High Frequency of X Chromosome Abnormalities in Women With Short Stature and Elevated Liver Enzymes

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1 JCEM ONLINE Advances in Genetics Endocrine Research High Frequency of X Chromosome Abnormalities in Women With Short Stature and Elevated Liver Enzymes Dominique Roulot, Valérie Malan, Marianne Ziol, Agnès Linglart, Valérie Bourcier, Michel Beaugrand, and Brigitte Benzacken Unité d Hépatologie (D.R.), Groupe Hospitalier Paris-Seine-St Denis, Hôpital Avicenne, Assistance Publique-Hôpitaux de Paris, Bobigny, France; Service d Hépato-Gastroentérologie (V.B., M.B.), Service d Anatomo-Pathologie (M.Z.), and Service d Histologie-Embryologie et Cytogénétique, Biologie de la Reproduction (B.B.), Groupe Hospitalier Paris-Seine-St Denis, Hôpital Jean Verdier, Assistance Publique-Hôpitaux de Paris, Bondy, France; Université Paris 13, Sorbonne Paris Cité (D.R., M.Z., M.B., B.B.), Bobigny, France; Département de Génétique (V.M.), Université Paris Descartes, INSERM Unité 781, Hopital Necker-Enfants Malades, Assistance Publique-Hôpitaux de Paris, Paris, France; Service d Endocrinologie et Diabétologie de l Enfant (A.L.), Groupe Hospitalier Paris Sud, Hôpital Bicêtre, Assistance Publique-Hôpitaux de Paris, and Université Paris Sud, INSERM Unité 936, Le Kremlin-Bicêtre, France Context: Paucisymptomatic forms of Turner s syndrome (TS), in which short stature is the predominant clinical abnormality, remain underdiagnosed. Abnormal liver tests are extremely frequent in adult TS patients reflecting various types of hepatic lesions. Objective: The objective of the study was to investigate whether unexplained elevated liver enzymes in women with short stature could reveal X chromosome abnormalities of undiagnosed TS. Design and Participants: Thirty-one consecutive short stature women displaying elevated liver enzymes and no previous diagnosis of TS were compared with 31 age-matched controls in a prospective study. Liver biopsy was performed in 26 patients. Main Outcome Measures: Systematic karyotype analysis and fluorescence in situ hybridization. Results: X chromosome abnormalities were found in 27 patients and one control (87.0% vs 3.2%, P.0001), including a 45,X/46,XX mosaicism in 24 patients and isochromosome of the long arm in three. Liver histological analysis showed architectural changes in 17 patients with nodular regenerative hyperplasia in 12. Biliary lesions were present in 13 patients and liver steatosis in 20. Conclusions: X chromosome abnormalities indicative of cryptic TS are extremely frequent in shortstature women with unexplained elevated liver enzymes. In short-stature women, abnormal liver tests should lead to systematic karyotype analysis. (J Clin Endocrinol Metab 99: E1592 E1596, 2014) ISSN Print X ISSN Online Printed in U.S.A. Copyright 2014 by the Endocrine Society Received January 6, Accepted April 3, First Published Online April 14, 2014 Turner s syndrome (TS) is a genetic disorder due to the complete or partial lack or to structural abnormalities of an X chromosome. It is one of the most frequent sex chromosome disorders, affecting approximately 1 in live female births (1). The most common features of TS are short stature and ovarian dysgenesia, although several associated pathological conditions affecting various organs have been described (2). When growth retardation and ovarian insufficiency are present, the diagnosis of TS is easily suspected based on clinical examination and confirmed by karyotype analysis. However, TS often remains undiagnosed, especially when short stature is the only clinical abnormality. Abbreviations: ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; BMI, body mass index; GGT, -glutamyl transferase; NRH, nodular regenerative hyperplasia; RH, regenerative hyperplasia; TS, Turner s syndrome;, times the upper limit of normal. E1592 jcem.endojournals.org J Clin Endocrinol Metab, August 2014, 99(8):E1592 E1596 doi: /jc

