von hippel - lindau disease ( vhl ; mim # 193300 ) is a hereditary multi - systemic tumour syndrome that pre - disposes affected individuals to haemangioblastomas of the central nervous system and retina , pheochromocytomas , clear - cell renal carcinomas , adenomas and carcinomas of the pancreas , paragangliomas , renal and pancreatic cysts , papillary cystadenomas of the epididymis and , rarely , cystadenomas of the endolymphatic sac and broad ligament . vhl affects approximately 1 in 36,000 newborns and is transmitted in an autosomal dominant manner with a penetrance of more than 90% by the age of 65 years [ 1 , 2 ] . vhl is a tumour suppressor gene located on chromosome 3p2526 [ 3 , 4 ] . the gene consists of three exons , is highly conserved across species , and is ubiquitously expressed in both foetal and adult tissues [ 5 , 6 ] . expression of the vhl gene is not restricted to the organs affected in vhl [ 3 , 79 ] . the vhl protein pvhl ) has been implicated in a variety of functions , including transcriptional regulation , posttranscriptional gene expression , extracellular matrix assembly , protein folding and ubiquitination as reviewed by kaelin . an increasing number of germline mutations have been reported in vhl - affected individuals [ 1114 ] , and genotype - to - phenotype correlations are now emerging . somatic mutations in vhl have also been detected in several types of sporadic and hereditary tumours [ 1618 ] . phenotypes vary among families , reflecting genotypic differences [ 1 , 12 , 14 , 19 ] . clinically , vhl is classified in type 1 or type 2 based on the absence or presence of pheochromocytomas . the occurrence of renal cell carcinoma ( rcc ) allows a further distinction between type 2 a ( low risk of rcc ) and type 2 b ( high risk of rcc ) . some type 2 families develop pheochromocytomas only , with no other neoplastic findings of vhl ( vhl - type 2 c ) . vhl tumours , including pheochromocytomas and paragangliomas , may appear clinically to be sporadic but represent milder cases of vhl , with the attenuated phenotype resulting from either a mild impairment in function of the mutated pvhl or somatic mosaicism . the latter is a condition in which genetically different cells coexist in tissues of the same individual , and the intratumoural mixture of vhl - mutated and vhl - non - mutated cells clearly can modulate the resulting phenotype . we have analysed the vhl gene in the available members of a vhl family in which the pro - band presented with bilateral pheochromocytomas and multiple paragangliomas . her father showed what we believe is a very mild and relatively late - onset vhl phenotype . in this study , we describe the somatic mosaicism of the pro - band 's father and we reviewed the literature for all the described cases of vhl - mosaicism . the clinical features of the female pro - band have been reported in our previously published study , briefly , the young girl now 26 years old , underwent surgery at 11 years of age to resect a pheochromocytoma associated with hypertension . at age 18 years , she underwent further surgery to remove a pheochromocytoma in the contralateral adrenal gland and two concurrent paragangliomas of the abdominal aorta and urinary bladder . one year ago , she was also found to have a right - sided , extra - axial , 1.6-cm supratentorial frontal meningioma ( fig . post - operatively , neuroendocrine serum markers ( plasma free metanephrines , chromogranin a , neuron- specific enolase , and gastrointestinal hormones carcinoembryonic antigen [ cea ] and calcitonin ) have remained negative . a family history obtained from her parents , one brother and one sister was uninformative , except for the father 's history . mr image of the pro - band 's brain.the arrow indicates a 1.6-cm - diameter right - sided supratentorial frontal meningioma . the patient 's father , now 51 years old , was found to have an angioma of the glans penis and had had surgery for a mandibular cyst and epididymal cystadenomas at age 43 years . indeed , abdominal ultrasonography and total - body magnetic resonance imaging ( mri ) revealed a 2.3-cm cyst in his right kidney . his blood pressure and levels of plasma free metanephrines , fractionated urinary metanephrines , chromogranin a were normal . genomic dna was extracted from peripheral blood lymphocytes ( pbls ) of the pro - band and her four first - degree relatives . to assess the possibility that the pro - band 's father had an attenuated vhl phenotype caused by mosaicism , somatic dna was extracted from his oral epithelial cells , hair roots and skin fibroblasts . the entire coding sequence of the vhl gene was pcr - amplified with 36-cycle reactions using conditions and primers , with minimum variations , described previously . pcr products were analysed by denaturing high - performance liquid chromatography ( dhplc ) using a wave 2100 dna fragment analysis system ( transgenomic wave system , omaha , ne ) at column temperatures recommended by the wavemaker version 4.1.31 software ( transgenomic ) and at melt temperatures determined by the dhplc melt software ( http://insertion.stanford.edu/melt.html ) . nucleic acids were separated in the column according to size and degree of denaturation in a gradient of two buffers ( a : 0.1 m triethylammonium acetate , [ teaa , ph 7 ] ; b : 0.1 m teaa , 25% acetonitrile ) . dhplc analysis was performed at a melt temperature of 60c at a constant flow rate of 0.9 ml / min using a linear gradient of acetonitrile . to 13.75% , increased over 5 min . to 16.25% , was kept constant for 1 min . , increased over 1 min . to 25% , was kept constant for 1 min . ( wash ) , decreased over 1 min . to 8.75% , and was kept constant for 1 min . heteroduplex molecules can be detected as an additional peak , or shoulder , in the chromatogram . amplimers with abnormal denaturing profiles were purified ( microcon pcr , millipore , bedford , ma ) and sequenced bidirectionally using an abi bigdye terminator cycle sequencing kit v.3.1 and an abi prism 310 genetic analyser ( both from applied biosystems , foster city , ca , usa ) . sequencing results were analysed using the sequencing analysis v.3.6.1 and autoassembler v.2.1 software packages ( both from applied biosystems ) . for semiquantitative analysis , the pcr products were cloned into a vector ( topo ta cloning , invitrogen carlsbad , ca , usa ) according to the manufacturer 's instructions , directly amplified from bacterial colonies , and sequenced . the clinical features of the female pro - band have been reported in our previously published study , briefly , the young girl now 26 years old , underwent surgery at 11 years of age to resect a pheochromocytoma associated with hypertension . at age 18 years , she underwent further surgery to remove a pheochromocytoma in the contralateral adrenal gland and two concurrent paragangliomas of the abdominal aorta and urinary bladder . one year ago , she was also found to have a right - sided , extra - axial , 1.6-cm supratentorial frontal meningioma ( fig . post - operatively , neuroendocrine serum markers ( plasma free metanephrines , chromogranin a , neuron- specific enolase , and gastrointestinal hormones carcinoembryonic antigen [ cea ] and calcitonin ) have remained negative . a family history obtained from her parents , one brother and one sister was uninformative , except for the father 's history . mr image of the pro - band 's brain.the arrow indicates a 1.6-cm - diameter right - sided supratentorial frontal meningioma . the patient 's father , now 51 years old , was found to have an angioma of the glans penis and had had surgery for a mandibular cyst and epididymal cystadenomas at age 43 years . indeed , abdominal ultrasonography and total - body magnetic resonance imaging ( mri ) revealed a 2.3-cm cyst in his right kidney . his blood pressure and levels of plasma free metanephrines , fractionated urinary metanephrines , chromogranin a were normal . genomic dna was extracted from peripheral blood lymphocytes ( pbls ) of the pro - band and her four first - degree relatives . to assess the possibility that the pro - band 's father had an attenuated vhl phenotype caused by mosaicism , somatic dna was extracted from his oral epithelial cells , hair roots and skin fibroblasts . the entire coding sequence of the vhl gene was pcr - amplified with 36-cycle reactions using conditions and primers , with minimum variations , described previously . pcr products were analysed by denaturing high - performance liquid chromatography ( dhplc ) using a wave 2100 dna fragment analysis system ( transgenomic wave system , omaha , ne ) at column temperatures recommended by the wavemaker version 4.1.31 software ( transgenomic ) and at melt temperatures determined by the dhplc melt software ( http://insertion.stanford.edu/melt.html ) . nucleic acids were separated in the column according to size and degree of denaturation in a gradient of two buffers ( a : 0.1 m triethylammonium acetate , [ teaa , ph 7 ] ; b : 0.1 m teaa , 25% acetonitrile ) . dhplc analysis was performed at a melt temperature of 60c at a constant flow rate of 0.9 ml / min using a linear gradient of acetonitrile . to 13.75% , increased over 5 min . to 16.25% , was kept constant for 1 min . , increased over 1 min . to 25% , was kept constant for 1 min . ( wash ) , decreased over 1 min . to 8.75% , and was kept constant for 1 min . heteroduplex molecules can be detected as an additional peak , or shoulder , in the chromatogram . amplimers with abnormal denaturing profiles were purified ( microcon pcr , millipore , bedford , ma ) and sequenced bidirectionally using an abi bigdye terminator cycle sequencing kit v.3.1 and an abi prism 310 genetic analyser ( both from applied biosystems , foster city , ca , usa ) . sequencing results were analysed using the