newborn screening for mucopolysaccharidoses

NEWBORN SCREENING FOR MUCOPOLYSACCHARIDOSES J.R. Alonso-Fernández J. Fidalgo C. Colón

Methabolopathies Laboratory, Paediatrics Departament, University of Santiago de Compostela Hospital Complex

LINE OF CALIBRATION FOR GAG PATTERNS (mg/dL) 0 0 10 10 25 25 70 70 100 100

Abs 1

Abs 2

Abs 3

Abs 4

Abs 5

Abs 6

Abs 7

1.426

1.537

1.140

1.187

1.112

1.065

1.309

1.427

1.525

1.123

1.107

1.141

1.094

1.393

1.298

1.464

1.153

1.039

1.167

1.025

1.251

1.241

1.449

1.137

1.015

1.080

1.024

1.368

1.141

1.220

0.947

0.912

1.083

1.044

1.283

1.183

1.403

0.967

0.869

0.993

0.969

1.201

0.907

0.984

0.703

0.632

0.619

0.592

0.809

0.922

0.863

0.690

0.650

0.734

0.579

0.903

0.562

0.848

0.650

0.502

0.524

0.463

0.697

0.641

0.689

0.460

0.457

0.582

0.431

0.687

Average 1.256 1.194 1.087 0.756 0.585

table 1. Data for each of the 7 runs used to construct the line of calibration. We use Chondroitin -6sulfate (typeC) to calibrate the test.

1,400 1,200

y = -0.0069x + 1.2565

1,000

Abs

Pattern concentrations used to calibrate the test are: 010-25-70-100 mg Ch-6S / dL impregnated on WHATMAN 903 paper.

R2 = 0.998

0,800 0,600

we measure the decrease in absorbance of the DMB reagent, at 584nm of wavelength, instead of measuring the increase in absorbance of the DMB-GAGs complex, a decrease in the blue colour related with the increase in the glycosaminoglycans concentration. The absorbance is read 5 min. After adding the DMB working solution to the eluates.

0,400 0,200 0,000 0

20

40

60

80

100

120

mg/dl GAGs

Figure 1. Line of calibration for the Glycosaminoglycans.

CUANTITATIFICATION OF OTHER MUCOPOLYSACCHARIDES Glycosaminoglycans

Other glycosaminoglycans tested with this method: chondroitin-4sulfate, chondroitin -6sulfate, dermatan sulfate, heparan sulfate, hialuronic acid and heparin.

Concentration (mg/dl) assay 1

Concentration (mg/dl) assay 2

Ch -4S 25 mg/dl

27.048

21.138

Ch- 4S 70 mg/dl

50.026

84.588

DS 25 mg/dl

48.262

21.022

DS 70 mg/dl

99.757

80.036

HS 100 mg/dl

80.983

72.754

Heparina 25 mg/dl

26.729

40.564

Heparina 70 mg/dl

64.613

78.894

4.557

0.000 (undetectable)

Ac.Hialurónico mg/dl

25

Ac.Hialurónico 70 15.709 24.353 Hialuronic acid gave the lowest mg/dl signal with the same concentrations, so we could not Table 2. Data from other glycosaminoglycans quantified with this method. detect MPS type IX.

LINE OF CALIBRATION FOR CREATININE PATTERNS (mg/dL) 0 0 40 40 100 100 280 280 400 400

Abs 1

Abs 2

Abs 3

Abs 4

Abs 5

Abs 6

Abs 7

0.088

0.150

0.147

0.170

0.094

0.090

0.091

0.110

0.147

0.165

0.178

0.109

0.099

0.092

0.347

0.413

0.404

0.383

0.353

0.296

0.344

0.341

0.421

0.425

0.389

0.351

0.317

0.320

0.685

0.967

0.770

0.685

0.740

0.559

0.558

0.642

0.691

0.841

0.800

0.687

0.531

0.557

1.730

1.622

1.714

1.566

1.738

1.613

1.625

1.572

1.661

1.655

1.665

1.638

1.551

1.694

2.198

1.993

2.201

2.052

2.059

2.122

2.148

1.942

2.116

2.240

2.258

2.018

2.189

2.149

Average 0.124 0.365 0.694 1.646 2.120

Table 3. Data for each of the 7 runs used to construct the line of calibration. By measuring creatinine concentrations we can correct the differences in concentration that exist between the newborn urine samples and to compensate the probable diferences in the elution and in the paper impregnation.

2.000

1.500

Abs

The Absorbance is read 20 min after adding the reagents.

