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Departments of Endocrinology and Metabolism (R.M., G.H.) and Ophthalmology (P.B.), Hôtel Dieu Hospital, Beirut, Lebanon; Department of Internal Medicine (J.W., A.P.), Division of Endocrinology, Diabetes, and Metabolism, Washington University School of Medicine, St. Louis, Missouri 63110; Department of Endocrinology and Metabolism (S.A., I.S.), American University Hospital, Beirut, Lebanon; and Laboratory of Molecular Biology and Cytogenetics (J.L.), Saint Joseph University of Beirut, Beirut, Lebanon
Address all correspondence and requests for reprints to: Dr. Georges Halaby, Department of Endocrinology and Metabolism, Hôtel Dieu Hospital, Beirut, Lebanon. E-mail: mjhalaby{at}sodetel.net.lb.
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Abstract |
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In this study we describe 31 Lebanese WFS patients belonging to 17 families; this, to our knowledge, is the largest number of patients reported in one series so far. Criteria for diagnosis of WFS were the presence of insulin-dependent diabetes mellitus and optic atrophy unexplained by any other disease. Central diabetes insipidus was found in 87% of the patients, and sensorineural deafness confirmed by audiograms was present in 64.5%. Other less frequent features included neurological and psychiatric abnormalities, urodynamic abnormalities, limited joint motility, cardiovascular and gastrointestinal autonomic neuropathy, hypergonadotropic hypogonadism in males, and diabetic microvascular disease. New features, not reported in previous descriptions, such as heart malformations and anterior pituitary dysfunction, were recognized in some of the patients and participated in the morbidity and mortality of the disease. Genetic analysis revealed WFS1 gene mutations in three families (23.5%), whereas no abnormalities were detected in mitochondrial DNA.
In conclusion, WFS is a devastating disease for the patients and their families. More information about WFS will lead to a better understanding of this disease and hopefully to improvement in means of its prevention and treatment.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Full characterization of all clinical and biological features of WFS is difficult because, with the exception of a few series, the number of patients in most reports is small. The pathogenesis of the disease is still unknown; one candidate gene has been mapped to chromosome 4p and was recently cloned and localized by Inoue and colleagues (4). It was shown to encode a transmembrane protein called wolframin whose function is not yet determined. Manifestations of WFS are variable and there is evidence for a genetic heterogeneity with some reports suggesting a role of mitochondrial DNA in the pathogenesis of this syndrome (5, 6, 7, 8).
Our study includes 31 Lebanese patients affected with WFS all derived from consanguineous marriages. By investigating these patients, we aimed to provide more information on the clinical manifestations and genetics of the disease for a better understanding of its pathophysiology that may lead to improvement in genetic counseling and therapy.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In all patients we performed a water restriction test followed by arginine vasopressin administration (4 µg Desmopressin iv) to diagnose diabetes insipidus (DI). DI was considered to be complete when maximal urine concentration was less than 300 mosm/kg and partial when maximal urine concentration was 300–800 mosm/kg. All patients also had a complete neurological examination by a specialist as well as an audiogram.
Peripheral diabetic neuropathy was assessed by monofilament and vibratory testing; autonomic neuropathy was looked for by measuring differences in blood pressure and heart rate between lying and standing positions. Diabetic nephropathy was assessed by 24-h microalbuminuria and plasma creatinine.
C-peptide levels were measured by RIA in 20 patients (baseline and 2, 5, and 10 min after glucagon stimulation). Islet cell antibodies and anti-GAD65 antibodies were measured, respectively, by immunofluorescence and RIA in patients who had diabetes for less than 5 yr (seven patients).
Brain magnetic resonance imaging (MRI) and cystomanometry were done in 19 patients. All patients were examined by a cardiologist, and echocardiography was done in those in whom a heart murmur was detected.
Pituitary function tests were performed in 20 patients who had good/acceptable glycemic control (HbA1c 
8%), tests included assessment of corticotropic and somatotropic axis by measurement of plasma IGF1 (IGF1-IRMA, Nichols Institute Diagnostics, San Clemente, CA) as well as measurement of plasma cortisol (Cort-CT2 RIA, CIS-Biointernational, Gif-sur-Yvette, France) and GH (ELSA-HGH immunoradiometric, CIS-Biointernational, detection limit 0.04 ng/ml) at baseline and 30, 60, and 120 min after insulin hypoglycemia. ACTH was measured by immunoradiometric assay (ELSA-ACTH, CIS-Biointernational) in patients found to have deficient cortisol response to hypoglycemia. Gonadotropic function was assessed by measurement of serum total testosterone (Diagnostic Products Corp., Los Angeles, CA) in postpubertal boys, estradiol (ESTR-US-CT RIA, CIS-Biointernational) in girls, and FSH and LH (IRMA-FSH, IRMA-LH, BYK-Sangtec Diagnostica, Dietzenbach, Germany) at baseline and 30, 60, and 120 min after GnRH (200 µg iv). Lactotropic and thyrotropic functions were documented by measurement of serum free T4 (Amerlex-MAB/competitive radioimmunometric, Amersham, Aylesbury, UK), TSH (IRMA-mat TSH, BYK-Sangtec Diagnostica), and prolactin (PRL) (IRMA-mat PRL, BYK-Sangtec Diagnostica) at baseline and 15, 30, and 60 min after TRH (500 µg iv).
