Estudio de la prevalencia de mutaciones en línea germinal (síndrome de Birt-Hogg-Dubé) y somáticas del gen FLCN en pacientes con fibrofoliculomas o tricodiscomas cutáneos y/o neumotórax recidivantes y/o bilaterales.
Loading...
Files
Identifiers
Publication date
2016
Reading date
08-02-2016
Authors
Advisors
Monteagudo Castro, Carlos
Jordá Cuevas, Esperanza
Journal Title
Journal ISSN
Volume Title
Publisher
Metrics
Export
Abstract
Antecedents and objectives: In 1977, Birt et al. described a series of patients with multiple papular skin-colored, dome-shaped lesions located on the face, neck, and trunk. Histologically, these lesions corresponded to fibrofolliculomas, trichodiscomas and soft fibromas. This was the first description of what later came to be known as Birt-Hogg-Dubé syndrome, a genodermatosis that exhibits autosomal dominant inheritance. In addition to skin lesions, patients with Birt-Hogg-Dubé syndrome may present a series of extracutaneous lesions. In recent years, it has been shown that these patients are at a greater risk of developing renal cancer, spontaneous pneumothorax and lung cysts. In 2002, the FLCN gene was identified as being associated with Birt-Hogg-Dubé syndrome. The loss of FLCN expression observed in almost all histologic subtypes of renal carcinoma implicates this gene in the pathogenesis of this type of tumor and provides further support for its function as a tumor suppressor gene. A second somatic mutation is necessary for renal cancer in patients with Birt-Hogg-Dubé syndrome to develop; this mutation leads to loss of mRNA expression of FLCN in these tumors. Contrary to what occurs in renal tumors, no loss of heterozygosity had been detected in skin or lung lesions. However, in the Fourth Birt-Hogg-Dubé Symposium in 2012, Toro et al. obtained DNA from fibrofolliculomas of patients with BHDS finding loss of heterozygosity at the FLCN locus in most of the stromal tissue samples. In every case of LOH, the germline FLCN mutation was retained, and the wild-type sequence was lost suggesting that bialellic inactivation could be implicated in the development of cutaneous lesions.
The present study aimed to detect germline mutations in FLCN in patients with fibrofolliculomas and/or trichodiscomas, and, with spontaneous pneumothorax and/or lung cysts; evaluate biallelic inactivation in FLCN in lung and skin lesions, evaluate somatic mutations in FLCN in skin and lung samples, evaluate BHD promoter methylation status and in patients with fibrofolliculomas or trichodiscomas, and/or spontaneous pneumothorax, evaluate mRNA expression of FLCN in fibrofolliculomas and trichodiscomas, and in lung and renal lesions and evaluate the presence of skin and/or lung lesions and germline and somatic mutations in FLCN in first-degree relative with Birt-Hogg-Dubé syndrome.
Methods: A retrospective, descriptive study of the clinic, histologic and immunohistochemical characteristics in patients diagnosed of fibrofolliculomas and/or trichodiscomas and/or bilateral spontaneous pneumothorax in the Hospital Clínico Universitario of Valencia from 1995 to 2012 was performed. The study included a total of 39 patients (22 males and 17 females).
All participants were subjected to dermatological investigation, chest X-rays or high-resolution computerized tomography scan and abdominal ultrasonography. Analysis of the FLCN gene was performed on genomic DNA extracted from peripheral leukocytes. PCR and direct sequencing for the detection of mutations in coding exons and adjacent flanking intronic regions were performed with Sanger and next generation sequencing. In addition, FLCN gene analysis was performed in fibrofolliculomas, trichodiscomas and lung lesions. DNA was obtained from macrodissected paraffin-embedded tumor samples and then subjected to direct sequencing. Immunohistochemistry of paraffin-embedded fibrofolliculomas, trichodiscomas, lung samples, renal carcinomas and normal kidney was performed for demonstrating the presence and location of folliculin using the rabbit polyclonal antibody (FL-342) against folliculin. Germline methylation of FLCN was performed according to manufacturer´s instructions (Zymo Research) with the available EZ-96 DNA Methylation-GoldTM KitTM.
Results: Our study included a total of 39 individuals. There were 22 men and 17 women with a median age of 53,26 years. Seventy-two percent of participants had multiple white or skin colored papules over the axilla, face, neck, scalp, groin, upper extremities, trunk and/or lower extremities histologically confirmed as fibrofolliculomas or trichodiscomas. Twenty-one percent of patients with BHDS were devoid of cutaneous lesions.
Forty-one percent of BHDS patients had lung cysts on CT imaging with bilateral cysts in a 33,% of them. The left lung had the highest frequency of pneumothorax (46,7%). Approximately 40% of patients had a pneumothorax in the right lung only with the median age of 43,27 years. The average number of pneumothoraces per patient was two. Incidence of pneumothorax was higher in men than in women, at a ratio of 3:1. Fifty-three percent of BHD patients had kidney cysts involving both kidneys in a 50% of cases.
In immunostaining, we found that in cutaneous and lung lesions FLCN immunostaining was retained in the cytoplasm but was stronger in the nucleus, predominantly localized in the nucleolus, whereas FLCN immunoreactivity was virtually absent in both cytoplasm and nucleus of kidney carcinomas, and present exclusively in the cytoplasm in normal kidney tubules.
Germline BHD mutations were present in 73,7% of patients with Birt-Hogg-Dubé syndrome. Mutation c.1286insC in exon 11 was the most frequent site of mutation, accounting for 35,7% of all the mutations detected. Regarding, somatic mutations, the exon 14 was the most frequent site of somatic mutations, followed by exons 5 and 6.
A seventy-five percent of patients diagnosed of BHDS had somatic mutations in at least one of their biopsies and a 66,7% of them had somatic mutations in all of the samples studied. Forty-two percent of patients with BHDS showed biallelic FLCN gene inactivation. In regard to cutaneous biopsies, biallelic inactivation was found in 5 of 12 (41,7%) patients and 5 of 15 (33,3%) samples. In regard to lung biopsies, a third of samples showed biallelic FLCN gene inactivation. No methylation of the FLCN/BHD promoter was found in any of the samples.
Conclusions: Skin lesions are missing in one-fifth of patients with BHDS. In contrast with the currently accepted hypothesis of haploinsuficiency as the only pathogenic mechanism for the development of cutaneous and pulmonary lesions in the BHDS, our results indicate that FLCN biallelic inactivation, by mechanisms other than metilation, with allelic loss, is a common phenomenon in cutaneous lesions, and to a lesser extent in lung lesions, may represent an analogous pathogenic mechanism to that already accepted for renal tumors in this syndrome.