Abstract
Objectives. 1) To determine the extent of short stature in patients with Fanconi anemia (FA); 2) to determine the extent and nature of endocrinopathy in FA; 3) to assess the impact on height of any endocrinopathies in these patients; and 4) to study the correlation, if any, between height, endocrinopathy, and FA complementation group.
Study Design. Fifty-four patients with FA, 30 males and 24 females from 47 unrelated families, were prospectively evaluated in a Pediatric Clinical Research Center. The patients ranged in age from 0.1–31.9 years, with the mean age at assessment 8.6 years.
Results. Endocrine abnormalities were found in 44 of the 54 FA patients tested (81%), including short stature, growth hormone (GH) insufficiency, hypothyroidism, glucose intolerance, hyperinsulinism, and/or overt diabetes mellitus. Twenty-one of 48 (44%) participants had a subnormal response to GH stimulation; 19 of 53 (36%) had overt or compensated hypothyroidism, while 8 of 40 participants had reduced thyroid-hormone binding. Two patients were diabetic at the time of study; impaired glucose tolerance was found in 8 of 40 patients (25%), but most surprisingly, hyperinsulinemia was present in 28 of 39 (72%) participants tested. Significantly, spontaneous overnight GH secretion was abnormal in all patients tested (n = 13). In addition, participants demonstrated a tendency toward primary hypothyroidism with serum tetraiodothyronine levels at the lower range of normal, while also having thyrotropin (thyroid-stimulating hormone) levels at the high end of normal.
Sixteen patients were assigned to FA complementation group A, (FA-A), 12 to FA-C, and 5 to FA-G; 10 of the 12 participants in FA-C were homozygous for a mutation in the intron-4 donor splice site of theFANCC gene. Patients in groups FA-A and FA-G were relatively taller than the group as a whole (but still below the mean for the general population), whereas those in FA-C had a significantly reduced height for age. GH response to stimulation testing was most consistently normal in participants from FA-G, but this did not reach statistical significance. The tendency toward hypothyroidism was more pronounced in participants belonging to complementation groups FA-C and FA-G, whereas insulin resistance was most evident in patients in FA-G, and least evident in those in FA-C.
Short stature was a very common finding among the patients with a mean height >2 standard deviations below the reference mean (standard deviation score: −2.35 ± 0.28). Patients with subnormal GH response and those with overt or compensated hypothyroidism were shorter than the group with no endocrinopathies. The heights of those participants with glucose or insulin abnormalities were less severely affected than those of normoglycemic, normoinsulinemic participants, although all were significantly below the normal mean. The mean height standard deviation score of patients with entirely normal endocrine function was also >2 standard deviations below the normal mean, demonstrating that short stature is an inherent feature of FA.
Conclusion. Endocrinopathies are a common feature of FA, primarily manifesting as glucose/insulin abnormalities, GH insufficiency, and hypothyroidism. Although short stature is a well-recognized feature of FA, 23 patients (43%) were within 2 standard deviations, and 5 of these (9% of the total) were actually above the mean for height for the general population. Those patients with endocrine dysfunction are more likely to have short stature. These data indicate that short stature is an integral feature of FA, but that superimposed endocrinopathies further impact on growth. The demonstration of abnormal endogenous GH secretion may demonstrate an underlying hypothalamic-pituitary dysfunction that results in poor growth.
- endocrine
- function
- Fanconi anemia
- height
- short stature
- GH deficiency
- hypothyroidism
- diabetes mellitus
- glucose intolerance
- insulin resistance
- FA =
- Fanconi anemia •
- IFAR =
- Integrated Fanconi Anemia Registry •
- GH =
- growth hormone •
- RIA =
- radioimmunoassay •
- AUC =
- area under the curve •
- IGF =
- insulin-like growth factor •
- TSH =
- thyroid-stimulating hormone •
- T4 =
- tetraiodothyronine •
- T3 =
- triiodothyronine •
- TRH =
- thyrotropin-releasing hormone •
- SD =
- standard deviation •
- SDS =
- standard deviation score •
- IVS4 =
- intron-4 splice site mutation •
- NS =
- nonsignificant •
- TBG =
- thyroid-hormone binding globulin •
- TNF =
- tumor necrosis factor •
- IL =
- interleukin
Fanconi anemia (FA) is a rare autosomal, recessive, multisystem disease associated with excess chromosomal breakage.1 The syndrome is characterized by a wide variety of clinical manifestations, including congenital anomalies, progressive pancytopenia, and an increased predisposition to malignancy, especially acute myelogenous leukemia.2,3 The physical anomalies most commonly associated with FA are short stature, radial aplasia, and hyperpigmentation. All these manifestations may be present and conversely, some patients lack malformations.4–6Diagnosis is facilitated by the unique hypersensitivity of FA cells to DNA cross-linking agents such as diepoxybutane.7,8 FA is genetically heterogeneous and is postulated to arise from mutations in any of at least 7 genes.9,10 The first gene associated with FA to be mapped and cloned was FANCC, assigned to 9q22.39,11; the major gene for FA, FANCA, was mapped to 16q24.3 and was cloned in 1996.12–15 More recently, FANCE, FANCF, and FANCG have been cloned and mapped to chromosome bands 6p21.2–21.3, 11p15, and 9p13, respectively.16–18 However, the specific function of the proteins encoded by these genes remains unclear.
