more than 95% of men with CF are infertile as a result of azoospermia caused by congenital bilateral agenesis of the vas deferens (CBAVD); it occurs in men without pulmonary or gastrointestinal manifestations of CF.

Cystic fibrosis is caused by mutations in the CFTR gene (cystic fibrosis transmembrane regulator), detected for the first time in 1989. The CFTR gene is located on the long (q) arm of chromosome 7 (7q31.2) [4, 5].

More than 1800 mutations in the CFTR gene have been identified [6]; many of which are so rare as to be called ‘private’ as they are only present within individual families. Moreover, the CFTR mutation detection rate varies by test method and ethnic background. The most common mutation, called F508del, is a deletion of one amino acid at position 508 in the CFTR protein. The resulting abnormal channel breaks down shortly after it is made, so it never reaches the cell membrane to transport chloride ions. The F508del mutation, accounts for two-thirds of all cystic fibrosis alleles worldwide. This mutation is particularly frequent in people of northern European ancestry (70% in Anglo-Saxon countries and 50% in the Mediterranean area).

The phenotypic variability is determined by the heterogeneity of mutations in the CFTR gene, but also by many other factors, such as modifier genes, epigenetic regulation, environment and timeliness in therapy [7, 8].

Carrier screening for cystic fibrosis involves analysis for common mutations in the CFTR gene from people with no personal history, or family history, of the disease.

This analysis shows whether a person is a carrier, at risk (one in four) of having a baby with cystic fibrosis if their partner is also a carrier. Carrier screening has been recommended by the American College of Medical Genetics and American College of Obstetricians and Gynecologists (ACMG/ACOG) [9] and the Human Genetics Society of Australasia (HGSA) [10] and has been established in the USA, Australia, and parts of Europe [11]. Accurate identification of CF mutations results in more applicable programs for prevention, diagnosis, and treatment of CF. In a study carried out in some areas of Northern Italy, carrier screening was associated with a decrease in the incidence of CF [12].

Previous reports about CF patients born in the Basilicata region (southern Italy) have identified types and incidence of the most common mutations of the CFTR gene [13, 14].

In this study are reported the results of a molecular screening of CF in patients enrolled from January 2009 to October 2013 using DNA samples. Patients were referred to our laboratory for CF screening for infertile couples [15]. In addition, we identified the gene mutations present in patients affected by cystic fibrosis in the pediatric population of Basilicata.

In this way the incidence and typology of CFTR mutations in Basilicata were characterized and related to previous reports on CF patients born in the same area.

Results

In the 964 infertile couples examined, 132 subjects (69 women and 63 men) were heterozygous for one of the mutations of CFTR, with a carrier occurrence of 6.85% (Table 1). The carrier occurrence in infertile couples is significantly higher than the hypothetical value of the general population (4%). The statistical analysis was performed according to the ‘hypothesis test for a proportion’. Our data indicated an expectation of 6.85% with a sample of 1,928 subjects. The Z-test revealed a value of 6.38% with a P value <0.0001. Nevertheless, mutation I148T, at first classified as a mutation, now is considered an ‘innocent’ polymorphism, able to be pathogenetic only if present in the same gene (on the same chromosome) with another polymorphism (3199del6). Hence it is necessary, in presence of polymorphism I148T, to detect the presence of polymorphism 3199del6. The polymorphism 3199del6 was not detected in all patients with the I148T variant.
Table 1

Number of subjects tested who were carriers of the cystic fibrosis transmembrane regulator gene

Mutation

Men

Women

Total

G551D

1

2

3

R553X

0

1

1

F508del

35

32

67

N1303K

7

8

15

I148T

4

9

13

G542X

3

6

9

DI507

2

0

2

L1077P

0

2

2

D1152H

1

6

7

W1282X

2

0

2

2183 AA>G

3

0

3

1259insA

0

1

1

4016insT

1

0

1

I507del

1

0

1

2789+5G>A

1

0

1

4382delA

0

2

2

G1244E

1

0

1

621+3A>G

1

0

1

Total

63

69

132

Among the infertile couples, three men, two of whom were brothers, were azoospermic with CBAVD. The molecular analysis of the CFTR gene revealed that the two brothers, aged 26 and 29 years old respectively, were both ‘compound heterozygotes G542X/D1152H’, while the third one, aged 37 years old, was ‘compound heterozygotes G542X/E831X’.

With regard to the 76 patients with cystic fibrosis and a positive sweat test, all have two CFTR-mutated genes (Figure 1).
Figure 1

76 patients with cystic fibrosis and positive sweat test, all have two genes mutated. SNPs: Single nucleotide polymorphism.