Blood samples were taken from all patients as well as from their parents and siblings for DNA extraction. DHPLC and direct DNA sequencing were performed to screen the coding region of the WFS1 gene in index patients of each family. The method consisted of direct sequencing of PCR products using M13 Dye terminator kits (Applied Biosystems, Foster City, CA) and an Applied Biosystems 373 automated sequencer (4). Mitochondrial DNA (mtDNA) was also screened for rearrangements and point mutations (6). Linkage analysis using linkage software (Genehunter: Daly M, Kruglyak L, Pratt S, Houstis N, Reeve M, Kirby A, Lander E. GeneHunter v.2.0 ß, Ward Systems Group, Inc., Frederick, MD) and microsatellite markers linked to the 4p16.1 locus (6) was done in families in whom no abnormality in the WFS1 gene was found.
Our study was approved by the ethical committee of the Chronic Care Center, and written informed consent was signed by the patients (or the parents for those under 18 yr of age).
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The median age of the patients was 16 yr (range 8–39 yr), the sex ratio (male/female) was 1.38, the median age of onset of IDDM was 6 yr (range 4–12 yr) with median duration of 10 yr (range 2–27 yr). The median age of onset of OA was 9 yr (range 5–20 yr) with median age of diagnosis at 15 yr. Diabetes insipidus, whether partial or complete, was confirmed in 87% of the patients; in these patients the median age of onset of polyuria was 9 yr (range 4–26 yr). Sensorineural deafness confirmed by audiograms was present in 64.5% of the patients with median age of clinical onset of 16 yr (range 7–24 yr).
Anti-GAD65 and islet cell antibodies were negative in all of the seven patients who had diabetes mellitus for less than 5 yr, whereas C-peptide levels (both basal and stimulated) were low [<0.1 ng/ml (< 0.03 nmol/liter)] in all patients.
According to diabetic microangiopathic complications (Table 1
), all-stage retinopathy (nonproliferative and proliferative) was found in 16.6% of patients who had diabetes for 5–10 yr, 42.8% of patients who had diabetes for 10–20 yr, and all patients whose diabetes was of more than 20 yr duration. Proliferative retinopathy was detected only in patients belonging to the latter subgroup. The prevalence of nephropathy (incipient or overt) in these three subgroups was 16.6, 57.1, and 75%, respectively, and that of peripheral neuropathy 33.3, 57.1, and 100%, respectively.
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One patient had surgery for congenital ventricular septal defect; five other patients presented with systolic ejection murmurs at the upper left sternal border and were found to have pulmonary valvular stenosis on echocardiography. Seven patients complained of limited joint motility, which was particularly evident in the interphalangeal and metacarpophalangeal articulations.
Neurological abnormalities were detected in 22 of the 31 patients (70.9%) (Table 2). The most common neurological feature was the absence of nauseal reflex followed by truncal ataxia, cold intolerance, and nystagmus. All of the 19 patients who had brain MRI were found to have moderate to severe optic tract atrophy (which was detected in all cases by funduscopy); other MRI abnormalities were the absence of posthypophysis signal, cerebellar and brain stem atrophy, and in advanced cases cortical atrophy (Table 3). There was a direct correlation between the frequency and severity of neurological manifestations, MRI abnormalities, and patient age (P < 0.005).
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Urodynamics showed abnormalities in 73.7% of the 19 patients in whom they were performed (13 of 19), these abnormalities consisted of increased bladder capacity and decreased sensitivity, incomplete bladder emptying, bladder instability and bladder atony. Abnormal urodynamics were found in 3 patients without evidence of diabetes insipidus.
Abnormality of one or more pituitary secretion was found in 75% (15 of 20) of patients who were investigated. Deficient somatotropic response, i.e. peak GH after hypoglycemia less than 7 ng/ml (<325 pmol/liter) in children and less than 3 ng/ml (<140 pmol/liter) in adults, was found in 45% (nine of 20) of patients, and one patient showed borderline response [peak GH = 7.75 ng/ml (360 pmol/liter)] (Table 4). Plasma IGF1 level was decreased in all of the patients with deficient response, compared with normals for age and sex, but was within the normal range in the patient with borderline response. All of the patients with deficient GH response had heights at or below the fifth percentile; however, severe growth retardation was not found in any of them (no height below the third percentile). Corticotropic function was deficient, i.e. plasma cortisol less than 20 µg/dl after hypoglycemia, in 20% (four of 20) of patients (Table 4); ACTH levels in these patients were also low (<20 pg/ml).