Mutations in FANCA account for ∼65% of FA patients enrolled in the International Farconi Anemia Registry (IFAR)13; mutations in FANCC account for ∼15%,19,20 and FANCG ∼10% (Auerbach, unpublished data). Thus, the genetic cause of FA can be ascertained in the vast majority of patients. We have studied a group of 54 individuals enrolled in the IFAR to assess the severity of short stature and to detect the presence of any contributing endocrine dysfunction, including growth hormone (GH) insufficiency, glucose intolerance/insulin resistance, thyroid disease, and disorders of prolactin production. We have compared endocrine outcome and growth failure with the presence of mutations in FANCA,FANCC, and FANCG, correlating phenotype with genotype in the patients within defined genetic groups.
METHODS
Patients
All participants with FA enrolled in the IFAR and who were being clinically assessed at the Rockefeller University were invited to participate in a study of anthropometric and hormonal studies. Fifty-four patients, 30 males and 24 females (from 47 unrelated families), agreed to have the anthropometric studies, and 53 of these consented to all or part of the hormonal testing. They were prospectively studied for endocrine function at the New York Presbyterian Hospital Children's Clinical Research Center. Although participants ranged in age from 0.1 to 31.9 years, most participants were young children, with 47 of the 54 participants (87%) between the ages of 2 and 16 years.
Diagnostic criteria for FA consisted of a positive test for sensitivity to the DNA–cross-linking agent, diepoxybutane.4,7,8 Most participants demonstrated clinical manifestations of the disease (hematologic abnormalities and/or congenital malformations characteristic of FA), but these were not necessary for the diagnosis/study inclusion. Twenty-four patients were receiving medical treatment for bone marrow failure: 18 patients were on androgen therapy and 2 patients had undergone bone marrow transplantation. The remaining patients were not receiving androgen therapy. None of the patients had been frequently transfused, therefore, hemochromatosis was not a confounding variable. Anthropometric patient data are summarized inTable 1. All studies were approved by the Institutional Review Boards of both Weill Medical College of Cornell University and the Rockefeller University. Written informed consent was obtained before the study from the participant and/or a parent.
Anthropometry
Endocrinologic Evaluation
Standing heights were measured using a Harpenden stadiometer. Forty-seven patients were evaluated for GH production by clonidine stimulation (150 μg/m2;21 10 of these also had arginine-stimulated GH testing (0.5 g/kg).22,23 Serum GH was determined by using the Quantitope radioimmunoassay (RIA) kit (Sanofi Diagnostics Pasteur, Inc, Chaska, MN). A peak value ≥10 ng/mL (10 μg/L) was considered to exclude classical GH insufficiency. Overnight GH values were performed on the second hospital night to allow for acclimation to the hospital environment. Thirteen patients had spontaneous overnight GH secretion measured every 20 minutes from 8pm through 8am(save for 2 patients who had blood measured every 60 minutes because of young age and small blood volume). Data from the overnight study was subjected to deconvolution analysis using the CLUSTER pulse detection algorithm,24 and subsequently evaluated for mean, baseline, number of secretory bursts (peaks), sum of peaks, area under the curve (AUC), as well as the sum of peaks and AUC above the baseline. Insulin-like growth factor 1 (IGF-1) levels were obtained in 44 of these patients and assessed by RIA after extraction to remove the binding proteins (Nichols Institute/Quest Laboratories, San Juan Capistrano, CA.) or Smith-Kline Beecham Laboratories (King of Prussia, PA). Hand and wrist radiographs were performed in 39 patients, and bone ages were assessed using the methods of Greulich and Pyle.25 All hand and wrist epiphyses that were not grossly abnormal were included in the bone-age assessment. Thyrotropin (also called thyroid-stimulating hormone [TSH]) was measured by ultrasensitive RIA in 53 patients with full thyroid function testing (tetraiodothyronine [T4]), triiodothyronine (T3), and T3-resin uptake in 41. Thirty-eight of these patients also underwent thyrotropin-releasing hormone (TRH) stimulation testing (7 μg/kg) and 32 of the participants had prolactin levels determined by enzyme immunoassay (Boehringer Mannheim, Indianapolis, IN). Oral glucose tolerance testing was performed in 40 patients to detect abnormalities of glucose metabolism (1.75 g of glucose/kg, maximum 75 g). Criteria for diabetes mellitus and impaired glucose tolerance are those of the American Diabetes Association guidelines.26 An insulin to glucose ratio of >0.25 for prepubertal children (>0.33 for postpubertal participants) at any point during testing was considered indicative of hyperinsulinemia/insulin resistance.27 Blood glucose levels were measured by the clinical chemistry laboratories of the New York Presbyterian Hospital. Insulin assays were performed using the Incstar RIA kit (Incstar Corp, Stillwater, MN).
Statistical Analysis
Height determinations were expressed as the number of standard deviations (SD) from the age and sex adjusted normal values28 to give SD scores (SDS). Target heights were defined as the adult stature corresponding to the mean of the parents' height SDS. Predicted heights were calculated by the Roche-Wainer-Thissen method29 using actual height and weight, parental heights, and bone age.
All values shown represent the mean ± standard error. Univariate analyses have been performed using linear correlation coefficients; the Wilcoxon Sum-of-Ranks test was used to compare values between series and the Student's t-test was used to compare the means between series. Significance was established at P < .05.
Statistical analysis of participants post-bone marrow transplant was not significantly different from that of participants pre-bone marrow transplant, therefore these 2 groups were combined for analysis. A similar analysis of participants receiving androgen therapy showed statistically significant differences only of total T4 levels, baseline glucose, and baseline insulin levels, although none of these differences were physiologically significant. For all other analyses, these groups were combined.