Discussion

The aim of this study was to characterize, at molecular level, the most common CFTR mutations in the population of the Basilicata region. We have defined the type and frequency of mutations found and put them in comparison with data from previous studies (Table 2) [13, 14]. In this study, we detected mutations that had not been revealed in the two previous studies [13, 14]. In infertile couples from Basilicata, the occurrence of CF carriers is significantly higher than in the general population (6.85% vs. 4%). It was not clear if a screening for the general population would be viable or effective, so we invited the carriers in the families of affected people to participate in the research.
Table 2

Comparison between the results obtained in this study and those obtained in a previous study

 

Castaldo et al.[14]

Mutations observed in the present study

F508del

55.8% (29)

48.62% (141)

N1303K

3.8% (2)

9.31% (27)

G542X

3.8% (2)

8.96% (26)

W1282X

3.8% (2)

1.03% (3)

2183AA>G

5.8% (3)

2.76% (8)

R1162X

0

0

1717-1G>A

1.9% (1)

0

T338I

0

0

R347P

0

0.69% (2)

711+5G>A

0

0

852del22

5.8% (3)

1.03% (3)

4382delA

0

0.69% (2)

1259insA

0

0.34% (1)

4016insT

0

0.34% (1)

R553X

0

0.34% (1)

R1158X

0

0

L1077P

0

1.03% (3)

I502T

0

0

3849+10kbC>T

1.9% (1)

0.34% (1)

D579G

0

0.69% (2)

G1244E

3.8% (2)

0

G1349D

0

0.34% (1)

2789+5G>A

0

1.03% (3)

711+1G>T

0

0

L1065P

0

0

2522insC

0

0

E585X

0

0

G85E

0

0

G178R

0

0

D1152H

0

3.10% (9)

I148T-3195del6

0

0

I148T (alone)

0

4.48% (13)

R334W

0

0

DI507

0

0.69% (2)

I1005R

0

0

3272-26A>G

0

0

2711delT

0

0

L558S

1.9% (1)

0.34% (1)

W1063X

0

0

D110H

0

0

S549R (A>C)

1.9% (1)

0.69% (2)

2184insA

0

0

3131del22

0

0

R709N

0

0

A349V

0

0

4015insA

0

0

Y849X

1.9% (1)

0.34% (1)

G551D

0

1.03% (3)

621+3A>G

0

0.34% (1)

E831X

0

0

I507del

0

0.69% (2)

IVS8 TG12/t5

0

1.03% (3)

H139R (A->G)

0

0.34% (1)

1248+1G>A

0

0.34% (1)

R74W;V201M;D1270N

0

0.69% (2)

S1455X

0

0.34% (1)

dele 2,3 (21kb)

0

0.34% (1)

991del5

0

0.34% (1)

UNKNOWN

7 %(4)

4.83% (14)

F508C

0

0.69% (2)

TOTAL

52

290

In CF, contrary to other genetic diseases (for example beta thalassemia), the identification of carriers is possible only through molecular research of CFTR mutations. A recent report has shown the costs of managing CF for the Italian National Health System [16]. The results reports a high cost for the chronic and evolutive aspects of the disease. As mentioned before, molecular screening of CF is highly recommended in the USA by the National Institutes of Health Consensus Development Conference Statement on genetic testing for cystic fibrosis [17]. The target of the screening could be men and women of reproductive age in the periconceptional period. A European consensus conference in 2009 has concluded that for complete information for couples, each national health system could implement a screening program. In Italy, the region of Veneto started a complete screening program for the general population some years ago. The results have shown a significant reduction of the incidence of the disease. More recently, an experimental periconceptional program of CF and thalassemia screening allowed for the identification of 94% of CFTR gene mutations, with respect to 80% in the rest of Italy. Moreover, this screening program involved a high number of couples [18].

The success of a genetic screening program is mainly the outcome of educational support informing the population about the disease, thereby offering the opportunity to make an informed decision about reproduction.

In this study, the aim was to establish the a priori risk of CF carrier status in the Basilicata population and to offer a reliable screening test to couples planning a pregnancy, allowing them to make informed decisions.

Conclusions

This study shows that, in the Basilicata region (Italy), it was observed that the CFTR phenotype is caused by a small number of mutations.

The knowledge of the frequency of mutations prevalent in CFTR genes in the province of Matera allows development of kits for their detection.

Our aim is to develop a kit able to detect not less than 96% of CTFR gene mutations so that the relative risk for the couples screened is superimposable with respect to the general population.

