Received: March 6, 2018
Accepted: April 16, 2018
Mediterr J Hematol Infect Dis 2018, 10(1): e2018031 DOI 10.4084/MJHID.2018.031
This article is available on PDF format at:
| This is an Open Access article distributed
under the terms of the Creative Commons Attribution License
(https://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
β-thalassemia major (β-TM)
is among the most common hereditary disorders imposing high expenses on
health-care system worldwide. The patient's survival is dependent on
lifetime blood transfusion which leads to iron overload and its
toxicity in various organs including endocrine glands. This article
provides an overview of endocrine disorders in beta-TM patients. This
single center investigation enrolled 28 β-TM
patients (16 males, 12 females) regularly transfused with packed red
cell since early years of life. For each patient were determined: age,
sex, number of transfusions received, history of splenectomy and
anthropometric parameters. Evaluation of hormonal status including
growth, gonadal, thyroid, adrenal cortex, and parathyroid glands was
done for all patients. Dual-energy X-ray absorptiometry was used to
diagnose low bone mass. Assessment of iron overload status was
performed by measuring the serum ferritin concentration and the results
of magnetic resonance imaging T2*.
Growth retardation was found in 16 of the 28 studied patients
(57%).Thirteen among them had delayed puberty. Spontaneous puberty was
achieved in 16 cases. Growth hormone (GH) deficiency was found in 10
cases (35%). Seventeen among the studied patients (60%) developed
disorders of glucose homeostasis. Subclinical hypothyroidism was found
in six patients (21%). Intensive chelation therapy had allowed the
reversibility of this complication in five cases. Adrenal Insufficiency
was observed in 9 cases (32%). Hypoparathyroidism has occurred in one
case. Ten of the 28 studied patients had low bone mass (35%).
Twenty-three of the 28 studied patients (82%) had at least one
Patients and Methods
For each patient were specified demographic and clinical data (family history, age, sex, origin, consanguinity, age at diagnosis, age at the first blood transfusion, anthropometric parameters); transfusion requirements and complications related to secondary hemochromatosis; chelating therapy (date of onset, type of chelation, modalities).
The size was taken using the DETECTO metal gauge. The target size was calculated as the average of the parents' heights plus 6.5 cm for boys or minus 6.5 cm for girls. Adult height was considered to be attained when growth during the preceding year was less than 1 cm, with a bone age of over 15 years. Pubertal stages were assessed according to Tanner and Marshall. Arrested puberty is characterized by a lack of pubertal progression over a year or more. Short stature is defined as height less than two standard deviations (SDs) below the mean for age and gender. Body mass index (BMI) was calculated as weight (kg) divided by the square of the height (m2) using reference charts for boys and girls. Blood glucose was determined using the glucose oxidase method on a Beckman Glucose Analyzer. Carbohydrate metabolism disorders were assessed according to the American Diabetes Association (ADA).
All patients underwent hormonal evaluation testing including somatotropic, gonadotropic, corticotropic, thyrotropic and parathyroid glands. Evaluation of the GH/IGF-1 axis was performed by GH stimulation tests as well as the insulin-like growth factor (IGF-1) and insulin-like growth factor-binding-protein (IGFBP) concentrations compared to norms for age and sex. The diagnosis of GH deficiency was established on an insufficient peak (less than 20 mIU /L) in response to two separate pharmacological stimuli (insulin tolerance and glucagon-propranolol tests). Subjects were arbitrarily classified according to GH peak in partial GH deficiency (GH peak between 10 and 20 mIU /L) and a total deficit if the values are less than 10 mIU/L. All subjects underwent a basal cortisolemia and after intramuscular injection of adrenocorticotropic hormone (Synacthen® test: 250 μg). An abnormal response (a serum cortisol peak below 550 nmol/L or an increment of less than 200 nmol/L from baseline or both) identifies adrenal insufficiency. Thyroid function was assessed by measuring free thyroxine (FT4) and thyrotrophic hormone (TSH). Subclinical hypothyroidism is defined as a combination of high TSH (≥ 5 mIU/L) with normal FT4 levels.
Skeletal age was evaluated according to Greulich-Pyle atlas. Bone mineral density (BMD) was performed by Dual-energy x-ray absorptiometry (DXA) on L1-L4 lumbar spine and total hips. Low bone mass was defined as Z-score values of –2.0 SDs or lower.