RESULTS
Twelve of the 34 patients whose complementation group status was known were found to have mutations inFANCC.19,20,30 Ten patients were homozygous for a mutation in the intron-4 donor splice site (IVS4 +4 A→T), and 2 patients were heterozygous for a guanine deletion in exon 1 at position 322 (322delG). In 1 of these, the second mutation was also in exon 1 (Q13X) whereas in the other, the second mutation is still undefined. All of the patients with IVS4 mutations were of Ashkenazi Jewish descent. The high proportion of patients with this mutation in the present study (19% vs 8% in the IFAR as a whole) is explained by the large proportion of this ethnic group living in the investigators' catchment area, and thus available for clinical investigation. Sixteen participants had mutations in FANCA 31; mutations in FANCF and FANCG were found in 1 and 5 participants, respectively. As a result of the small sample sizes, FA complementation group FA-F and FA-G were not analyzed as discrete groups.
Height
The mean height SDS at assessment for all the patients studied was −2.35 ± 0.28, which is significantly below normal for age and sex. Thirty-one of the 54 patients (57%) had a height >2 SDs below the mean, whereas 5 patients (9%) had a height >0 SD (ie, the mean for the general population). The mean target height SDS of the patients whose parental height data were available was +0.13 ± 0.18 (n = 27), which implies that the parents of this group are of average height. The actual height SDS of this subgroup, however, was −2.33 ± 0.39, indicating that the patients lost an average of 2.46 SD compared with their expected target height. Final adult height prediction could be estimated for 22 of these participants by the method of Roche-Wainer-Thissen, and was found to be −1.24 ± 0.33. The improvement from the actual height SDS to the predicted height SDS is explained by the patients' delayed skeletal maturity; the mean delay in bone age from the chronological age was 0.98 years. Weight SDS was below normal (−1.26 ± 0.24), but significantly better than the height SDS, indicating that insufficient caloric intake is not sufficient to explain the height deficit. Heights arranged by endocrinopathy are in Table 2.
Height SDS by Endocrinopathy
Three of the GH-deficient patients have received GH therapy. One of these patients is alive and well, a second patient developed fatal acute myelogenous leukemia, and the third died of complications after bone marrow transplant for aplastic anemia.
GH Secretion
GH insufficiency was defined as peak GH response to provocation (clonidine and/or arginine) <10 ng/mL, and was present in 22 of 48 (46%) patients tested. The mean height SDS of the patients with GH insufficiency was −2.66 ± 0.45, which was not significantly shorter than the GH-sufficient patients (−2.14 ± 0.38,P = nonsignificant [NS]) (see Table 2).
Forty-seven participants underwent clonidine stimulation, while 11 participants underwent arginine stimulation of GH (see Table 3). Twenty-six participants responded to clonidine stimulation (including 1 of the 3 adult participants), whereas none responded to arginine. For the FA participants overall, the mean maximally stimulated GH value (ie, to clonidine) was 12.6 ± 1.3 ng/mL (12.6 ± 1.3 μg/L) (Table 3), a level above the threshold of 10 μg/L commonly used to define GH insufficiency.32,33 In these patients, pubertal status affects maximally-stimulated GH levels in a paradoxical manner, with pubertal participants producing lower GH levels than prepubertal ones (8.8 ± 2.01 vs 13.7 ± 1.45, P ≤ .05). Additionally, in this population, females did not produce significantly more GH than males (12.4 ± 1.7 vs 12.9 ± 1.9,P = NS). No participant responded to arginine with a peak GH level of 10 μg/L or greater, although 6 of these 11 did respond to clonidine. Participants receiving androgen treatment did not have maximally-secreted GH levels different from individuals naı̈ve to androgens.
GH Parameters
Spontaneous GH Secretion
To assess hypothalamic-pituitary regulation, participants were evaluated for spontaneous overnight GH secretion. Data obtained from the overnight GH evaluations were subjected to deconvolution analysis, using the CLUSTER pulse detection algorithm. When assessing the overall pattern of GH secretion, all participants tested (13/13) manifested multiple aspects of neurosecretory GH deficiency. Parameters examined include the mean, the number of individual bursts of GH secretion (peaks), the sum of the peak amplitudes, the sum of the peak amplitudes corrected for the baseline (net sum of peak amplitudes), the AUC, and the AUC above the baseline (pulsatile AUC), and are summarized in Table 3. These data were compared with published normative data obtained from non-FA control participants only when those studies used equivalent hormone assays and also used the (identical) CLUSTER algorithm (with age and sex also matched wherever possible). Many of these values were found to be significantly low when compared with these published studies, including the mean GH level (2.3 ± 0.3 μg/L,P ≤ .00534), the number of GH peaks (2.8 ± 0.24 peaks, P ≤ .002,34 andP ≤ .0335), the sum of the peak amplitudes (11.0 ± 1.8, P ≤ .0335) and the AUC (761.4 ± 97.6 μg/L/12 hr, P ≤ .00000334 and P ≤ .00735). The mean peak amplitude was also abnormally low, (3.9 ± 0.5 μg/L35) and this observation approached statistical significance (P ≤ .1).