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> 120 ng/ml (5.5 nmol/liter)]. Three additional patients (two males, one female) had normal basal PRL but increased response to TRH [ > 120 ng/ml (5.5 nmol/liter)]. Galactorrhea was not observed in any patient, and none of these patient reported taking medications that could influence PRL secretion. No patient was diagnosed with clinical hypothyroidism; however, four patients (20%) had low-normal level of free T4 with normal TSH and no exaggerated TSH response to TRH. Examination of the eight WFS1 exons in our 17 families revealed three families (23.5%) with two different putative WFS1 mutations. At least one affected individual from the 14 families without WFS1 alterations were screened for potential mtDNA mutations, but none were identified. Subsequently, eight of the 14 families without WFS1 alterations were tested for linkage to chromosome 4p, and five of the eight families showed evidence consistent with linkage to the WFS1 locus (our unpublished data).
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Discussion |
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In the present study, we were not able to estimate accurately the prevalence of WFS in the Lebanese population because of the lack of population data, but in comparison with juvenile-onset autoimmune (type 1) diabetes mellitus, WFS seems to be much more prevalent in the Lebanese than in other populations (3, 11). In our opinion, this is explained mostly by the high frequency of consanguineous marriages over many generations; a racial predisposition cannot be excluded but is made less probable because affected families belonged to different Lebanese communities that have different racial origins.
With respect to family studies, another element that deserves notice is the high number of deaths of unknown etiology during infancy and childhood that we have documented. This may be due in many cases to undiagnosed acute complications of insulin-deficient diabetes mellitus, particularly diabetic ketoacidosis, and points out the importance of an early diagnosis in relatives of an index patient by providing adequate education to the parents.
Increased health risk, especially of hearing loss and diabetes (nonautoimmune insulin-dependent and noninsulin-dependent), has been reported in carriers of WFS1 gene abnormalities (12). Although our study was not aimed at estimating such risk, it is worthy to note that in our families, two cases of maternal deafness were detected by direct questioning and confirmed by audiograms, two cases of insulin-dependent nonautoimmune diabetes diagnosed in first-degree relatives of our patients, and a history of noninsulin-dependent type 2 diabetes in second-degree relatives in three families.
Death occurs prematurely in WFS: in one report 60% of the patients affected by this syndrome died by age 35 yr. The main causes of mortality reported in the literature are the neurological manifestations, especially respiratory failure due to brain stem atrophy and the complications of urinary tract atony (13). All our patients were alive at the time of the writing of this paper; however, many of them were already very debilitated and required frequent hospitalizations.
Results concerning the incidence of diabetic microvascular complications in WFS have been controversial (3, 14, 15, 16); in contrast to what was previously reported by some authors, our study does not support a lack of such complications in WFS. In fact, the overall incidence of retinopathy, neuropathy, and nephropathy in our study is not lower than the incidence usually described in autoimmune type 1 diabetes (17).
Few data are found in the literature about heart malformations in this syndrome. Indeed, cardiovascular autonomic neuropathy is a well-known complication of long-lasting diabetes, and signs consistent with this type of neuropathy (resting tachycardia, orthostatic hypotension) were detected in some of our patients; however, five of the patients (16.1%) also had valvular heart disease, particularly pulmonary stenosis, and one had a ventricular septal defect. This percentage of cardiac abnormalities is significantly higher than the one reported in the general population (P < 0.001) (18). One Turkish family with WFS was previously described in which some affected siblings had ventricular septal defects (19); thus, we believe that heart malformations are new features that may be present in association with this syndrome. The pathogenesis of such malformations is not known, but their presence in certain WFS families emphasizes the clinical heterogeneity of the disease and may help to better understand the function of WFS gene(s) product(s).
Limited joint motility was detected in some of our patients and has been previously described by other authors (20). Gastrointestinal dysmotility may be due to autonomic diabetic neuropathy or may represent an integral part of WFS (3) and probably plays a role in the morbidity or mortality of the disease by increasing the risk of food aspiration.
Urinary tract dilation and abnormal urodynamics were found in a high proportion of our patients. This finding is concordant with the literature in which urologic abnormalities are reported as frequent complications of WFS (3, 21, 22). These complications have been mainly attributed to the high output urine state resulting from DI; however, the presence of urologic manifestations in three of our patients who had no evidence of DI as well as the finding of a hyperreflexic and not atonic bladder in some patients confirm the contribution of diabetic neuropathy in the pathogenesis of urologic disturbances. The possibility of a primary bladder dysfunction in WFS has also been suggested (21).