Iron overload was assessed using the mean serum ferritin levels and the MRI T2*. Cardiac and liver T2* were assessed by a validated technique based on MRI relaxometry at 1.5 T. For the heart T2* images, all patients underwent a single breath hold multiecho bright blood sequence with variable echo times (TEs). For the liver, a single axial slice was obtained in the center of the organ using a multiecho sequence, and a single breath hold was used to obtain images with the same parameters. Excel spreadsheet was used for image analysis and measurement of T2*. Images were imported into a software for the region of interest (ROI) drawing. For the heart, signal intensity was obtained using an ROI drawn through the full thickness of the septum wall of the myocardial short axis image. For the liver, the signal intensity was also provided using an ROI covering the right lobe of the liver parenchyma and avoiding major vessels. The same ROI was copied across all images for each organ. Each image generated the values of both signal intensity (SI) and TEs which were manually inputted into an Excel spreadsheet. The mean signal intensity in each slice with varying TEs was used to fit the T2* curve using the formula SI = Ke–TE/T2* in the spreadsheet. A curve-fitting truncation model consisting of a monoexponential decay curve with a linear fit was applied. Excel was applied as previously described. Myocardial ion concentration (MIC) was evacuated using Carpenter curves. Liver iron concentration (LIC) was calculated using Hankins curves. Values of cardiac T2* (CT2*) <20 ms were considered to indicate cardiac siderosis which was classified as moderate (10 ms< CT2* <20 ms) and severe (CT2* <10 ms). LIC >3 mg/g dry weight (dw) was considered to indicate liver siderosis which was classified on mild (3 < LIC <7 mg/g dw). Moderate (7< LIC <15 mg/g dw) and severe (LIC >15 mg/g dw). Serum ferritin (SF) concentration was measured every 3 months using standard enzyme immunoassay. The 12-month mean SF value was considered.
The iron chelating treatments used were subcutaneous deferoxamine (Desferal®), administered in two repeated doses (40 mg/kg/day) 5-days-per-week, and oral chelators namely deferasirox in a single dose (20 - 40 mg/kg/day) and deferiprone in 3 daily taken (75 - 100 mg/kg/day). The combined treatment consisted of combining deferoxamine with oral iron chelation. A group of 13 healthy subjects was used as control.
Written informed consent was obtained from the patients or their parents.
Statistical analysis: All statistical procedures were performed using SPSS version 18.0. Results are presented in mean ± SDs. Pearson correlation analysis and unpaired T student`s test were used. P value < 0.05 was considered statistically significant.
Growth: sixteen (57%) of the studied patients had growth velocity standard deviation score less than -2SDs. Among them, thirteen had a pubertal delay. Bone maturation delay was present in all cases. Bone age delay and chronological age were over one year in all patients. In the absence of growth hormone deficiency, height changes with sex and age are illustrated in figure 1. Curves show that years, most children have a normal growth pattern up to the age of 9 years, and a reduced or absent height gain during puberty, which is more marked in boys than in girls. Circulating IGF-1 levels were significantly lower than controls (p = 0.00) (Figure 2).
|Figure 1. Mean changes in height for sex and age.|
|Figure 2. Distribution of IGF-I values by age and sex.|
Somatotropic function: GH provocation tests showed an average peak GH levels in 18 patients (40.22 ± 21.7 m IU/ L; range (21₋129). Ten patients (35%) had GH deficiency, among them a partial GH deficiency was found in 5 cases (16.14 mIU /L ± 2.87; range 3.7₋ 6.7) and a severe GH deficiency was demonstrated in 5 other cases (5.16 mIU / L ± 1.35; range 13.7 ₋ 19.7). Growth assessment for patients with complete GH deficiency showed that of the 3 patients who had attained the adult age the parental target size was reached in only one case. In patients with partial GH deficiency, statural growth was normal in 3 cases. Only one patient had a complete GH deficiency in contrast to normal IGF-1 level. Risk factors, probably related to the occurrence of GH deficiency, are: a history of splenectomy (p = 0.000), association with adrenal insufficiency (p = 0.042), low bone mass (p = 0.037). The levels of IGF-1 and IGFBP were significantly lower in patients with GH deficiency than those without GH deficiency (p: 0.008 and 0.037 respectively).