The AUC can be corrected for the baseline and the pulsatile AUC used as the basis of comparison. Correcting for the baseline illustrates that the pulsatile portion of GH secretion is quite low, accounting for only 29.5% of the total, rather than the 94% reported by Pozo et al.34
Analogous to the maximally stimulated GH levels discussed above, FA patients fail to manifest the increased spontaneous GH secretion associated with puberty. FA participants actually demonstrate a decreased spontaneous GH secretion with advancing puberty that approached statistical significance, with the mean peak amplitude decreasing considerably in pubertal participants compared with their prepubertal peers (4.6 ± 0.6 vs 2.8 ± 0.8,P ≤ .06). Correcting the mean peak amplitude for the baseline did not alter this relationship, (3.6 ± 0.6 vs 1.8 ± 0.8, P ≤ .09). These findings have serious implications regarding final adult height, as our expectation of improvement in growth with age is based on the delayed skeletal maturation in these patients and the use of final adult height prediction algorithms created for individuals with normal GH secretion.
Glucose/Insulin Homeostasis
Glucose and insulin abnormalities were common in the FA participants tested, and are shown in Table 4. At the time of the study, 10 of 40 (25%) participants had abnormal glucose tolerance tests, with type 2 diabetes mellitus in 2 participants (1 of whom was post-bone marrow transplantation), and impaired glucose tolerance in an additional 8 patients. Hyperinsulinemia, however, was noted in 28 of 39 participants (72%) tested, including 9 of the 10 hyperglycemic patients. Only 3 of the 10 hyperglycemic participants and 9 of the 28 hyperinsulinemic participants were on androgen therapy, again indicating that androgen therapy does not explain the abnormality. Baseline glucose and insulin levels were found to be higher in androgen-treated participants, but the degree of elevation was not physiologically significant.
Glycemic Control
Thyroid Function
Abnormalities of thyroid function were found in 19 of 53 (36%) patients tested. Two participants were previously shown to be hypothyroid and were receiving thyroid hormone replacement (and were therefore excluded from the following analyses). Thirteen patients (25%) had compensated (primary) hypothyroidism with normal T4 levels 7.1 ± 0.4 μg/dl (91.4 ± 5.1 nmol/L) (normal values: 4.5–11 μg/dl [64–154 nmol/L]) and slightly elevated TSH values (mean 5.8 ± 0.5 mU/L [normal values: 0.4–4.0 mU/L]); while 4 participants had low thyroxine values (mean: 3.6 ± 0.7 μg/dl [46.3 ± 9.0 nmol/L]) with normal TSH levels (mean 2.7 ± 0.9 mU/L). These findings could be secondary to pituitary/hypothalamic dysfunction, to reduced thyroid hormone binding, or to the Sick-Euthyroid Syndrome, a condition thought to be an adaptation to the catabolic state.36 Overall (n = 52), however, the mean total T4 was 7.2 ± 0.3 μg/dl (92.6 ± 3.9 nmol/L), the mean T3 level was 1.3 ± 0.1 μg/L (2.0 ± 0.2 nmol/L) (normal values: 0.6–1.5 μg/L [0.9–2.3 nmol/L]), while the mean free-T4index was 7.9 ± 0.3 (normal values: 5.5–11), indicating that reduced thyroid-hormone binding did not significantly alter the true thyroxine level (Table 5A). The mean height SDS of the hypothyroid patients was −3.4 ± 0.6, significantly less than that of the group with normal thyroid function, P≤ .005. TRH testing did not reveal any additional hypothyroid patients. The mean (baseline) TSH value among all participants was 3.5 ± 0.3 mU/L, a result within the normal range but clearly at the upper range of normal, which again, may suggest a hypothalamic-pituitary dysregulation of the thyroid axis. As expected, the total T4 of the androgen-treated participants was lower than that of the untreated participants, but the free-T4 index was not, indicating that androgen treatment did not result in the abnormalities of thyroid function noted here.
Eight of 40 participants (5 males, 3 females), including 1 with high TSH, had a reduced thyroid-hormone binding capacity. We evaluated the thyroid-hormone binding pattern in 6 of these patients and found that thyroxine binding globulin (TBG)-bound T4 was low in all patients studied, with a mean of 3.4 g/mL, (normal values: 4.2–8.8), whereas albumin-bound and prealbumin-bound T4 were normal (see Table 5B). In this group, only 2 participants were being treated with androgens, which are known to lower T4 binding. In the 6 participants studied, absolute TBG concentrations were low only in the 2 patients receiving androgen therapy, indicating that TBG-binding affinity, but not TBG concentration, is inherently abnormal in FA, independent of androgen treatment status. There was no obvious clinical pathology resulting from the low thyroid-hormone binding, and the mean height SDS of the reduced binding-capacity group was not significantly different from the group as a whole.
Prolactin levels obtained as part of the TRH test were significantly elevated in 1 of 34 patients studied; this patient was subsequently found to have a prolactinoma. Gonadotropin levels were appropriate for age in all participants tested. We also observed normal circadian cortisol levels in all participants, except for 1 young woman with incipient bone marrow failure.
Genotype-Phenotype Correlations
Sixteen participants were assigned to FA complementation group A (FA-A), 12 to FA-C, 5 to FA-G and 1 to group FA-F; 20 participants were not assigned to a specific complementation group. Height, thyroid function, glucose metabolism, and prolactin secretion were assessed and compared for groups FA-A, FA-C, and FA-G, with significant relationships found between complementation group and height, glucose, insulin, and thyroid-hormone levels (see Table 6).