sequencing analysis v.3.6.1 and autoassembler v.2.1 software packages ( both from applied biosystems ) . for semiquantitative analysis , the pcr products were cloned into a vector ( topo ta cloning , invitrogen carlsbad , ca , usa ) according to the manufacturer 's instructions , directly amplified from bacterial colonies , and sequenced . the vhl gene sequence was altered in both the proband and her father , though to different extents in each . dhplc analysis of vhl exon 3 pbls dna showed quantitative differences in the dna elution profiles between daughter and father ( fig . this finding suggested a heterozygous mutation in both relatives and mosaicism in the father . to test for mosaicism dna exon 3 amplicons extracted from the father 's buccal mucosa , hair roots , and skin fibroblasts showed different levels of intensity of the same altered dhplc peaks , although the intensity of the peaks in these tissues was comparable to that seen in the pbl dna ( fig . 2b ) . denaturing high - performance liquid chromatography ( dhplc ) analysis of exon 3 of the von hippel - lindau disease ( vhl ) gene in all the family members . ( a ) dhplc analysis of the pcr products of exon 3 of the vhl gene in the pro - band ( dhplc 3 ) , her father ( dhplc 1 ) , her mother ( dhplc 2 ) and her brother and sister ( dhplc 4 , 5 ) . in the first - degree pedigree of this family , the pro - band is indicated by number 03 , while her father , mother , sister and brother are indicated by the numbers 01 , 02 , 04 and 05 , respectively . dhplc analysis of the pro - band 's dna shows an extra peak that is barely visible ( but reproducibly so ) in her father 's dna . ( b ) dhplc analysis of dna extracted from a normal control ( nc ) sample , the father 's pbls , and the father 's oral mucosa ( tissue 1 ) , hair roots ( tissue 2 ) and fibroblasts ( tissue 3 ) . sequence analysis demonstrated that the abnormal elution profile ( abnormal peak ) seen in the proband and at various levels in the different cells from the pro - band 's father was the result of a missense mutation in exon 3 . this mutation was a g - to - a substitution at cdna nucleotide 695 , predicting the replacement of wild - type arginine with glutamine codon 161 of pvhl ( r161q ) . the mutation - related peak in the pbls dna of the father was smaller than the same peak in the daughter 's pbls dna ( compare fig . 3a with fig . amplicon cloning revealed that only a few clones contained the g - to - a mutant allele . in other words , in the dna extracted from the father 's circulating pbls , the ratio of wild - type to mutated gene was approximately 85:15 , instead of the 50:50 ratio expected in the absence of mosaicism . sequencing analysis of exon 3 of the vhl gene in dna from pbls of the pro - band ( a ) and her father ( b ) . to summarize , direct sequencing analysis suggested that only a small fraction ( about 15% ) of pbls contained the vhl mutation , lower than the typical 50% observed in a heterozygous individual . furthermore , only in the dna extracted from buccal cells , hair roots and cultured skin fibroblast cells was this mutation evident . the mosaic individual we described here presented at age 51 years with an angioma of the glans penis and a renal cyst . at age his daughter , in contrast , had a full - blown germline vhl gene mutation at a much younger age ( 11 years ) . in the mosaic subject , the late disease onset and mild vhl phenotype might have been mediated by the presence of two different cell populations , a prevailing population with a normal vhl gene and a smaller one with a mutated vhl gene . very few abnormal cells are likely to be present in the father 's chromaffin cells , thus explaining the absence of pheochromocytomas / paragangliomas , because decreased / lower likelihood to have a so - called second hit event with subsequent movement of mutated cells more towards homozygosity , as it has been shown in multiple endocrine neoplasia type 2 associated tumours [ 23 , 24 ] . the inaccessibility of the chromaffin cells in the father 's adrenal medulla and paraganglia precluded the experimental quantification the ratio of normal to mutated vhl . as a consequence of the atypical phenotype of vhl in the father , the presence of familial vhl was not recognized initially . in recent years , the role of pvhl in the regulation of hypoxia - inducible genes through the targeted ubiquitination and degradation of hif1 has been elucidated , leading to a model of how disruption of the vhl gene can result in highly vascularized tumours . when pvhl is absent or mutated , hif1 subunits accumulate , resulting in cell proliferation and neovascularization in vhl tumours . vhl is inherited in an autosomal dominant fashion , with about 80% of cases being familial and about 20% sporadic as a result of a de novo mutation . the family history can sometimes be falsely negative because of failure to recognize the