Puberty: spontaneous onset of puberty was obtained in 16 cases (9 boys and 7 girls) at a mean age of 15 years for boys (range 14-16 years) and 13 years for girls (range 11-15 years). Adult height was reached in 7 cases (5 boys and 2 girls) at a mean age of 20 years for boys (range 17-22 years) and 17 years for girls. Twelve of the studied patients (42%) had a delayed puberty and hypogonadism requiring lifelong hormone replacement therapy. Lack of pubertal progression was observed in 4 cases (2 girls and 2 boys), the absence of the onset of pubertal development in 6 cases (5 boys and one girl) and a primary amenorrhea in two other cases. Factors associated with pubertal disorders are transfusion requirements before chelating therapy (p = 0.042) and myocardial iron assessment by MRI T2* (p = 0.037).
Carbohydrate metabolism: seventeen of the studied patients (60%) had disturbances of glucose homeostasis with an impaired fasting glycemia in four cases, impaired glucose tolerance in eight and diabetes mellitus in five. All of them except one had been diagnosed after the age of 10. Diabetes was preceded by a pre-diabetic stage in all cases with an average of 7 years (1 - 8). The mean age at the time of diagnosis was 20 ± 3.4 years (12 - 15). No significant difference was seen between males and females in the prevalence of diabetes mellitus. All diabetic patients had a family history of type I or type II diabetes in their siblings, parents or grandparents. The mean body mass index of diabetic patients was 19 ± 2.41 kg / m² (16.24-25). Overweight was noted in one patient. Two cases had first presented with diabetic ketoacidosis. Islet cell antibodies, insulin autoantibodies, and anti-glutamate decarboxylase were negative in all cases. Sixteen patients were splenectomized. Serum ferritin level in β-TM patients with diabetes and those without a history of diabetes were not significantly different. Severe cardiac loading (CT2* ≤10ms) was present in seven patients, and 10 patients had a severe iron deposition in the liver (LIC >15 mg/g dw). All patients had metformin as an antidiabetic agent associated with combined intensive iron chelation therapy during a mean follow-up of 3 years (1-6 years). Risk factors of carbohydrate metabolism disorders were: age at onset of chelation therapy (p = 0.025) and ferritinemia; in fact, patients with carbohydrate metabolism disorders had a higher average ferritin level than those who did not, and the difference was statistically significant (p = 0.03).
Thyroid function: subclinical hypothyroidism was found in six patients (mean age 17 ± 3.14 years range 14 – 16) (mean TSH levels 5.97 ± 1.31 m IU /L range 5-9). Antithyroid antibodies were negative in all cases. Mean serum ferritin level was 1405.8 ± 441.93 µg / l. Severe cardiac loading (CT2* ≤10ms) were seen in 3 cases, and severe liver loading (LIC >15 mg/g dw) was observed in 4 cases. Only one patient required hormone replacement therapy with levothyroxine. In all other cases, combined chelation therapy allowed the normalization of thyroid hormone levels.
Adrenal function: the cortisol peak was normal in 19 patients after ACTH stimulation. Nine patients (32%) had adrenal insufficiency with a mean cortisol peak of 413.93 ± 82.76 nmol / L(range 309 - 463). No patient was symptomatic. A polyendocrinopathy was found in all cases. All patients had combined chelation therapy with deferoxamine and deferasirox or deferiprone. Splenectomy was performed in all cases. None of them had received corticosteroid replacement therapy. Mean serum ferritin was 1,391.88 ± 661µg / l. Severe hepatic and cardiac loading was present in three patients. Factors associated with the occurrence of adrenal insufficiency were age (p = 0.033), history of splenectomy (p = 0.031), number of transfusions received (p = 0.034), and associated GH deficiency (p = 0.042).
Phosphocalcic metabolism and low bone mass: out of 28 patients, only one girl was found to have hypoparathyroidism. The mean age at diagnosis was 17 years. The patient initially complained of extremity paresthesias, mean serum calcium was 1.6 mmol/L (range 1.7 -1.9). Serum parathyroid hormone (PTH) level was low: 2 pg / ml (normal: 12-72). Among the 28 studied patients, 16 had decreased bone mineral density. Low bone mass was found in ten patients predominantly male (7 patients). All patients had one or more associated endocrinopathy. Factors related to the development of low bone mass include: hypothyroidism (p = 0.007), GH deficiency (p = 0.037), decreased IGF-1 levels (p = 0.002), and iron overload on cardiac (p = 0.021) and liver (p = 0.002) T2* MRI.