Thyroid Function
Thyroid-Hormone Binding
Height SDS by Complementation Group
Participants from FA-A had improved heights (−1.55 ± 0.45 vs −2.72 ± 0.36, P ≤ .08), and lower 120 minute glucose levels during an oral glucose tolerance test 115 ± 4 vs 130 ± 8 mg/dL (6.4 ± 0.2 vs 7.2 ± 0.4 nmol/L),P ≤ .03. The overall glucose and insulin response during the oral glucose tolerance test demonstrate that these individuals have moderate insulin resistance, not statistically different from the group as a whole. Additionally, mean T4 and TSH were not significantly different from the group as a whole (7.3 ± 0.4 vs 7.7 ± 0.3 μg/dl [94.0 ± 5.1 vs 99.1 ± 3.9 nmol/L], P = NS and 3.1 ± 0.5 vs 3.4 ± 0.3, P = NS, respectively) indicating only a baseline tendency toward primary hypothyroidism.
Individuals from FA-C have a significantly reduced height, (−3.84 ± 0.86 vs −1.93 ± 0.25, P ≤ .01), and a significantly lower peak insulin level (26.9 ± 7.7 vs 90 ± 15.4 μU/mL [207 ± 55 vs 645 ± 110 pmol/L],P ≤ .03) with a peak blood sugar not significantly different from the group as a whole (132 ± 9 vs 150 ± 7 mg/dL [7.3 ± 0.5 vs 8.3 ± 0.4 nmol/L], P= NS). Peak glucose to insulin ratios were also significantly higher (ie, less insulin resistant) in individuals from FA-C (10.6 ± 3.1 vs 4.46 ± 0.9, P ≤ .03). Individuals in group FA-C demonstrate a mildly increased tendency toward primary hypothyroidism with T4 identical to the group as a whole (7.3 ± 0.8 vs 7.7 ± 0.3 μg/dl [94.0 ± 10.3 vs 99.1 ± 3.9 nmol/L], P = NS), but with a modestly elevated TSH level, which did not reach statistical significance (4.1 ± 0.8 vs 3.4 ± 0.3, P = NS).
Ten of the 12 individuals in FA-C were homozygous for the IVS4. These individuals had an even worse height outcome (−4.50 ± 0.88,P ≤ .0006). Peak insulin values were lowered even further (15.3 ± 4.4 μU/mL [109 ± 31 pmol/L],P ≤ .01), resulting in an even higher glucose to insulin ratio, (13.3 ± 3.6, P ≤ .02), while the blood sugars remained in the normal range (120 ± 8 vs 150 ± 7 mg/dL [6.7 ± 0.4 vs 8.3 ± 0.4], P= NS). Thyroid status is analogous to the FA-C group as a whole, but more exaggerated with the T4 approximately unchanged (7.2 ± 1.0 vs 7.7 ± 0.3 μg/dl [92.7 ± 12.9 vs 99.1 ± 3.9 nmol/L], P = NS) but the TSH slightly higher (4.4 ± 0.9 vs 3.4 ± 0.3, P= NS) than the FA-C group as a whole.
Patients from group FA-G had a moderately compromised height (−2.01 ± 0.78 versus −2.41 ± 0.31, P = NS), with a trend toward higher peak GH levels (20.7 ± 7.9 vs 12.6 ± 1.3 ng/mL, P = .11). These patients have a lower baseline blood sugar (73 ± 5 vs 87 ± 4 mg/dL [4.1 ± 0.3 vs 4.8 ± 0.2 nmol/L], P ≤ .03), while having a lower peak glucose to insulin ratio (2.0 ± 0.9 vs 4.46 ± 0.9, P = NS), indicating a higher than average degree of insulin resistance. This group also had a significantly low T4 level, (5.5 ± 0.8 vs 7.7 ± 0.3 μU/dl [70.8 ± 10.3 vs 99.1 ± 3.9 nmol/L], P ≤ .04).
DISCUSSION
FA is a genetically heterogeneous disorder of unknown pathophysiology, although the first of the genes responsible for the syndrome (FANCC) was identified in 1992.9,11 FA is presumed to arise from abnormal processing of DNA damage, but cytoplasmic (in addition to nuclear) localization has been demonstrated for both FANCC and FANCA protein activity37–39 as well as the FANCA/FANCG complex.40,41 Although first recognized as possessing a typical constellation of phenotypic abnormalities in 1927,42 it is now clear that affected individuals with abnormal chromosomal fragility after exposure of cells to DNA cross-linking agents may lack some or all of the typical physical and hematologic traits that characterize FA.4,5 Short stature is a common finding in FA, with the mean height SDS of −2.37, although the severity of the short stature varies widely.
In this study, endocrinopathy was a frequent finding, with 44 of the 54 participants (81%) demonstrating at least 1 abnormal finding: 72% showed hyperinsulinemia, 25% had impaired glucose tolerance or overt diabetes mellitus; 44% of patients tested had a subnormal response to GH stimulation, 100% had abnormal spontaneous GH secretion profiles; 36% had thyroid hormone deficiency, and 20% had reduced thyroid hormone binding. The patients with low GH responses tended to have a greater degree of growth retardation than the group as a whole and stature was significantly worse for those with hypothyroidism. Participants with either glucose or insulin abnormalities had a better height than the euglycemic/normoinsulinemic group, but these individuals were also significantly below the mean.
The group of patients with no demonstrable endocrinopathy (ie, with adequate stimulated GH secretion, normal thyroid function, and no glucose or insulin abnormalities) had a mean height SDS of −2.06 ± 0.32, demonstrating that a significant degree of short stature is typical of FA and seems to be an inherent feature of the disease. Although the control of statural growth is a complex process controlled by many endocrine and nonendocrine processes, these results suggest that GH insufficiency and hypothyroidism–superimposed on a baseline of short stature–may further contribute to the evolution of short stature in FA. Our finding that 13 of 13 patients evaluated for spontaneous GH secretion manifest multiple aspects of neurosecretory GH deficiency suggests that this may be contributing to the baseline short stature in this syndrome.