2.500

1.000

y = 0.005x + 0.1641 2

R = 0.9963

the creatinine concentrations used as patterns to calibrate the results are: 0-40100-280-400 mg creatinine / dL impregnated on the same paper as the Ch-6S.

0.500

0.000 0

50

100

150

200

250

300

350

mg/dL Creatinina

Figure 2. Line of calibration for the creatinine.

400

450

ELUATE PREPARATION

4.4 cm 4 discs of 6 mm diameter are punched from both urine samples and patterns impregnated on paper (WHATMAN 903) with a special puncher, introducing them in a 96 well-microtiter plate of 4.4 cm in height

Then, the discs are shaken for 40minutes after the addition of 300 µL of distilled water in each well to elute both urine samples and pattern discs.

The resulting eluates are introduced in a 96-well microtiter plate with a U shaped bottom

REAGENTS REAGENTS FOR CREATININE QUANTIFICATION:

REAGENTS FOR GLYCOSAMINOGLYCANS QUANTIFICATION:

saturated picric acid solution (diluted 5 times)

DMB working solution:

7.5 g/L of sodium hydroxide solution

The DMB working solution was prepared by diluting 1:4 the DMB stock solution with formic acid – sodium formiate 0.2 M, pH 3.5 (Whitley, 1989, modified).

ABSORPTION SPECTRA OF DMB DYE AND DMB-GAG COMPLEX. Maximum point of absorbance for DMB

difference in Abs between the DMB-GAG complex and the DMB dye

Maximum point of absorbance for DMB-GAG complex

R.Humbel and S.Etringer, 1974 It doesn’t matter that the reagent absorbance is higher than the maximum of the complex absorbance when measuring the decrease in absorbance.

G.Panin et al., 1986 The difference in Absorbance between the maximum point of Abs for DMB and the maximum point of Abs for DMB-GAG complex, is greater than the maximum point of Abs for DMB-GAG complex.

DETECCTION LIMIT AND QUANTIFICATION LIMIT BLANK SAMPLES 1 2 3 4 5 6 7 8 9 10

GAGs mg/dL 0 0 0 0 0 0 1,15 1,62 0 0

Creatinine mg/dl 0 0 0 0 0 0 0 0 0 0

Table 4. The 10 blank (non impregnated paper), used to determine both the detection limit and the quantification limit. Est.Deviation ( S.D. ) Average DETECTION LIMIT +3 QUANTIF.LIMIT +10

GAG Creatinine concentr. concentr. 0,537 0 0,203 0

1,970 5,930

0 0

Table 5. Data from both the detection limit and the quantification limit.

METHOD VALIDATION: Interassay Precision pattern of 25mg/dL dissolved in urine samples 1 2 3 4 5 6 7 8 9 10 11 12

GAG Abs concentr. 0,940 24,940 0,907 30,020 1,469 27,244 1,420 34,922 0,982 18,255 0,975 19,394 1,137 36,782 1,160 33,460 0,653 31,779 0,698 22,080 1,176 27,589 1,153 30,586

CREATININE Abs concentr. 0,568 73,753 0,551 70,341 0,456 56,182 0,450 62,340 0,472 75,043 0,468 74,393 0,407 59,337 0,538 84,243 0,493 71,683 0,491 71,264 0,494 69,990 0,528 76,715

coefficient 0,33815574 0,42677702 0,48492239 0,56018808 0,24326041 0,26069812 0,61988343 0,39718575 0,44332926 0,30983217 0,394187 0,39869395

Table 6. Data from 12 runs used to calculate the intertest precision. 25 mg / dl Est.Dev. Average Coef.Var.

GAG Abs 0,238 1,056 0,225

Concentr.

5,729 28,089 20,398

CREATININE Abs Concentr. coefficient 0,044 7,484 0,107 0,493 70 0,406 9,051 10,625995 25

Table 7. Data from the C.V., average and S.D. for the 25 mg/dL pattern.

Interassay precision: pattern of 70mg/dL dissolved in urine samples 1 2 3 4 5 6 7 8 9 10 11 12

DMB Abs concentr. 0,663 68,181 0,546 86,440 1,304 53,248 1,286 56,043 0,674 63,846 0,681 62,809 0,957 62,794 0,991 57,898 0,514 61,322 0,520 60,043 0,972 54,602 0,993 51,897

CREATININE Abs concentr. 0,552 70,483 0,640 88,272 0,456 55,842 0,450 54,623 0,466 73,972 0,468 74,278 0,477 72,512 0,462 69,832 0,457 63,714 0,485 69,878 0,515 74,181 0,495 70,089

coefficient 0,96735122 0,97923725 0,95354684 1,02600723 0,86310785 0,84560128 0,86597725 0,8291079 0,96244671 0,85925575 0,73606381 0,74043374

Table 8. Data from 12 runs used to calculate the intertest precision. 70 mg / dl Est.Dev. Average Coef.Var.