Extending knowledge about the molecular pathology, on the one hand, allows better delineation of the mutations in the CFTR gene, and on the other, dramatically increases the predictive power of molecular testing.

Methods

In this study, we investigated 964 infertile couples (1.928 subjects examined; 3.856 alleles studied) born and living in Basilicata, from 2009 to December 2013 (five years). Moreover, we evaluated gene mutation of CFTR in 76 children born in Basilicata (152 CF alleles examined) with a positive sweat test (>60mmol/L). Written informed consent was obtained from the subjects examined for publication of this study in compliance with the Helsinki Declaration.

We applied the following tests to each patient after a venous blood sample was collected (in EDTA-K3).

Molecular analysis of the CFTR gene was performed following these steps:
  • DNA isolation, starting from 25μl of blood, using the Promega extraction kit (DNA IQ™ System, cod.C6701; Promega Italy S.r.l., Milan, Italy).

  • Polymerase chain reaction (PCR) and reverse hybridization. The procedure includes two steps: PCR amplification using biotinylated primers and hybridization of amplification products to a test strip containing allele-specific oligonucleotide probes immobilized as an array of parallel lines. Bound biotinylated sequences are detected using streptavidin-alkaline phosphatase and color substrates. The amplification and the reverse hybridization on a strip were obtained with the use of commercial kits produced by Nuclear Laser Medicine S.r.l., Settala (MI), Italy (cod. AC023/AC025 and AC089): genetic tests aimed at checking 60 mutations in the CFTR genes. The mutations analyzed are listed in Table 3. The test has a sensitivity and a specificity of more than 99%. With a direct analysis of 60 mutations of the CTFR gene, with reverse dot blot, it is possible to detect 90% of the most common CF alleles in Southern Italy (the regions of Campania, Puglia, Basilicata and Molise).

  • The patients who tested negative or with a single mutation detected by reverse dot blot and with a clinical suspicion of atypical cystic fibrosis were analyzed with a complete scanning of the codificant region, through amplification and direct sequencing of 27 exones of the CFTR gene. In patients negative for reverse dot blot and in whom there were no clinical signs of cystic fibrosis, sequencing of the CFTR gene was not necessary. DNA sequencing is not essential, since the detection of innocent polymorphisms is not important to control the disease. The sequence of oligonucleotides for each exone, with the annealing temperatures (T°A) and the length in base pair (bp) of the amplified product, are reported in Table 4. The amplification conditions for the 27 exones change according to the annealing temperature (T°A.), which depends on the oligonucleotides used, which in turn are specific for analysis of each exone. The amplification report is shown in Table 5.

Table 3

List of 60 mutations in the cystic fibrosis transmembrane regulator gene (specificity 100%)

F508del

I507del

F508C

621+1G>T

D110H

E585X

G1349D

I502T

1706del17

1677delTA

R117H

H139R

1898+1G>A

4015delA

G542X

1717-1G>A

Q552X

852del22

G178R

1898+3A>G

 

G551D

S549R(A>C)

2183AA>G

T338I

991del5

1898+5G>T

 

N1303K

4016insT

3849+10kb C>T

R347P

R334W

2184insA

 

G85E

711+5G>A

711+1G>T

1259insA

R347H

2522insC

 

2789+5G>A

W1282X

G1244E

R1066H

R352Q

3120+1G>A

 

I148T

3199del6

S912X

R1158X

1717-8G>A

R1066C

 

R1162X

4382delA

D1152H

L1077P

D579G

3272-26A>G

 

L1065P

R553X

PoliT: 5T, 7T, 9T

1874insT

3659delC

 
Table 4

Sequence of oligonucleotides for each exon, with the annealing temperatures (T°A) and the length in base pair (bp) of the amplified product

Exon

Sequence of primers

T°A

bp

1

GAGAAAGCCGCTAGAGCAAA(CF1F)

55°C

394

TCCTTTACCCCAAACCCAAC(CF1R)

2

TCCAAATCTGTATGGAGACCA(CF2F)

55°C

603

TCAGTGTGAAAATGAGATGTTCC(CF2R)

3

TCTGGCTGAGTGTTTGGTGT(CF3F)

55°C

399

TTTGGAGTTGGATTCATCCTTT(CF3R)

4

AAACTTGTCTCCCACTGTTGC(CF4F)

55°C

453

GGCCTGTGCAAGGAAGTATT(CF4R)

5

GTGCCTAGATGCTGGGAAAT(CF5F)

55°C

393

AAAACTCCGCCTTTCCAGTT(CF5R)

6a

TGCTATGTGCTCCATGTAATGA(CF6AF)