Before this study, there have only been a few isolated case reports of GH deficiency43–45 and of hypothyroidism46 in patients with FA, suggesting that more care should be taken to exclude these problems in individuals with this condition. The frequent finding of bone-age retardation leads to the expectation that, in the absence of clinical deterioration, final adult height might not be impacted as severely as relative height during childhood, although our calculations still predict the loss of 1.24 SDs. In so far as these data are meaningful, they presume that the delay in bone age will result in a prolonged period of growth, and this may not necessarily occur in patients with FA. Our findings of decreased GH secretion in pubertal compared with prepubertal participants suggests that the expectation of improved growth with time is unsupported. The validity of height prediction algorithms for patients with syndromes of abnormal growth such as FA is questionable.
GH Secretion
Clonidine is believed to induce GH release by stimulating α2-adrenergic receptors in the hypothalamus, which then results in increased GHRH tone.21 Conversely, arginine causes GH secretion principally by lowering somatostatin tone at the level of the hypothalamus.47 The disparate GH response to various provocations seen in these patients may be attributable to the modes of action of these 2 compounds. The GH response to clonidine indicates that the hypothalamus maintains α-adrenergic responsivity, and that both the hypothalamus and pituitary retain normal functional abilities. These patients may not respond well to arginine because FA patients have low baseline somatostatin tone which, when lowered further, does not significantly induce the release of previously produced GH. Indeed, it is likely that with a low baseline GHRH tone, minimal GH is translated, and therefore only minimal GH is available for release by agonists such as arginine. Maximally-stimulated GH was 12.6 ± 1.25 ng/mL (12.6 ± 1.25 μg/L) for FA participants overall, with no significant difference between males and females (12.4 ± 1.71 vs 12.9 ± 1.89 respectively, P = NS). When grouped by pubertal status, however, significant differences were found. In contrast to the physiologic augmentation of GH secretion with puberty,48in our study pubertal participants had significantly lower maximally stimulated GH, relative to the prepubertal children (8.8 ± 2.01 vs 13.7 ± 1.45, P ≤ .05). Importantly, on an absolute basis, the maximally stimulated GH in these pubertal participants was below the threshold of 10 ng/mL (10 μg/L) commonly used to define GH insufficiency.32,33 There is no general agreement as to the nature of the changes in maximally-stimulated GH values with puberty, as assessed by response to clonidine stimulation. Previous studies have demonstrated both increases with puberty48–50 as well as decreases.32,51,52However, these studies do not suggest that (in the absence of GH insufficiency) maximally stimulated GH decreases below normal at any point. The reduced GH secretory ability may result in an overestimation of final adult height, based on a delayed skeletal maturation and the presumption that a longer growth period will ameliorate some of the short stature noted early in life. The diminished GH secretion after puberty may suggest a sex steroid-induced deterioration in the underlying pathology of FA, in contrast to the well-described sex steroid-induced augmentation of GH secretion in control participants.53
Spontaneous GH Secretion
Thirteen participants underwent an evaluation of spontaneous nocturnal GH secretion to assess hypothalamic-pituitary regulation. In many conditions, maximally stimulated GH levels may not correlate with growth,54 while spontaneous overnight GH secretion has been advocated as a physiologically relevant measure of true GH secretory ability.34,48,55,56 To minimize the variability of GH secretion associated with the sleep pattern, our participants were allowed a day of acclimation with no stimulation tests performed before the overnight study. In addition, in an attempt to standardize the data obtained from the overnight GH evaluations and allow for improved comparison to published values, we performed deconvolution analysis using the CLUSTER algorithm.24 Multiple parameters were examined, including the mean, the baseline, the number of peaks, the sum of the peaks, the area under the curve, as well as these same parameters corrected for the baseline. Comparison was then made to published reports of similar studies in non-Fanconi control participants. Only studies using equivalent GH assays and the identical pulse-algorithm (CLUSTER) transformed data were used for comparison.34,35,57 Such comparison demonstrates that 13/13 participants studied manifested impaired spontaneous GH secretion.
The mean overnight GH level in the patients with FA was 2.32 ng/mL (2.32 μg/L), significantly lower than the mean of 3.5 ng/mL reported in normally growing controls by Pozo34 (P≤ .005). When analyzing the pattern of GH, no participant seemed to secrete GH appropriately. Patients had few, if any, peaks of GH secretion, tending to secrete GH in a tonic low-level rather than in discrete bursts. FA patients had 2.77 secretory bursts per 12 hours, significantly fewer than either the 4.4 bursts reported by Roemmich (P ≤ .03)35 and the 3.5 bursts reported by Pozo (P ≤ .002).34 The mean amplitude of the secretory bursts was lower in the FA patients than in controls (3.94 compared with 7.82 ng/mL)35 with a trend toward statistical significance. (P < .1). Great importance has been placed on the sum of the peak amplitudes in analyzing spontaneous overnight GH secretory profiles. In our participants, the sum of the peak amplitudes, 10.95 ng/mL, was significantly lower than that reported in the literature of 34.2 ng/mL35(P ≤ .03) and 32.1 ng/mL.57 In contrast to the findings of Hindmarsh et al55 in non-FA participants, we found no improved correlation of growth in these patients by correcting the sum of peak amplitudes for the baseline.