disorder in some family members , reduced penetrance , intrafamilial variability of clinical expression , death of the affected parent before the onset of symptoms or late onset of the disease in the affected parent . another reason why vhl may go unrecognized in either parent , so that the disease in the child is erroneously considered to be sporadic , is somatic mosaicism . first described vhl mosaicism in 2 ( 5% ) of 42 unrelated families , both of which lacked a history of vhl . the two patients ( one man and one woman ) had clinical evidence of vhl , but no vhl mutations were detected in the initial genetic test performed on dna from their pbls ; in contrast , their clinically affected offspring tested positive for vhl mutations in their pbls . another case of parental mosaicism was described by murgia et al . in kindred in whom this pro - band presented at age 26 years with cerebellar haemangioblastoma , retinal haemangioma , multiple bilateral renal cysts and bilateral pheochromocytomas . in contrast , his asymptomatic ( mosaic ) mother , whose pbls dna showed a barely visible single - strand conformation polymorphism bandshift identical to that seen in her son , presented with only a small pheochromocytoma and renal microcysts by age 48 years . in both of the above studies , dna was also extracted from tissues , such as buccal cells and skin fibroblasts to confirm the somatic mosaicism . in similar families , the mosaic individual may be mildly or minimally affected , generally tends to have less severe disease than his or her offspring , and may be asymptomatic or present with less severe features of the disease . disease severity varies among mosaic individuals depending on whether mutations occur early or late in embryogenesis . asymptomatic carriers have been described in a number of heritable tumour syndromes reviewed by zlotogora   and in many other heritable diseases , such as hutchinson - gilford progeria . mosaicism has also been demonstrated to be the cause of many cases of clinical recurrence . in a mosaic individual , the co - existence of mutated cells with even a small population of normal cells is an important parameter in predicting phenotype and overall prognosis and can increase the difficulty of obtaining a correct diagnosis of vhl . vhl in a mosaic individual may be difficult to recognize merely on clinical grounds , but it should always be considered when evaluating patients with isolated vhl - related tumours or parents of affected individuals . under these conditions , such individuals should be analysed for low - level mutations by emerging dna analysis techniques . it is presumed that the genotype - phenotype correlation in vhl reflects the degree to which the functions of pvhl are quantitatively and qualitatively altered by different mutations . a number of mutation carriers have been described , but not in sufficient numbers to define mutation - based phenotypes . furthermore , the percentages of symptomatic and asymptomatic vhl mutation carriers and the most important variable affecting disease penetrance and severity ( age at diagnosis , sex distribution , genetic co - factors or environmental modifiers ) have yet to be evaluated . this would allow the development of unified surveillance guidelines for vhl patients or those at risk for this disease . in summary , we have provided molecular evidence of somatic mosaicism in the father of a patient with full vhl . because of the incomplete penetrance of the disease the diagnosis of vhl was first not recognized . because of penetrance age related in vhl , a long - term follow up is warranted . counselling of patients and closely related family members must take a central place in the management of hereditary multi - organ cancer syndromes , such as vhl . a careful and complete clinical examination in the parents of each patient affected by an apparently de novo vhl germline mutation is warranted . the evaluation of the parents of a pro - band with an apparent de novo vhl gene mutation should include molecular genetic testing if the vhl disease - causing mutation in the pro - band is known . if the disease - causing vhl mutation in the pro - band is unknown , both parents should be offered a complete and extensive clinical and images examination , such as neurological test including mri of the craniospinal axis ; ophthalmologic evaluation ; measurement of plasma free metanephrines , chromogranin a , and fractionated urinary metanephrines ; and abdominal ultrasonography , mri or computed tomography . the real incidence of mosaicism in vhl remains uncertain , but such a phenomenon has important consequences for molecular testing , clinical diagnosis and genetic counselling , in terms of prediction of phenotype and risk of recurrence after the initial diagnosis . the real incidence of mosaicism will hopefully yield better data on the real incidence of de novo vhl mutations .