GAG CREATININE Abs Concentr. Abs Concentr. coefficient 0,269 8,794 0,052 8,504 0,089 0,842 61,594 0,493 70 0,885 31,983 14,279 10,571001 12,183 10,072

Table 9. Data from the C.V., average and S.D. for the 70 mg/dL pattern.

Intraassay Precision: pattern of 25 mg/dL dissolved in urine samples 1 2 3 4 5 6 7 8 9 10 11 12

GAG Abs concentr. 1.111 25.290 1.176 16.194 1.153 19.360 1.131 22.456 1.206 12.144 1.189 14.397 1.168 17.286 1.104 26.175 1.153 19.443 1.162 18.116 1.176 16.263 1.165 17.770

CREATININE Abs concentr. 0.429 51.655 0.462 58.346 0.416 49.092 0.461 58.143 0.432 52.224 0.442 54.299 0.450 56.027 0.439 53.648 0.427 51.289 0.443 54.502 0.403 46.387 0.438 53.607

coefficient 0.48959673 0.27755518 0.39435756 0.38622518 0.23253738 0.26514908 0.30853249 0.48789713 0.37908509 0.33238537 0.35060297 0.33148762

Table 10. Data from 12 runs used to calculate the intratest precision. 25 mg/dL Est. Dev. Average Coef.Var.

GAG Abs Concentr. 0,028 3,992 1,158 18,741 2,494 21,303

CREATININE Abs Concentr. coefficient 0,0163 3,321 0,076 0,437 53 0,352 3,74 6,235 21,815

Table 11. Data from the C.V., average and S.D. for the 25 mg/dL pattern.

Intraassay Precision: Pattern of 70 mgdL dissolved in urine samples 1 2 3 4 5 6 7 8 9 10 11 12

GAG Abs concentr. 0.870 58.590 0.900 54.374 0.637 90.798 0.910 52.978 0.821 65.294 0.845 62.060 0.830 64.064 0.893 55.383 0.944 48.278 0.891 55.673 0.929 50.338 0.965 45.472

CREATININE Abs concentr. coefficient 0.486 63.288 0.92576271 0.449 55.722 0.97580135 0.521 70.407 1.28961819 0.434 52.631 1.00659208 0.488 63.736 1.02445161 0.493 64.692 0.95931306 0.483 62.597 1.02343808 0.439 53.790 1.02961273 0.430 51.899 0.93023429 0.467 59.404 0.93720345 0.396 44.963 1.11952991 0.433 52.550 0.86531368

Table 12. Data from 12 runs used to calculate the intratest precision.

70 mg/dL Est.Dev. Average Coef.Var.

GAG Abs Concentr. 0,081 11,324 0,869 58,608 9,422 19,322

CREATININE Abs Concentr. coefficient 0,033 6,899 0,105 0,46 58 1,007 7,378 11,901 10,503

Table 13. Data from the C.V., average and S.D. for the 70 mg/dL pattern.

STUDY OF THE PATHOLOGICAL SAMPLES: PATIENT I.D

PATHOLOGY

AGE

NOTES

GAG mg/dl

Creatinina mg/dl

mg GAG/g Creat.

LE12806

MPS I

12 MONTHS

11.21

26.45

424

MU117706

MPS II

20 MONTHS

29.80

71.17

419

OM119506

MPS II

4 YEARS

29.78

53.05

561

SA121205

MPS III

3 YEARS

21.45

45.12

475

BR15403

MPS IV

2 YEARS

18.08

80.89

223

AR111101

MPS VI

7 YEARS

7.76

1.24

6265

BO13502

MPS VII

6 YEARS

38.44

106.38

361

PF28805

MPS IS

22 YEARS

First control post ERT

23.10

216.65

107

PF4131006

MPS IS

23 YEARS

16.19

158.42

102

AC117202

MPS IS

23 YEARS

Last control post ERT Pre-Treatment

5,89

80.54

73

AC221106

MPS IS

26 YEARS

Pre-Treatment

7.32

31.92

229

PF18305

MPS IS

22 YEARS

Pre-Treatment

4.27

46.86

91

PF310106

MPS IS

23 YEARS

Post -Treatment

5.83

96.76

60

PI21106

NORMAL

6 MONTHS

0

6.67

0

Table 14. Pathological samples, used to test method sensitivity and specificity, from Argentina