The AUC in the FA patients was remarkably lower than that reported by others, 761 versus 3184 ng/mL/12 hr35 (P≤ .007) and 2032 ng/mL/12 hr34 (P ≤ .000003). As a result of the high baseline GH levels in the FA participants, the AUC above the baseline—the pulsatile AUC—constitutes a lower proportion of the total than in controls (30% vs 94%34). Additionally, this corrected AUC is lower in females than in males, and decreases with onset of puberty. Both of these findings are contrary to findings in non-FA control participants.35,58
Height
Height SDS was not found to correlate with maximally-stimulated GH, serum IGF-1, or IGF-1 SDS (either for chronological or bone age). We also found no correlation between height SDS and maximal spontaneous GH levels, AUC, or any other tested parameter of overnight GH secretion. The imperfect association between IGF-1 level and height SDS may be related to any of the multiple known modifiers of IGF-1 levels, including nutrition59 and disease-state.60–62 That the GH and IGF-1 values are not as severely affected as is the height demonstrates that in this population, neither parameter is a sufficiently sensitive surrogate marker for growth. Recent experiments by Popovic et al63have demonstrated that participants with FA have greatly elevated levels of a type 1 collagen marker (P1CP) that do not respond normally to GH, paradoxically decreasing in response to GH. In the face of poor growth, this suggests that these individuals undergo futile biosynthesis of collagen.
The factor most significantly associated with a deficit in height in this group was the presence of the IVS4 mutation. In 22% of our patients who are known to have this mutation, mean height SDS was −4.50, which was markedly reduced as compared with the group as a whole (P ≤ .0006, Table 6). In addition, these individuals more commonly had primary hypothyroidism with low T4 and elevated TSH levels. Patients with this mutation have already been noted to have a more severe clinical phenotype consisting of multiple congenital malformations20,64 and early onset of hematologic abnormalities, and may represent a more profound genetic defect.
Short stature in FA cannot be explained based on endocrinopathy alone. Hormonal deficiencies were not observed in all patients tested, and neither do we believe that hormonal replacement therapy will completely normalize growth. The number of FA patients on GH is too few to analyze with any statistical significance.65
These data are consistent with a hypoactive hypothalamus as the underlying cause of the poor growth in FA. The lack of normal endogenous GH secretion (marked by low pulsatility, low maximal spontaneous GH levels, and globally decreased GH secretion), combined with normal response to clonidine stimulation, is consistent with low GHRH tone. Activation of the GHRH receptor not only induces the release of previously produced GH, but also stimulates the de novo synthesis of GH (and the GHRH receptor as well),66 which would lead to the prediction that in the face of low GHRH tone, there would be decreased GH responsivity to both GHRH and other stimuli. The poor response of GH to arginine stimulation suggests that the baseline somatostatin tone is low. The combination of low GHRH tone together with low somatostatin tone would be expected to yield both the clinical and biochemical picture seen in FA.
Glucose/Insulin Homeostasis
Abnormalities of glucose homeostasis were extremely common in the FA participants studied. Hyperglycemia (either impaired glucose tolerance or diabetes mellitus) was found in 25% of participants tested, while hyperinsulinemia was found in 72% of the participants tested. Patients from FA-A were euglycemic, but demonstrate moderate hyperinsulinemia. Participants from FA-C had higher baseline insulin levels but the lowest incremental rise after glucose stimulation, while maintaining euglycemia throughout the 2-hour study. The improved peak glucose to insulin ratios may indicate a lower degree of insulin resistance, or alternatively reflect impaired insulin secretion (ie, incipient insulinopenia). Individuals from FA-G were also euglycemic, but demonstrated profound hyperinsulinemia.
Thyroid Function
Patients with FA have a low-normal T4, and a high-normal TSH level. This combination is consistent with several explanations: a combined central and peripheral (impending) hypothyroidism, a discrepancy between peripheral thyroid hormone concentrations and the central sensing or feedback mechanisms, or a TSH molecule with decreased bioactivity.
Reduced thyroid hormone binding was unexpectedly common in this cohort (20%), as the prevalence in the general population is ∼1 in 5000 live births.67 Although reduced thyroxine binding is not usually clinically significant, it may lead to false diagnosis of hypothyroidism. Reduced thyroid hormone binding might be explained by abnormal hepatic function with reduced formation of any combination of thyroid-hormone binding globulin (TBG), albumin, or prealbumin, which all carry thyroid hormones in the circulation. A specific TBG gene defect is unlikely, as TBG is located on the X chromosome, and none of the 6 FA genes assigned chromosomal locations thus far is on the X chromosome. In an effort to better characterize the nature of the abnormal thyroid hormone binding, we analyzed the T4-binding profile in a subset of patients. In 6 of 6 patients tested, TBG-bound T4 was low, whereas albumin-bound and prealbumin-bound T4 were normal. Absolute TBG levels were found to be normal in participants naı̈ve to androgen treatment, but low in the patients receiving androgen therapy. This suggests that the thyroid-hormone binding affinity of TBG is inherently abnormal in FA. Such an abnormality could be attributable to altered TBG-sialylation, a posttranslational modification known to modulate T4-binding affinity.