2 doi: /jc jcem.endojournals.org E1593 Liver involvement is frequent in patients with TS. Abnormal liver enzymes were reported in up to 80% of the patients over the age of 35 years (3) and may reflect hepatic vascular disorders, fatty liver disease, and biliary involvement (4). The pathophysiological mechanisms of these lesions remain obscure and the increase in liver enzymes may precede TS diagnosis. Because both short stature and abnormal liver tests can be the only phenotypical expression of TS, the association of these two features might be predictive of X chromosome abnormalities. In this study we prospectively investigated the frequency of X chromosome abnormalities in short stature women with unexplained elevated liver enzymes and analyzed the associated liver pathological changes. Patients and Methods Fifty consecutive female patients below or equal to 155 cm in height (or 160 cm in case of discordance with sibling height 10 cm), referred to our department over a time period of 2 years for persistent liver test abnormalities (more than 6 months), and short stature controls matched for age, were investigated as described in Supplemental Patients and Methods. This section also contains the description of the karyotype study, the histological data, and the statistical analysis. None of the patients were previously diagnosed with TS. Results Characteristics of patients Clinical and biochemical characteristics of the patients are listed in Table 1. Patient mean age was years vs years in the control group. Patient mean stature was cm ( and SD score relative to general and TS populations, respectively) vs cm for the control group ( and SD score relative to general and TS populations, respectively) (Supplemental Figure 1). Pa- Table 1. Clinical, Biological Data and Karyotypes in Women With Short Stature and Elevated Liver Enzymes Patient Number Age, y Height, cm BMI, kg/m 2 Associated Diseases AST, ALT, AP, GGT, Karyotype D, AH, DL ,X,i(X)(q10) a AH N mos 45,X[9]/47,XXX[5]/46,XX[86] b HT N 4.0 mos 45,X[7]/46,XX[93] Absence N N mos 45,X[11]/47,XXX[2]/46,XX[87] DL N N N 1.8 mos 45,X[11]/46,XX[89] D, AH mos 45,X[26]/46,XX[74] AH, DL mos 45,X[11]/47,XXX[5]/46,XX[84] AH N mos 45,X[8]/47,XXX[2]/46,XX[90] AH, DL N 9.5 mos 45,X[8]/47,XXX[4]/46,XX[88] AH, DL N N N 1.2 mos 45,X[20]/47,XXX[6]/46,XX[74] AH, DL N N mos 45,X[10]/47,XXX[5]/46,XX[85] AH, DL N 2.0 mos 45,X[15]/46,XX[85] DL, HT N 3.0 mos 45,X[18]/47,XXX[2]/46,XX[80] AH, DL, HT mos 45,X[10]/46,XX[90] AH, DL N N mos 45,X[15]/46,XX[85] AH 1.2 N N 3.7 mos 45,X[7]/47,XXX[1]/46,XX[92] DL mos 45,X[9]/46,XX[91] Absence N N N 3.0 mos 45,X[8]/47,XXX[1]/46,XX[91] DL mos 45,X[7]/47,XXX[1]/46,XX[92] HT N 4.0 mos 45,X[2]/46,X,i(X)(q10)[98] a AH mos 45,X[7]/46,XX[93] D, AH, HT mos 45,X[82]/ 46X,r(X)[9]/46,XX[9] c AH N N mos 45,X[16]/47,XXX[4]/46,XX[80] AH ,X,i(X)(q10) a DL N mos 45,X[11]/46,XX[89] Absence N 2.8 mos 45,X[28]/46,XX[72] D, DL N N mos 45,X[8]/47,XXX[3]/46,XX[89] Absence N N N ,XX Absence ,XX Absence ,XX AH N ,XX Abbreviations: AH, arterial hypertension; D, diabetes; DL, dyslipidemia; HT, Hashimoto thyroiditis; N, normal. Mean values SD for all patients included the following: age, years; height, cm; BMI, kg/m 2 ; AST, ULN; ALT, ULN; GGT, ULN; AP ULN. a Xi(X)(q10), isochromosome for the X long arm. b mos 45,X [9], mosaïcism with 45,X in 9% and 47,XXX in 5% of the tested cells. c 46,r(X)[9]: ring chromosome X in 9% of the cells.