Neurological and psychiatric abnormalities are well-known components of WFS (3, 23, 24, 25, 26); they were detected in a high percentage of our patients (70.9%), and their frequency and severity were positively correlated with the patients age. The most common neurological feature in our series was the absence of the nauseal reflex. This feature has not been reported previously and is therefore a new finding that may predispose to pulmonary aspiration and play a role in respiratory complications of WFS. The other neurological signs were similar to those described in the literature and reflect underlying brain atrophy, which was confirmed by MRI imaging. In fact, moderate atrophy, especially of cerebellum and brain stem, was found on MRI even in patients who had no clinical evidence of neurological problems at the time of the study.
Psychiatric illness was diagnosed in 22.6% of patients. This proportion is higher than the one in Barrett’s series (3); other authors, however, have reported even higher frequencies (25). Also, many studies found an increased risk of psychiatric disease in WFS carriers (27, 28, 29, 30, 31, 32).Only two cases of confirmed psychiatric disease were detected in first-degree relatives of our patients; however, no conclusion could by drawn from this study because psychological tests were not performed and on direct questioning people are often reluctant to admit such problems.
Male hypogonadism due to primary gonadal failure is a well-known feature of WFS (3, 33). Primary hypogonadism was detected in four (22.2%) of our male patients. Ovarian failure has not been reported previously and was not detected in any of our patients. The reason for testicular and not ovarian involvement is not elucidated but is probably related to expression/function of the gene(s), which are abnormal in WFS.
Very few data are available concerning pituitary function in patients with WFS. In one report two girls were found to have short stature and GH deficiency (34), but according to our knowledge, no pituitary exploration was performed in any other study. The high incidence of alteration in one or more pituitary secretions in our patients is not surprising considering the neurodegenerative nature of Wolfram disease, the level of the dysfunction (whether hypothalamic or hypophysial) is difficult to determine with certainty; however, the exaggerated PRL response to TRH and the presence of symptoms, which are usually associated with hypothalamic injury such as temperature dysregulation and cold intolerance (see Table 2
), are in favor of a hypothalamic origin.
Hypothalamo-hypophysial dysfunction is correlated with older age of the patients and the presence of neurological complications and MRI abnormalities, particularly brain stem atrophy and dilatation of the third ventricle. Defective GH secretion is the most frequent alteration documented in our patients; later onset of GH deficiency may explain the absence of severe short stature in these patients. Whether GH supplementation is beneficial in such patients remains to be determined. Corticotropic deficiency is also found in a significant proportion of our subjects. In our opinion, the awareness that this deficiency could be associated with WFS is of vital importance.
Genetic studies of WFS have been performed by several groups of researchers, and the results have provided evidence of genetic heterogeneity (35, 36, 37). Defects in mtDNA have been detected in some studies but were absent in others as it was the case in our series (3, 5, 6, 7, 38). Linkage of WFS to markers on chromosome 4p has been reported during the last decade (39). More recently a candidate gene (WFS1) was mapped to the 4p16.1 and isolated (40). In addition, mutations in this gene have been identified in patients affected with Wolfram disease (4, 41, 42, 43, 44). WFS1 encodes a transmembrane protein suggested to play a role in the survival of certain populations of cells, particularly neuronal and endocrine cells (4, 45). We identified two putative WFS1 mutations in three of 17 of our families; however, noncoding regulatory mutations cannot be ruled out in which linkage to 4p16.1 was determined in five of eight of the families in which WFS1 coding mutations were not identified. As previously mentioned, three of our eight families showed evidence that was inconsistent with linkage to the WFS1 locus, thus suggesting the presence of a second WFS gene.
We were not able to find any distinguishing feature in the individuals in whom WFS1 mutations were detected and those patients without WFS1 mutations, but all of them were characterized by the presence of the four components of the disease (DIDMOAD) and by a more severe evolution during follow-up.
In conclusion, our study is one of the largest in the literature concerning Wolfram syndrome. It provides more information on the clinical manifestations of the disease that may permit an earlier diagnosis and help to prevent some of the complications. The recognition of new clinical features not reported in previous descriptions, especially pituitary dysfunction, certain neurological manifestations, and heart abnormalities, may reduce the morbidity and mortality of WFS. In addition, this study allows a better understanding of the genetics of the disease and emphasizes its phenotypic and genotypic heterogeneity. All this information will open new horizons to future genetic and molecular investigations that may lead to a better elucidation of the pathophysiology of WFS and hopefully to improvement in means of prevention and treatment of this devastating disease.
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Acknowledgments |
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Footnotes |
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Received December 30, 2002.
Accepted January 15, 2004.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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