Associated endocrinopathies: among the studied patients five had no endocrine disorder, and 23 (82%) had at least one endocrinopathy with one endocrinopathy in six cases, two in eight cases, three in four cases and four in five cases. The mean age of these patients was 18.4 ± 1.3 years. The prevalence rates of endocrine disorders are shown in Table 1. There was a significant difference between mean serum ferritin in thalassemic patients with endocrine complications (1660 ± 1208 μg /l) and those without endocrinopathies (1166 ± 823 μg/l): p = 0.01.
|Table 1. Endocrine disorders according to their number|
- Telfer PT, Warburton F, Christou S,
Hadjigavriel M, Sitarou M, Kolnagou A, Angastiniotis
M. Improved survival in thalassemia major patients on switching from
desferrioxamine to combined chelation therapy with desferrioxamine and
deferiprone. Haematologica 2009; 94 (12):1777-1778. http://dx.doi.org/10.3324/haematol.2009.009118 PMid: 19815834
- De Sanctis V, Roos M, Gasser T, Fortini M, Raiola G, Galati MC. Italian Working Group on Endocrine Complications in Non-Endocrine Diseases. Impact of long-term iron chelation therapy on growth and endocrine functions in thalassaemia. J Pediatr Endocrinol Metab 2006; 19(4):471-480. PMID:16759032
- Sempé. Sempé M, Pédron G, Roy-Pernot MP. Auxologie méthode et séquences. Théraplix. Paris; 1979.
- Tanner J.M. Growth at adolescence. 2nd Edition Blackwell Scientific Publications; 1962.
- Diagnosis and classification of diabetes mellitus. American diabetes association. Diabetes Care 2010; 33: 62-69. http://dx.doi.org/10.2337/dc10-S062 PMid: 20042775
- Brabant G, von zur Mühlen A, Wüster C, Ranke MB, Kratzsch J, Kiess W, Ketelslegers JM, Wilhelmsen L, Hulthén L, Saller B, Mattsson A, Wilde J, Schemer R, Kann P. German KIMS Board. Serum insulin-like growth factor I reference values for an automated chemiluminescence immunoassay system: results from a multicenter study. Horm Res 2003; 60:53-60. http://dx.doi.org/10.1159/000071871 PMid:12876414
- Greulich WW, Pyle SI. Radiographic atlas of skeletal development of the hand and wrist. 2nd Ed. Stanford University Press; 1959.
- Fernandes JL, Sampaio EF, Verissimo M, Pereira FB, da Silva JA, de Figueiredo GS, Kalaf JM, Coelho OR . Heart and liver T2 assessment for iron overload using different software programs. Eur Radiol 2011; 21(12):2503–2510. http://dx.doi.org/10.1007/s00330-011-2208-1 PMid:21842212
- Carpenter JP, He T, Kirk P, Roughton M, Anderson LJ, de Noronha SV, Sheppard MN, Porter JB, Walker JM, Wood JC, Galanello R, Forni G, Catani G, Matta G, Fucharoen S, Fleming A, House MJ, Black G, Firmin DN, St Pierre TG, Pennell DJ. On T2* magnetic resonance and cardiac iron. Circulation 2011; 123(14):1519–1528. http://dx.doi.org/10.1161/CIRCULATIONAHA.110.007641 PMid:21444881
- Hankins JS, Mccarville MB, Loeffler RB et al. R2* magnetic resonance imaging of the liver in patients with iron overload. Blood 2009; 113(20):4853–4855. http://dx.doi.org/10.1182/blood-2008-12-191643 PMid:19264677
- Di Tucci AA, Matta G, Deplano S, Gabbas A, Depau C, Derudas D, Caocci G, Agus A, Angelucci E. Myocardial iron overload assessment by T2* magnetic resonance imaging in adult transfusion dependent patients with acquired anemias. Haematologica 200 ; 93(9):1385–1388. http://dx.doi.org/10.3324/haematol.12759 PMid:18603557