3 E1594 Roulot et al Short Stature, X Chromosome, Liver Involvement J Clin Endocrinol Metab, August 2014, 99(8):E1592 E1596 tient mean body mass index (BMI) was kg/m 2 ; 25 of 31 (81%) were overweight (BMI 25 kg/m 2 ). None of the patients showed dysmorphic syndrome or associated malformations, except one patient displaying congenital deafness (number 22). Fourteen patients had no pregnancy, primary amenorrhea being documented in eight of these. Two other patients reported miscarriage during their only pregnancy. Several associated diseases, mainly cardiovascular, were found in the patients group. Arterial hypertension was present in 17 patients (54.8%), with documented early onset (before 20 y) in three patients. Coronary atherosclerosis was reported in six patients (13%). Previous echocardiographic examination had revealed vascular abnormalities and/or cardiac valvulopathy in six patients: dilation of ascending aorta in two cases, associated with aortic insufficiency in one case; mitral insufficiency in four patients, associated with coronary aneurysma in two cases. Type 2 diabetes was present in four patients (12%) and dyslipidemia in 13 (42%). Autoimmune diseases were observed in six patients (19%), including five cases of Hashimoto thyroiditis and one case of systemic lupus erythematosus. In comparison, only two subjects in the control group were treated for arterial hypertension and one for type 2 diabetes. Serum bilirubin levels were normal in all patients. The median value of aspartate aminotransferase (AST) was times the upper limit of normal (), that of alanine aminotransferase (ALT) was , that of -glutamyl transferase (GGT) was and that of alkaline phosphatase (AP) was Moderate elevation of serum AP and GGT, associated with mild to moderate increase in AST and/or ALT, was found in 14 patients. Four patients had increased serum levels of AP and GGT with normal aminotransferases, nine patients had increased serum aminotransferases and/or GGT with normal serum AP levels and four patients had an isolated rise of GGT values. Hypergammaglobulinemia was present in one patient (number 19), who showed portal hypertension, documented by the presence of esophageal varices. Antinuclear antibodies were found in two patients (number 24 and number 28), and antithyroperoxidase were present in five patients (patients 3, 13, 14, 20, and 22). None of these five patients had hypothyroidism at the time of the study. Abdominal ultrasound echography showed hyperechoic appearance of the liver in 13 cases and the presence of typical hemangiomas in four. One patient (number 19) exhibited features of portal hypertension associated with dysmorphic liver and enlarged spleen. Karyotype analysis Karyotype abnormalities were found in 27 of the 31 patients (87%), including X chromosome monosomy or structural abnormalities of chromosome X, all known to be associated with TS (Table 1). A mosaïcism, mos 45,X/ 46,XX (Supplemental Figure 2) was found in 24 patients (89% of all chromosomal abnormalities): 14 of these 24 patients displayed a more complex form of mosaïcism: mos 45,X/47,XXX /46,XX; apart from one patient showing only 9% of 46,XX cells, 23 patients exhibited more than 72% of 46,XX cells. Structural abnormalities of the short arm of the X chromosome were observed in four patients. The karyotype of three patients (patients 1, 20, and 24) (Supplemental Figure 2) revealed an isochromosome of the long arm of the X chromosome 46,X,i(X)(q10); in one case the isochromosome was associated with a mosaïcism. One patient (number 22) showed a ring chromosome X in 9% of analyzed cells, associated with a mosaïcism: mos 45,X[82]/46,r(X)[9]/46,XX[9]. In contrast, only one of the 31 healthy controls (3.2%) displayed abnormal X chromosome with a mosaïcism of 45,X and 47,XXX affecting, respectively, 8% and 2% of the examined cells (mos 45,X[8]/47,XXX[2]/46,XX[90]). Liver histopathological analysis A liver biopsy was performed in 26 patients including two with a normal karyotype. For the 24 patients with chromosomal abnormalities, histopathological data are summarized in Table 2. Liver architectural changes (Supplemental Figure 3) were observed in 17 patients (65%), including nodular regenerative hyperplasia (NRH) in 12 and regenerative hyperplasia (RH) in five. Biliary involvement was present in 13 patients (50%): eight patients displayed slight cholangitis, one a marked form of cholangitis and one ductopenia. Isolated cholangiolar proliferation was observed in three patients. Nine patients had portal vasculature abnormalities consisting of aberrant vessels adjacent to portal tracts. Macrovesicular steatosis was observed in 20 patients (75%). Steatosis was mild in 11 cases, moderate in four, and severe in five. Steatohepatitis was observed in only one patient (number 6). In the two patients without chromosome X abnormalities (number 29 and number 30), no histopathological lesions were observed, except isolated steatosis in patient number 29, likely associated with her increased BMI (40 kg/m 2 ). Discussion We report an unexpectedly high frequency of chromosome X abnormalities (87%) in women with short stature