55°C

415

TGCATAGAGCAGTCCTGGTT(CF6AR)

6b

TGCCCATCTGTTGAATAAAAG(CF6BF)

55°C

411

CCCATGAAAGTGAATTTGTGC(CF6BR)

7

TTCCATTCCAAGATCCCTGA(CF7F)

55°C

404

GCACATTTTTGCAAAGTTCA(CF7F)

8

GAATCCTAGTGCTTGGCAAAT(CF8F)

55°C

404

GATCCTCCTTCCAGTTCTACCA(CF8R)

9

GGCCATGTGCTTTTCAAACT(CF9F)

55°C

389

CTCCAAAAATACCTTCCAGCA(CF9R)

10

TGAATCCTGAGCGTGATTTG(CF10F)

55°C

435

TTCATGTGTTTGCAAGCTTCTT(CF10R)

11

GAAGGAAGATGTGCCTTTCAA(CF11F)

55°C

395

CCAAGATACGGGCACAGATT(CF11R)

12

TCAGTGAATCGATGTGGTGAC(CF12F)

55°C

419

ATGAGGCGGTGAGAAAAGGT(CF12R)

13-1

TCATGCTATCAGAATTCACAAGG(CF13F1)

56°C

575

GGGAGTCTTTTGCACAATGG(CF13R1)

13-2

CTGGAGAGTTTGGGGAAAAA(CF13F2)

56°C

449

AAATACCCCCAAGCGATGTA(CF13R2)

14a

CAATGGTGGCATGAACTGT(CF14AF)

55°C

437

GTGGTTCTACTTGTTGATTTTTCAG(CF14AR)

14b

TGGCTTTCTTGTGAGGTTCA(CF4BF)

55°C

446

TGCTTGGGAGAAATGAAACA(CF14BR)

15

GTCGCCAAATAACGATTTCC(CF15F)

55°C

406

AGGTTCAACAAAGGGCACAT(CF15R)

16

TTTGGGTTCTGAATGCGTCT(CF16F)

55°C

388

GGCCAGGTAAGCAGTTCTGA(CF16R)

17a

CTCACCAACATGTTTTCTTTGA(CF17AF)

55°C

399

CCAAAATGAAGTCACATGGTCA(CF17AR)

17b

GAATGGCACCAGTGTGAAAA(CF17BF)

55°C

682

CAATCTGTGTGCATCGGTTT(CF17BR)

18

TGTGCCCTAGGAGAAGTGTG(CF18F)

55°C

335

TGACAGATACACAGTGACCCTCA(CF18R)

19

GCCCGACAAATAACCAAGTG(CF19F)

55°C

399

GCAAGCAGTGTTCAAATCTCA(CF19R)

20

CCAATTCCTTATGCCCAGTT(CF20F)

55°C

408

TGGCTAAGTCCTTTTGCTCA(CF20R)

21

TGATGGTAAGTACATGGGTGTTTC(CF21F)

57°C

578

GGAGCCATACCAGTGAGGAG(CF21R)

22

TCAAATGGTGGCAGGTAGTG(CF22F)

55°C

382

TCACCATGAAGCAGGCATAA(CF22R)

23

CCCATGGTTGAAAAGCTGAT(CF23F)

55°C

417

TGAGTAAAGCTGGATGGCTG(CF23R)

24

GCCTTCTGTCCCAGATCTCA(CF24F)

60°C

362

GAGCAAATGTCCCATGTCAA(CF24R)

Table 5

Amplification report

Temperature

Time

Cycles

95°C

5’

1

95°C

30”

35

T° A

30”

72°C

20”

72°C

5’

1

Consent

Written informed consent was obtained from all of the patients (including legal guardians of the children) for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Our local institutional Ethics Committee approved this study.

Declarations

Acknowledgements

The authors thank the ‘Association Gian Franco Lupo’ (ONLUS: a non-profit organization of social utility).

Authors’ Affiliations

(1)
Unit of Cytogenetics and Molecular Genetics, ‘Maddonna delle Grazie Hospital’
(2)
Department of Biochemistry and Molecular Biology Ernesto Quagliariello, University of Bari
(3)
Center of Reproduction and Andrology (CREA)
(4)
Cystic Fibrosis Centre, AOR San Carlo Hospital
(5)
Regional Center of Pharmacovigilance, Basilicata Region
(6)
Unit of Obstetrics and Gynecology, AOR San Carlo Hospital
(7)
Department of Surgical Science, University of Parma, Parma Hospital
(8)
Unit of Clinical Chemistry, ‘Madonna delle Grazie Hospital’

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Copyright

© Dell'Edera et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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