- Ouederni M , Ben Khaled M , Mellouli F , Ben Fraj E , Dhouib N , Yakoub IB , Abbes S , Mnif N , Bejaoui M .Myocardial and liver iron overload, assessed using T2* magnetic resonance imaging with an excel spreadsheet for post processing in Tunisian thalassemia major patients. Ann Hematol 2017; 96(1):133-139. http://dx.doi.org/10.1007/s 00277-016-2841-5 PMid:27730342
- Soliman A T, Khalafallah H, Ashour R. Growth and factors affecting it in thalassemia major. Hemoglobin 2009; 33(S1):S116–S126. http://dx.doi.org/10.3109/03630260903347781 PMid:20001614
- Skordis N, Kyriakou A. The multifactorial origin of growth failure in thalassaemia. Pediatr Endocrinol Rev 2011; 8 Suppl 2:271-277. PMid:21705977
- Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis 2010;5:11. http://dx.doi/10.1186/1750-1172-5-11 PMID: 20492708
- Kyriakou A, Skordis N.Thalassaemia and Aberrations of Growth and Puberty. Mediterr J Hematol Infect Dis. 2009; 1(1): e2009003. PMID: 21415985
- De Sanctis V, Skordis N, Galati MC, Raiola G, Giovannini M, Candini G, Kaffe K, Savvides I, Christou S . Growth hormone and adrenal response to intramuscular glucagon test and its relationship to IGF-1 production and left ventricular ejection fraction in adult B-thalassemia major patients. Pediatr Endocrinol Rev 2011; 8 Suppl 2:290-294. PMid:21705980
- Pincelli AI, Masera N, Tavecchia L, Perotti M, Perra S, Mariani R, Piperno A, Mancia G, Grassi G, Masera G. GH deficiency in adult B-thalassemia major patients and its relationship with IGF-1 production. Pediatr Endocrinol Rev 2011; 8 Suppl 2:284-289. PMid:21705979
- De Sanctis V, Elsedfy H, Soliman AT, Elhakim IZ, Pepe A, Kattamis C, Soliman NA, Elalaily R, El Kholy M, Yassin M. Acquired hypogonadotropic hypogonadism (AHH) in thalassaemia major patients: An underdiagnosed condition? Mediterr J Hematol Infect Dis 2016, 8(1): e2016001. http://dx.doi.org/10.4084/MJHID.2016.001
- De Sanctis V, Soliman AT, Elsedfy H, Albu A, Al Jaouni S, Anastasi S, Bisconte MG, Canatan D, Christou S, Daar S, Di Maio S, El Kholy M, Khater D, Elshinawy M, Kilinc Y, Mattei R, Mosli HH, Quota A, Roberti MG, Sobti P, AL Yaarubi S, Canpisi S, Kattamis C. Review and recommendations on management of adult female thalassemia patients with hypogonadism based on literature review and experience of ICET-A network specialists. Mediterr J Hematol Infect Dis 2017, 9(1): e2017001. http://dx.doi.org/10.4084/MJHID.2017.001
- Borgna-Pignatti C, De Stefano Ρ, Zonta Μ, Vullo C, De Sanctis V, Melevendi C, Naselle A, Masera G, Terzoli S, Gabitti V, Piga A.Growth and sexual maturation in thalassemia major. J Pediatr 1985 ; 106: 150-156. PMid:3965675
- Bejaoui M, Guirat N. Beta thalassemia major in a developing country: epidemiological, clinical and evolutionary aspects. Mediterr J Hematol Infect Dis 2013;5(1):e2013002. http://dx.doi.org/10.4084/MJHID.2013.002 PMid:23350015
- Thuret I, Pondarré C, Loundou A, Steschenko D, Girot R, Bachir D, Rose C, Barlogis V, Donadieu J, De Montalembert M, Hagege I, Pegourie B, Berger C, Micheau M, Bernaudin F, Leblanc T, Lutz L, Galactéros F, Siméoni MC, Badens C. Complications and treatment of patients with β-thalassemia in France: results of the National Registry. Haematologica 2010; 95(5):724-729. http://dx.doi.org/10.3324/haematol.2009.018051 PMid: 20007138
- Toumba M, Sergis A, Kanaris C, Skordis N . Endocrine complications in patients with thalassaemia major. Pediatr Endocrinol Rev 2007; 5: 642-648. PMId:18084158
- Gamberini MR, Fortini M, De Sanctis V, Gilli G, Testa MR.. Diabetes mellitus and impaired glucose tolerance in thalassaemia major: incidence, prevalence, risk factors and survival in patients followed in the Ferrara Center.Pediatr Endocrinol Rev 2004 ; 2 (Suppl 2): 285-291. PMid:16462713
- Au WY, Lam WW, Chu W, Tam S, Wong WK, Liang R, Ha SY . A T2* magnetic resonance imaging study of pancreatic iron overload in thalassemia major. Haematologica 2008; 93:116-119. http://dx.doi.org/10.3324/haematol.11768 PMid:18166794
- Bas M, Gumruk F, Gonc N, et al. Biochemical markers of glucose metabolism may be used to estimate the degree and progression of iron overload in the liver and pancreas of patients with β-thalassemia major. Ann Hematol 2015;94:1099-2104. http://dx.doi.org/10.1007/s00277-015-2342-y PMid:25740381
- Phenekos C, Karamerou A, Pipis P, Constantoulakis M, Lasaridis J, Detsi S, Politou K. Thyroid function in patients with homozygous β-thalassemia. Clin Endocrinol (Oxf) 1984; 20:445–450. PMid:6424976
- Magro S, Puzzanio P, Consarino C, Galati MC, Morgione S, Porcelli D, Grimaldi S, Tancre D, Arcuri V, De Santis V. Hypothyroidism in patients with thalassemia syndromes. Acta Haematol (Basel) 1990; 84:72–76. http://dx.doi.org/10.1159/000205032 PMid:2120889
- Depaz G, Deville A, Coussement N, Manassero J, Mariani R. Thyroid function in thalassemia major. Ann Pediatr (Paris) 1985 ; 32:809–811. PMid:4091451
- Landau H, Matoth I, Landau-Cordova Z, Goldfarb A, Rachmilewitz EA, Glaser B. Cross-sectional and longitudinal study of the pituitary-thyroid axis in patients with thalassaemia major. Clin Endocrinol (Oxf) 1993 ; 38:55–61.PMid:8435886
- De Sanctis V, Elsedfy H , Soliman AT., Elhakim IZ , Kattamis C, Soliman NA, Elalaily R. Clinical and Biochemical Data of Adult Thalassemia Major patients (TM) with Multiple Endocrine Complications (MEC) versus TM Patients with Normal Endocrine Functions: A long-term Retrospective Study (40 years) in a Tertiary Care Center in Italy. Mediterr J Hematol Infect Dis 2016; 8(1): e2016022. http://dx.doi.org/10.4084/MJHID.2016.022 PMid: 27158435
- Huang KE , Mittelman SD, Coates TD, Geffner ME, Wood JC. A significant proportion of thalassemia major patients have adrenal insufficiencydetectable on provocative testing. J Pediatr Hematol Oncol. 2015; 37(1):54-59. http://dx.doi.org/10.1097/MPH.0000000000000199 PMid:24942024
- De Sanctis V , Soliman AT, Canatan D , Elsedfy H, Karimi M , Daar S , Rimawi H , Christou S, Skordis N , Tzoulis P, Sobti P, Kakkar S, KilincY, Khater D, Alyaarubi SA, Kaleva V, Lum SH, Yassin MA, Saki F, Obiedat M, Anastasi S, Galati MC, Raiola G, Campisi S , Soliman N, Elshinawy M, Al Jaouni S, Di Maio S, Wali Y, Elhakim IZ, Kattamis C. An ICET- A survey on Hypoparathyroidism in Patients with Thalassaemia Majorand Intermedia: A preliminary report. Acta Biomed 2018; 16;88(4):435-444. http://dx.doi.org/10.23750/abm.v88i4.6837 PMid:29350657
M, Citarella S, Filosa A, De Michele E, Palmieri F, Ragozzino A,
Amendola G, Pugliese U, Tartaglione I, Della Rocca F, Cinque P, Nobili
B, Perrotta S. Endocrine function and bone disease during long-term
chelation therapy with deferasirox in patients with β-thalassemia
major. Am J Hematol 2014; 89(12):1102-1106. http://dx.doi.org/10.1002/ajh.23844 PMid:25197009