4 doi: /jc jcem.endojournals.org E1595 Table 2. Liver Histological Findings in Women With Short Stature and Elevated Liver Enzymes Patient Number Architectural Changes Perisinusoidal Fibrosis Portal Fibrosis (Metavir) Portal Inflammation Vascular Abnormalities Steatosis, % Cholangitis Cholangiolar Proliferation 1 NRH Absent 1 Absent Moderate 1 10 Slight Present 2 NRH Absent 0 Absent Absent 1 10 Absent Absent 3 NRH Absent 1 Absent Absent 0 Absent Present 4 NRH Absent 1 Slight Absent 1 10 Slight Present 5 RH Absent 0 Slight Absent Slight Present 6 Absent Present 1 Slight Absent Absent Absent 7 NRH Absent 1 Absent Marked 0 Slight Present 8 RH Absent 1 Marked Marked 0 Slight Present 9 NRH Absent 0 Slight Absent Absent Present 11 NRH Absent 0 Absent Moderate 1 10 Absent Absent 13 Absent Present 1 Absent Marked Slight Absent 14 RH Absent 1 Slight Moderate 1 10 Slight Absent 16 NRH Present 0 Absent Absent 0 Absent Absent 17 RH Absent 0 Absent Absent 1 10 Absent Absent 18 RH Absent 0 Absent Moderate 0 Absent Absent 19 NRH Absent 0 Absent Absent 1 10 Absent Absent 20 Absent Present 1 Absent Absent Absent Absent 21 Absent Absent 1 Slight Absent Slight Absent 22 Absent Absent 2 Slight Moderate Absent Absent 23 NRH Absent 1 Slight Absent 0 Absent Present 24 NRH Absent 2 Absent Moderate 1 10 Ductopenia Absent 25 NRH Absent 1 Slight Moderate 1 10 Absent Absent 26 Absent Present 1 Absent Absent 60 Absent Absent 27 Absent Absent 1 Absent Absent 1 10 Marked Absent 29 Absent Present 1 Absent Absent Absent Absent 30 Absent Present 0 Absent Absent 1 10 Absent Present Data are on 26 patients undergoing liver biopsies of 31 patients with elevated liver enzymes. and unexplained elevated liver enzymes. Chromosomal abnormalities consisted of both X monosomy (either complete or mosaïcism) and X structural rearrangements, mosaïcism being the most frequently observed. A correlation of X chromosome loss and increasing age has been reported in the general population (5). Accordingly, the expected rate of X monosomy should have been approximately 2.8% in both patient and control groups of our study (ie, age range y). Although the rate of monosomy in the control group (3.2%) is close to the expected value, the 77% rate of X monosomy in the patient group (24 of 31 patients) is much higher than expected. Features consistent with TS were present in some of our patients. Approximately 30% of women with X chromosome abnormalities reported primary amenorrhea; 45% had no pregnancies or reported miscarriages. Arterial hypertension was particularly frequent in the patient group (54.8%). Atherosclerosis was diagnosed in 13% of the patients, aortic dilation was present in two cases and valvulopathy in four among the 16 individuals undergoing echocardiography. Arterial hypertension and cardiovascular abnormalities, including valvulopathies, have been reported with high frequency in TS (6). Type 2 diabetes mellitus and hypothyroidism of autoimmune origin, which are also common among TS women (7, 8), were present in nine patients. Overall, nearly all of the 27 patients with X chromosome abnormalities showed at least one clinical symptom that can be observed in TS and might represent minor phenotypic forms of TS. The severity of karyotypic changes and clinical features are correlated in TS patients (9). Interestingly, most of our patients displayed mosaicism with a 46,XX contingent, likely explaining why they were taller, on average, than in classical TS: 152 cm vs 143 cm average height of TS patients diagnosed during childhood in France (10). All liver biopsies obtained for 24 patients showed histological lesions associating or not steatosis in 75% of the cases, liver architectural changes in 65%, and biliary lesions in 50%. Although less severe on average, these lesions are representative of those described in women with early-diagnosed TS (4). Not surprisingly, in the previous study, karyotypes mainly consisted of complete X monosomy. Mechanisms of hepatic lesions associated with X chromosome abnormalities are unknown except for steatosis. BMI values superior to 25 kg/m 2 and insulin resistance syndrome, a common cause of hepatic steatosis, were frequently reported in TS patients (11). Biliary lesions in our patients could have an autoimmune origin. A higher frequency of X chromosome loss was reported in women with primary biliary cirrhosis, compared with controls matched for age (12). Autoimmunity is more frequent in TS patients than in normal females (13). Interestingly, five patients of our study were treated for Hashimoto thyroiditis. Finally, 65% of the patients of the present study displayed liver architectural changes, including NRH in 12 patients, and RH in five. NRH or RH probably reflects primary liver vascular involvement because it is believed that these lesions correspond to adaptive changes caused

