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Original Studies |
Nephrology and Endocrinology and Metabolism Divisions, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; and Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania
Address all correspondence and requests for reprints to: Nuzhet O. Atuk, Department of Medicine, University of Virginia, Health Sciences Center, Box 133, Charlottesville, Virginia 22908.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The early occurrence of retinal manifestations in childhood, and the predominant accompaniment of pheochromocytoma occurring between 7 yr of age and early adulthood without renal cell carcinoma is characteristic of members of a large kindred (McC), which we have studied since 1964 (7, 8, 9). Since then, we have updated and expanded this family study, and confirmed that in affected members pheochromocytoma and retinal angiomas are common and occur early, and that renal cell carcinoma, pancreatic adenoma, and hemangioblastoma are rare. We studied 14 members of the McC family for the presence of a causative mutation and associated clinical features.\.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Figure 1
is an updated pedigree of
three families. The number of affected members, their VHLD
manifestations, and age of diagnosis are listed in Table 1.
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). Any family member who
had documented retinal angiomatosis (RA) or pheochromocytoma was
considered to meet the criteria for VHLD (1). Any child with an
affected parent was also considered at high risk to inherit the disease
and was followed periodically with urinary catecholamine measurements
and ophthalmological examinations. In these three families, 7
unaffected and 7 affected members volunteered for genetic analysis. The
clinical studies were approved by the Human Investigation Committee,
and patients gave their written informed consent. Studies were
performed at the University of Virginia General Clinical Research
Center. We screened 50 unselected members (age 2–70 yr) of the three
families of the kindred (III-1, III-2, and III-3), by measuring urinary
norepinephrine (NE), epinephrine (E), metanephrines (MN), and
vanylmandilic acid (VMA) concentrations and by ophthalmoscopy. Blood
pressure was measured every 4 h over 24 h. The measurements
of plasma NE, E, and dihydroxyphenyl glycol levels were added in later
studies for early detection and recurrence of pheochromocytoma (9). If
there was a positive finding for VHLD in the screening studies, an iv
pyelogram with laminography was performed and later, abdominal
computerized tomography scan, 131I-MIBG scintigraphy;
magnetic resonance imaging of the central nervous system (CNS) and
abdomen were performed as indicated clinically. Analytic assays
Urinary NE, E, MN, and VMA concentrations were measured as
described previously (8). Table 2 shows
the sensitivity and specificity of these measurements in our
laboratory. Tumors were assayed for NE and E by homogenizing 0.5-1.0 g
tissue in 10 mL cold trichloracetic acid, centrifuged to obtain a clear
supernatant solution, and extracts were assayed fluorometrically (10).
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The results were analyzed using the nonparametric Spearman rank correlation tests and Student’s paired t test when applicable. Values are expressed as mean ± SEM.
DNA sequence analysis
Genomic DNA was extracted from peripheral blood leukocytes using a commercially available kit (Promega, Madison, WI). The VHLD gene was amplified by PCR using exon specific primers (4). Sequencing was performed using a commercially available cycle sequencing kit and dye-labeled terminators according to manufacturer’s directions (Perkin- Elmer, Norwalk, CT). Sequences were analyzed on an ABI automated sequencer. Mutations were confirmed by sequencing both DNA strands.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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).
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Urinary excretion of NE + E was elevated in 16 of 17 patients, MNs were
increased in 15 of 17, and VMA was elevated in 13 of 17 patients. NE +
E excretion at the time of diagnosis of pheochromocytoma was
significantly higher in younger patients (Fig. 3a). There was no correlation between
tumor size and age of tumor resection. However, younger patients had
significantly higher tumor NE + E concertrations per gram of tissue
(P = 0.01). Notable was the finding that, in 2 patients who
had more than 1 tumor, the tumor catecholamine content (4 samples per
tumor) was similar for all tumors form the same individual (Fig. 3b).
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). Of the 10 individuals tested who were
past the age at which symptoms usually first appear, all had a genotype
consistent with their clinical presentation (i.e. affected
individuals had the mutation, whereas unaffected individuals did not).
Four children under age 10 also have the mutation, are currently
asymptomatic, and have no evidence of disease (Table 3).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Before the development of methods to identify the mutations, dection of disease was dependent on regular screening examinations (ophthalmologic examinaton, urinary catecholamine measurements), usually yearly. Because the onset of symptoms occurred as early as 5 yr and as late as 25 yr, 20 yr of regular follow up is necessary. The current cost of yearly screening is $650 (excluding time lost from work) for a total of $13,000 for 20 yr. In contrast, the current cost of genetic analysis is $260; if the mutation is not present, the cost savings is $12,740/person over 20yr.
The higher urinary NE + E concentration in resected tumors from younger individuals indicates that these tumors have greater synthetic activity and likely decreased catecholamine degradation. Monoamine oxidase activity appears to be related to age, with greater activity in normal older adults compared with younger adults (13). The biological significance of higher catechol concertration in urine and tumors of younger patients in this study is unknown but emphasizes of measuring both cahecholamine levels and metabolites and the need for adequate preoperative medical preparation. Additionally, these findings may reflect the influence of unknown modulators of gene activity.
The mutation identified in this family results in the nonconservative substitution of a hydrophobic amino acid (alanine) for a hydrophilic one (serine) and is not one of the two known polymorphisms present in the coding region (12). Although this mutation has not been reported previously, a G to A at nucleotide 658, resulting in substitution of a threonine for an alanine, has been reported in at least one other VHLD patient (6). Missense mutations have been correlated with VHLD type 2A, i.e. VHLD with pheochromocytoma, but without renal cell carcinoma (6). It is likely that the substitution of a serine for an alanine affects the folding of the VHLD protein and thus interferes with its reported function (14, 15).
This longitudinal study of a large kindred with VHLD demonstrates the benefits of regular examinations and biochemical screening to identify and treat affected members. We suggest that patients with the mutation be followed at least annually with an ophthalmological examination and 24- h urinary measurement of catecholamines. Furthermore, we advocate a screening magnetic resonance imaging for CNS hemangioblastoma in the midteens, and baseline ultrasonography and computerized tomography scanning of the kidney’s in the mid-20’s. Development of any symptoms at any time mandates immediate evaluation. The ability to identify the causative mutation, particularly in childhood, now enables identification of family members who are at risk for the disease, for whom genetic counseling should be considered, and for directed studies to detect and treat the disease at an early stage. This genetic approach is more cost effective and avoids unnecessary testing in subjects who do not have the mutation.
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Acknowledgments |
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Footnotes |
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Received January 24, 1997.
Revised September 19, 1997.
Accepted October 1, 1997.
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References |
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