The cause of hormonal insufficiency is unclear. GH insufficiency is generally presumed to be of hypothalamic or pituitary origin, but the hypothyroidism was generally accompanied by elevated TSH levels and, therefore, seems to be of thyroidal origin, although hypothalamic-pituitary dysregulation leading to abnormal central responsiveness cannot be excluded. The cause of hyperinsulinemia is generally thought to be caused by insulin resistance, a problem of the end-organ. The causal link between the GH, thyroid, and glucose/insulin abnormalities may be attributable to the basic cellular defect in FA, but until this has been elucidated, we can only postulate a unifying explanation. We suggest that the endocrinopathies are a secondary effect, mediated by cytokines such as trinitrofluorenone tumor necrosis factor [TNF]-α, interleukin (IL)-1, IL-6, and INF-γ. Multiple recent associations of FA proteins and cytokines bolster this hypothesis. Increased cytokine activity was inferred from the demonstrations of high-level constitutive cytokine-dependent protein expression in FA cells, which rose further on cytokine stimulation.68,69 Human (and murine) hematopoietic progenitor cells from FA-C (and Fa-c) participants were shown to have increased sensitivity to TNF-α, INF-γ, and macrophage inflammatory protein-1α, as measured by both growth inhibition and increased apoptosis.70,71 Increased cytokine activity has also been shown to alter sialylation, potentially explaining the high incidence of TBG-deficiency in such individuals.72
Interestingly, individuals with such diverse systemic catabolic conditions such as cancer, acquired immunodeficiency disease, and trypanosomiasis have a similar clinical phenotype with multiple endocrinopathies and have been shown to have elevated cytokine levels.73–78 Both TNF-α and IL-6 dysregulation have been associated with insulin resistance,73,79 as well as abnormal thyroid function—decreased thyroglobulin and thyroid peroxidase (as measured by mRNA levels)80 and reduced serum T3 levels.81
CONCLUSION
We have found increased incidences of endocrinopathies in participants with FA, as well as significant phenotype-genotype correlations between complementation groups and specific endocrinopathies. Members of FA-A seem to have a relatively mild endocrine phenotype. They have more normal stature, moderate insulin resistance, and no increased tendency toward primary hypothyroidism. Members of FA-C, however, have greater impairment of stature and a greater tendency toward primary hypothyroidism, but the least insulin resistance. Those participants homozygous for the IVS4 ofFANCC seem to be a more exaggerated subset, with the same qualitative problems but with quantitative differences. Members of FA-G have moderately compromised heights, but demonstrate profound insulin resistance and a greater tendency toward primary hypothyroidism. The variability in the endocrinologic phenotype for each of these 3 complementation groups is of great interest, as recent studies provide some evidence that FANCA, FANCC, and FANCG interact to form a protein complex of unknown function.82 However, the numbers of patients in each complementation group is small in this study and additional participants must be examined to confirm these correlations. With the recent success of bone marrow or cord-blood transplantation in curing the fatal hematologic complications of FA,83,84 the long-term effects of the disease are now of greater importance, including the issue of final adult height. Replacement therapy for hypothyroidism is relatively straightforward, but GH therapy in these participants raises several medical and ethical dilemmas. FA patients are at an increased risk of malignancy, and in particular they are at 15 000 times greater risk of developing acute myelogenous leukemia.3,85,86 The question has been raised as to whether GH therapy could increase this risk. The incidence of new leukemia development in GH-treated patients without predisposing risk factors is believed not to be different to that of the general population.87–90 Indeed, some authors have postulated that some of the patients who developed leukemia on GH therapy may have been FA patients with short stature and no other abnormalities.91
Previous reports have suggested that diabetes mellitus occurs more commonly in patients with FA,92,93 but that the true incidence is unknown. Our data support the view that diabetes is common, with 2 patients diabetic at the time of evaluation, and 29 of 39 (74%) with abnormalities on oral glucose tolerance testing (10 of 40 participants with hyperglycemia and 28 of 39 with hyperinsulinemia). Although androgen therapy has been associated with insulin resistance,92,94 we have shown that the glucose intolerance/insulin resistance predates androgen treatment and is an inherent feature of FA. In this light, we find very interesting the reports by Swift et al95 and Morrell et al93of an increased incidence of glucose intolerance/diabetes mellitus in individuals heterozygous for FA, as well as other conditions associated with increased chromosomal breakage.
Although patients with FA are shorter than the general population and shorter than their parent-derived target heights, the mean stature of the group examined lay near the third percentile, and many individuals were not extremely short. Children with FA frequently have reduced GH secretion and responsiveness, hyperglycemia/hyperinsulinism, and hypothyroidism, which may further compromise their growth. The specific pattern and the characteristics of the endocrine disorders found in these participants are consistent with the secondary effects of cytokine-mediated endocrinopathies. Finally, we have found that specific endocrine-phenotypes exist for the patients in specific FA complementation groups. This is not surprising, as there seem to be distinct patterns of expression for Fanca and Fancc during embryonic development, as shown in the mouse.96,97 We suggest endocrine evaluation in all FA children because correction of these endocrinopathies may improve growth, final height outcome, and the overall quality of life.
ACKNOWLEDGMENTS
This work was supported in part by Grant R37-HL32987 from the National Institutes of Health (A.D.A.), the Genentech Center for Clinical Research and Education (M.P.W.) and by General Clinical Research Center Grants M01-RR06020 from the National Institutes of Health to the New York Presbyterian Hospital Children's Clinical Research Center and the Rockefeller University Hospital General Clinical Research Center, respectively.
We thank the nursing staff and all those that helped make this work possible.
Footnotes
- Received June 5, 2000.
- Accepted November 15, 2000.
- Address correspondence to Michael P. Wajnrajch, MD, Cornell University Medical Center, 525 E 68th St, Room M-624, New York, NY 10021. E-mail: mpwajnr{at}med.cornell.edu
Dr Gertner is now at Serono Laboratories, Norwell, Massachusetts.
REFERENCES
- Copyright © 2001 American Academy of Pediatrics
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