5 E1596 Roulot et al Short Stature, X Chromosome, Liver Involvement J Clin Endocrinol Metab, August 2014, 99(8):E1592 E1596 by heterogeneous distribution of the intrahepatic blood flow (14). Liver involvement in TS patients necessitates appropriate management and follow-up. Indeed, although liver involvement has long been considered as benign, a 6-fold increase of the relative risk of cirrhosis was reported in TS patients compared with the general population (15). Specific treatment may be required to prevent progression of hepatic lesions. Ursodeoxycholic acid therapy is beneficial in TS patients with biliary lesions and in case of portal hypertension, long-term -blocker treatment is required (16). Finally, hormone replacement therapy improves liver function (17, 18) in addition to its beneficial effects on the overall quality of life of these patients. In conclusion, in women with short stature and elevated liver enzymes of unknown origin, chromosome X abnormalities are highly frequent (87% in our series). Although the diagnosis of TS should be systematically suspected in women with short stature, this is far from being common practice (19). Our study confirms that TS is likely more prevalent and variable in presentation than previously suspected. Based on our findings, all short stature women with unexplained elevated liver enzymes should undergo a karyotype examination as a diagnostic procedure. Acknowledgments Address all correspondence and requests for reprints to: Professor Dominique Roulot, Unité d Hépatologie, Hôpital Avicenne, Bobigny Cedex, France. dominique.roulot@avc.aphp.fr. Disclosure Summary: The authors have nothing to declare. References 1. Saenger P. Turner s syndrome. Curr Ther Endocrinol Metab. 1997; 6: Ranke MB, Saenger P. Turner s syndrome. Lancet. 2001;358: Sylven L, Hagenfeldt K, Brondum-Nielsen K, von Schoultz B. Middle-aged women with Turner s syndrome. Medical status, hormonal treatment and social life. Acta Endocrinol. 1991;125: Roulot D, Degott C, Chazouilleres O, et al. Vascular involvement of the liver in Turner s syndrome. Hepatology. 2004;39: Guttenbach M, Schakowski R, Schmid M. Aneuploidy and aging: sex chromosome exclusion into micronuclei. Hum Genet. 1994;94: Ho VB, Bakalov VK, Cooley M, et al. Major vascular anomalies in Turner syndrome: prevalence and magnetic resonance angiographic features. Circulation. 2004;110: Nielsen J, Johansen K, Yde H. The frequency of diabetes mellitus in patients with Turner s syndrome and pure gonadal dysgenesis. Blood glucose, plasma insulin and growth hormone level during an oral glucose tolerance test. Acta Endocrinol. 1969;62: El-Mansoury M, Bryman I, Berntorp K, Hanson C, Wilhelmsen L, Landin-Wilhelmsen K. Hypothyroidism is common in Turner syndrome: results of a five-year follow-up. J Clin Endocrinol Metab. 2005;90: Sybert VP, McCauley E. Turner s syndrome. N Engl J Med. 2004; 351: Sempe M. Growth curves in untreated female gonadal dysgenesis. French reference data from birth to age 22 years. Ann Pediatr. 1997; 44: Bakalov VK, Cooley MM, Quon MJ, et al. Impaired insulin secretion in the Turner metabolic syndrome. J Clin Endocrinol Metab. 2004;89: Invernizzi P, Miozzo M, Battezzati PM, et al. Frequency of monosomy X in women with primary biliary cirrhosis. Lancet. 2004;363: Elsheikh M, Dunger DB, Conway GS, Wass JA. Turner s syndrome in adulthood. Endocr Rev. 2002;23: Wanless IR, Godwin TA, Allen F, Feder A. Nodular regenerative hyperplasia of the liver in hematologic disorders: a possible response to obliterative portal venopathy. A morphometric study of nine cases with a hypothesis on the pathogenesis. Medicine. 1980;59: Gravholt CH, Juul S, Naeraa RW, Hansen J. Morbidity in Turner syndrome. J Clin Epidemiol. 1998;51: Roulot D. Liver involvement in Turner syndrome. Liver Int. 2013; 33: Elsheikh M, Hodgson HJ, Wass JA, Conway GS. Hormone replacement therapy may improve hepatic function in women with Turner s syndrome. Clin Endocrinol (Oxf). 2001;55: Koulouri O, Ostberg J, Conway GS. Liver dysfunction in Turner s syndrome: prevalence, natural history and effect of exogenous oestrogen. Clin Endocrinol (Oxf). 2008;69: Stochholm K, Juul S, Juel K, Naeraa RW, Gravholt CH. Prevalence, incidence, diagnostic delay, and mortality in Turner syndrome. J Clin Endocrinol Metab. 2006;91: