Osteomalacia related toOsteomalacia related to

vitamin D deficiency vitamin D deficiency

in ain a

Patient Patient

with CVIDwith CVID

Case ReportCase Report

23 year-old woman diagnosed as having 23 year-old woman diagnosed as having CVID in 1999 was admitted to the hospital CVID in 1999 was admitted to the hospital by reason of evaluation of her general by reason of evaluation of her general status in November of 2005. status in November of 2005.

She did not have a complaint..She did not have a complaint..

Medical History Many hospitalizations caused by several recurrent Many hospitalizations caused by several recurrent

airway tract infections and diarheae since the age of 11 airway tract infections and diarheae since the age of 11 years.years.

She was given a diagnosis of She was given a diagnosis of bronchiectasia in 1999.bronchiectasia in 1999.

Significanty low immunoglobulin levelsSignificanty low immunoglobulin levels (IgG 225 mg/dl (700-1400)(IgG 225 mg/dl (700-1400) IgM < 24 mg/dl (70-40)IgM < 24 mg/dl (70-40) IgA < 16 mg/dl (40-230)IgA < 16 mg/dl (40-230)

Defective responses to vaccination with Defective responses to vaccination with Haemophilus Haemophilus influenzaeinfluenzae and tetanus toxoid, and tetanus toxoid,

Elimination of the causes related to secondary Elimination of the causes related to secondary immunodeficiencies prompted the immunodeficiencies prompted the diagnosis of CVIDdiagnosis of CVID and and she was started on IVIG 400 mg/kg/month in 1999. she was started on IVIG 400 mg/kg/month in 1999.

Medical history (2)Medical history (2)

LobectomyLobectomy operation was performed in 2000. operation was performed in 2000. She was also diagnosed with She was also diagnosed with diabetes mellitusdiabetes mellitus and has and has

been undergoing insulin therapy since 2000. been undergoing insulin therapy since 2000. Alaso diagnosed as having Alaso diagnosed as having polyneuropathy and polyneuropathy and

sensorineural hearing loss in 2003.sensorineural hearing loss in 2003. There was There was no history of consanguityno history of consanguity. . 2-3 times/year hospitalization frequency while under 2-3 times/year hospitalization frequency while under

therapy therapy

Examination On AdmissionExamination On Admission

UnremarkableUnremarkable apart fromapart from reduced sounds in the left reduced sounds in the left lower part of the lunglower part of the lung..

Blood pressure 110/80 mmHgBlood pressure 110/80 mmHg Temperature 36,5 centigradeTemperature 36,5 centigrade Pulse 90 beats/minutePulse 90 beats/minute 52 kg weight, BMI of 52 kg weight, BMI of 19,3319,33

Laboratory On AdmissionLaboratory On Admission

Quantitative immunoglobulin (Ig) Quantitative immunoglobulin (Ig) evaluation (Dade-Behring, nephelometry) evaluation (Dade-Behring, nephelometry) revealed:revealed:

IgG IgG 660 mg/dl (700-1400)660 mg/dl (700-1400)

IgA and IgM undetectable. IgA and IgM undetectable.

Lymphocyte Lymphocyte SubsetsSubsets

Patient Patient value (%)value (%)

Normal (%)Normal (%)

CD 3CD 3 8686 60-8560-85

CD 19CD 19 55 7-237-23

CD 4CD 4 3030 29-5929-59

CD 8CD 8 4848 19-4819-48

CD CD 3-/16+56+3-/16+56+

3,53,5 6-296-29

Evaluation of Lymphocyte Subsets by Flow-cytometry

The biochemical analyses revealed isolated The biochemical analyses revealed isolated high alkaline phosphatase high alkaline phosphatase ((472 U/L472 U/L, N. 90-, N. 90-260) 260) levelslevels..

Persistant finding since 1999 but no further Persistant finding since 1999 but no further evaluation had been performed..evaluation had been performed..

Other biochemical parameters including Other biochemical parameters including ssedimentation rate edimentation rate ((13 mm/h13 mm/h) and) and C- C-

reactive proteinreactive protein((0,3 mg/L0,3 mg/L, , N=0-5 mg/L)N=0-5 mg/L) were all normal were all normal except for:except for:

Albumin (g/dl) 4,1 Albumin (g/dl) 4,1 3,5-5 3,5-5

Globulin (g/dl) 2,2 Globulin (g/dl) 2,2 2,5-3,5 2,5-3,5

Vitamin B 12 (pg/ml) 243 Vitamin B 12 (pg/ml) 243 197-866 197-866

Folat (ng/ml) Folat (ng/ml) 8,16 8,16 3-16 3-16

Vitamin A (mg/L) Vitamin A (mg/L) 1,7 1,7 0,3-1,1 0,3-1,1

Beta-Carotene (micg/dl) 11,2Beta-Carotene (micg/dl) 11,2 10-80 10-80

Vitamin E (mg/L) 8,4 Vitamin E (mg/L) 8,4 6-18 6-18

Iron (micg/dl) 49 Iron (micg/dl) 49 50-140 50-140

Ferritin (mg/ml) 21,65 Ferritin (mg/ml) 21,65 13-150 13-150

Hct (%) Hct (%) 34,9 34,9 35-4535-45

HgbA1C (%) 6,0 HgbA1C (%) 6,0 < 6,5 < 6,5

Autoantibody profile including;Autoantibody profile including;

AntinuclearAntinuclear

AntimitochondrialAntimitochondrial

Anti-smooth muscleAnti-smooth muscle

LLiver-kidney microsomaliver-kidney microsomal

Gastric parietal cell Gastric parietal cell

Anti-neutrophil cytoplasmic antibodiesAnti-neutrophil cytoplasmic antibodies

Rheumatoid factor (RF)Rheumatoid factor (RF)

Anti-thyroid antibodies Anti-thyroid antibodies were were negative. negative.

The The high resolution high resolution computed tomography (CT) computed tomography (CT) of of thorax and CT of the abdomen thorax and CT of the abdomen did not did not revealreveal granulomatous diseasgranulomatous disease (liver size normal). e (liver size normal).

Endoscopy and biopsy revealed normal duodenal Endoscopy and biopsy revealed normal duodenal mucosa, there were mucosa, there were no intraepithelial no intraepithelial lymphocytes. lymphocytes.

Is there a bone metabolism Is there a bone metabolism abnormality ??abnormality ??

OOsteocalcinsteocalcin was found to be was found to be increasedincreased ( (35,5-35,5- N: 3,1-N: 3,1-13,7 ng/ml13,7 ng/ml))

DeoxypyridinolineDeoxypyridinoline was was normalnormal (6,3 (6,3 nM/mMnM/mM N:3,0-7,4) N:3,0-7,4) PParathyroid hormonearathyroid hormone (PTH) (PTH) was was normalnormal (49 pg/mL (49 pg/mL

N:15-65)N:15-65) Urinary calciumUrinary calcium was low (was low ( 2525 mg/24 h mg/24 h N: N:100-300)100-300) CalciumCalcium of 8,8 mg/dl (N:8,2-10,4) of 8,8 mg/dl (N:8,2-10,4) PhosphatePhosphate 3,3 3,3 mg/dlmg/dl (N: (N:2,3-4,52,3-4,5)) Magnesium Magnesium 2,2 2,2 mg/dl (N:mg/dl (N:1,5-2,61,5-2,6))

RadiologyRadiology

The X-ray examination of the cranium and extremities The X-ray examination of the cranium and extremities revealed revealed increased radiolucencyincreased radiolucency without without specific specific abnormalities suggestive of Paget’s disease.abnormalities suggestive of Paget’s disease.

The The bone densitybone density evaluation showed evaluation showed osteoporosis.osteoporosis. The The T scoreT score for the left for the left hiphip was was – 3.2– 3.2 and for the and for the lumbar lumbar

spinespine was was - 3.00- 3.00. . The bone The bone radionuclide scanradionuclide scan was found to be was found to be normalnormal..

TThe patient’s status he patient’s status was considered was considered preosteomalacic phasepreosteomalacic phase..

25 OH vit D25 OH vit D and and 1,25 (OH) 2 vit D levels1,25 (OH) 2 vit D levels were found to be were found to be lowlow..

25 OH vit D25 OH vit D 55 ng/ml (N: ng/ml (N: 7,6-75)7,6-75)

1,25 (OH) 2 vit D1,25 (OH) 2 vit D < 8 < 8 pg/ml pg/ml (N:29,6-65,1)(N:29,6-65,1)

Vitamin A Vitamin A 1,7 1,7 (N: (N: 0,3- 0,3-1,11,1 mg/L) mg/L)

Vitamin E 8,4 Vitamin E 8,4 (N: (N: 6-186-18 mg/L) mg/L)

AActive vitamin D ctive vitamin D (rocaltrol (rocaltrol 0,25 mcg 0,25 mcg bidbid and calcium (1 gr/day) therapy and calcium (1 gr/day) therapy waswas started. started.

Rehospitalized 6 months later, by reason of Rehospitalized 6 months later, by reason of breakdown of therapy..breakdown of therapy..

Osteocalcin Osteocalcin normalized (11 ng/ml)normalized (11 ng/ml) PTH increased (68 pg/ml) PTH increased (68 pg/ml) ALP increased (508 U/L)ALP increased (508 U/L)

25 OH vit D 25 OH vit D significantlysignificantly low ( 1,2 ng/ml) low ( 1,2 ng/ml) 1,25 OH vit D 1,25 OH vit D significantlysignificantly low (1,2 pg/ml) low (1,2 pg/ml)

Active vitamin D (rocaltrol 0,5 mcg Active vitamin D (rocaltrol 0,5 mcg bıd and calcium therapy 1 gr/day were bıd and calcium therapy 1 gr/day were commencedcommenced..

Six months later…Six months later…

ALPALP was was normalized ……..normalized …….. 239 U/L (90-260) 239 U/L (90-260) Urinary calcium increasedUrinary calcium increased…..117 mg/24 h)…..117 mg/24 h) PTHPTH was was decreaseddecreased no normal range……50,7 pg/ml no normal range……50,7 pg/ml

(15-65) (15-65) 25 OH vit D25 OH vit D was was increasedincreased to………… 5 ng/ml (7,6-65) to………… 5 ng/ml (7,6-65)

Still lower than normal…..Still lower than normal…..

Possible Reasons for Low Vit D LevelsPossible Reasons for Low Vit D Levels

LLiving to the north of latitude 37iving to the north of latitude 37 Dark-skinned people, vegeterians and veiled Dark-skinned people, vegeterians and veiled

womenwomen AAnticonvulsant or antituberculous drug use nticonvulsant or antituberculous drug use GGastrointestinal and pancreatico-biliary astrointestinal and pancreatico-biliary

diseasesdiseases

Celiac disease Celiac disease Vitamin D resistance related to Vitamin D resistance related to VDR mutation VDR mutation Vitamin D binding protein deficiency Vitamin D binding protein deficiency

were were ruled out..ruled out..

Vitamin D receptor expression (VDR) Vitamin D receptor expression (VDR) was evaluated and found to be was evaluated and found to be significanly decreasedsignificanly decreased in PBMC and in PBMC and hair follicle compared with control hair follicle compared with control group… group…

ControlControl CaseCase

PBMPBMCC

HFHF PBMCPBMC HFHF

ExpressiExpressionon

0,140,142424

37,98237,98255

0,01430,0143 0,69070,6907

SDSD 0,010,013030

18,94518,94544

95% CI95% CI 0,010,012727

18,56618,56611

VDR mRNA EXPRESSION of THE CASE and THE CONTROL

VDR Gene Polymorphism

She was found to be heterozygous regarding Apa, Bsm and Taq genes by High-Performance Liquid Chromatography (DHPLC)..

She was homozygous (FF) for Fok gene..

None of these polymorphisms related to None of these polymorphisms related to impaired VDR expression and function…impaired VDR expression and function…

We could not investigate We could not investigate intestinal VDR expressionintestinal VDR expression

Low intestinal VDR expression is related to resistance to Low intestinal VDR expression is related to resistance to vit D treatment and calcium absorption… vit D treatment and calcium absorption…

Clinical ConsequensesClinical Consequenses

VVitamin D deficiency might have a role in the itamin D deficiency might have a role in the pathogenesis of pathogenesis of polyneuropathypolyneuropathy in this patient. in this patient.

Untreated vit D deficiency might play a role as an Untreated vit D deficiency might play a role as an additive risk factoradditive risk factor in terms of in terms of tendency to tendency to malignancymalignancy in patients with CVID. in patients with CVID.

Increased tendency to Increased tendency to autoimmune diseases (type-1 autoimmune diseases (type-1 diabetes !)diabetes !)

Increased tendency toIncreased tendency to infections (tuberculosis) infections (tuberculosis) Increased tendency toIncreased tendency to sepsis sepsis Irreperable Irreperable bone loss and fracturesbone loss and fractures

Isolation of total RNA and cDNA Isolation of total RNA and cDNA synthesissynthesis

2 mL of blood and 10 follicles of hair sample of the case 2 mL of blood and 10 follicles of hair sample of the case were collected. were collected. Fifty microlitersFifty microliters of total RNA was isolated of total RNA was isolated from peripheral blood mononuclear cells (PBMC) and from peripheral blood mononuclear cells (PBMC) and hair follicle by using hair follicle by using High Pure RNA Isolation Kit (Roche, High Pure RNA Isolation Kit (Roche, Germany).Germany). Reverse transcriptionReverse transcription procedure was procedure was performed for performed for cDNA cDNA synthesis by using Transcriptor First synthesis by using Transcriptor First Strand cDNA Synthesis Kit according to the Strand cDNA Synthesis Kit according to the manufacturers’ instructions.manufacturers’ instructions.

Relative quantification of VDR Relative quantification of VDR Real-time quantitative RT-PCR analyses of Real-time quantitative RT-PCR analyses of VDR VDR were were

performed with Lightcycler instrument and software. performed with Lightcycler instrument and software. Glyceraldehyde-3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase (GAPDH GAPDH “housekeeping” gene) was chosen as an internal “housekeeping” gene) was chosen as an internal standard to control for variability in amplification. The standard to control for variability in amplification. The sequences of primers and probes used are shown in sequences of primers and probes used are shown in Table-3. PCR was performed by using TaqMan Master Table-3. PCR was performed by using TaqMan Master Kit (Roche Diagnostics) according to the instructions of Kit (Roche Diagnostics) according to the instructions of the manufacturer. The the manufacturer. The VDR VDR target probe was labeled at target probe was labeled at the 5’ end with the reporter dye molecule 6-the 5’ end with the reporter dye molecule 6-carboxyfluorescein (FAM). The carboxyfluorescein (FAM). The GAPDH GAPDH target probe target probe was labeled with 6- carboxyfluorescein. Both probes was labeled with 6- carboxyfluorescein. Both probes were labeled with the quencher fluor 6-were labeled with the quencher fluor 6-carboxytetramethylrhodamine (TAMRA) at the 3’ end. To carboxytetramethylrhodamine (TAMRA) at the 3’ end. To quantify VDR mRNA from PBMC and hair follicle, we quantify VDR mRNA from PBMC and hair follicle, we constructed a calibration curve (Error: 0.100 Efficency: constructed a calibration curve (Error: 0.100 Efficency: 1,790) using 1,790) using GAPDH GAPDH mRNA as an endogenous control.mRNA as an endogenous control.