(Néstor Chamoles neurochemistry laboratory, Buenos Aires)

THE 3 CASES FROM THE 3 CARRIER FAMILIES OF MUCOPOLYSACCHARIDOSES PATIENT RCD Mother Father DRO Mother Father Brother Cristina Q.P Father

GAG Abs GAG conc. Creat. Conc. Creat.Conc. COEFFICIENT 1,194 12,009 0,358 32,883 0,365 1,267 0 (undetectable) 0,822 135,282 0 (undetectable) 1,198 11,282 0,656 98,763 0,114 1,159 17,841 0,898 152,028 0,117 1,243 3,946 0,445 51,993 0,075 1,225 6,854 0,238 6,372 1,075 0 (undetectable) 0 (undetectable) 1,396 0,339 22,105 1,259 11,416 0,315 16,143 0,707 1,358 0,47 0,467 54,668 0,008

Table 15. Results from the 3 carrier families.

We confirmed the reliability of the method by an enzymatic assay for the DRO family.

BLOOD SAMPLES IMPRAGNATED ON PAPER

Enzymatic Activity (μmol/L/h)

DRO

0,0

Mother

1,9

Father

1,1

Table 16. Results from the DRO carrier family after the enzymatic assay which correlate with the urine test for this family.

SAMPLE FROM THE ERNDIM QUALITY PROGRAMME GAG Abs Concentrc. 1.174 14.475 1.168 15.341

CREATININE Abs Concentrc. 0,319 30.453 0,327 31.903

COEFFICIENT

0,475 0,48

Table 17. Patient (19) with MPS type VI.

•Increased GAG concentrations and a coefficients values. •the urine sample from this patient was tested as a duplicate in the same run, giving similar concentrations and coefficients.

ENZYME REPLACEMENT THERAPY (ERT): PRE- AND POST TREATMENT PATIENT JMM pre-ERT JMM 24 h JMM 48 h

GAG conc. Creat. Conc. Coefficient GAG Abs. (mg/dl) Creat. Abs (mg/dl) (mg / mg) 1.129 22.462 0,477 74.028 0,303 1.225 4.121 0,619 106.250 0,038 1.2970 (undetectable) 0,304 34.718 0(undetectable)

Table 18. Patient suffering from MPS type I (Hurler type), before and after the treatment administration.

we can observe in the table a decrease in the GAGs concentration after the administration of treatment and a decrease in the coefficient as well

POSITIVES RESULTS positive sample? concentrated urine sample? submitted to a TLC to determine the increased Mucopolysaccharide or Mucopolysaccharides in urine.

Ch-6S

Ch-4S

DS

HS

enzymatic assay (blood): which are the enzymes involved?

FIGURE SHOWING THE CUTOFF POINT ACCORDING TO AGE (Whitley et al., 1989) 10000

Coefficient (mg GAGs / g Creat)

Serie1

100

All the coefficients of the pathological samples from this study are above the cut-off point according to age

Serie2 Serie3

1 0

5

10

15

20

25

30

35

40

Age( (years) ) Figure 3. coefficient related to age. Series 1(pink): Pathological samples from Argentina. series 2 (green): the three cases from the three carrier families. series 3 (red): Quality control from the ERNDIM (MPS type VI).

FIGURE SHOWING THE COEFFICIENT ACCORDING TO AGE FOR A NEONATAL POPULATION Box-and-w hisker Means

We can conclude that exists a decrease in the coefficient value for a neonatal population according to age rises.

20

COCIENTE

15

10

This fact, is related with the maturation of the neonatal renal system.

Coefficient (mgGAGs/ mg Creat)

5

0 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 EDAD select Sólo Neonatales

Age (days)

Figure 4. coefficient related to age for the neonatal population of the study.

Red and Blue colours are the coefficient values which are out of the C.I. limits. The white ones are coefficients within the C.I. limits.

CONCLUSIONES: 1) We conclude that this method has an easy implementation in the neonatal screening programes that collect urine samples. 2) It’s not necessary to make sample dilutions to increase mucopolysaccharide concentrations with this procedure. This is very useful as newborn normally excrete a high amount of mucopolysaccharides in urine and even higher if they suffer from this disease. So, the samples from screening programmes are a suitable age range. 3) we tested the level of performance of the analytical procedure with samples from children and adults, and in both cases were good. 4) we also tested the level of performance of the analytical procedure by following enzyme replacement therapy (ERT), and they correlate with the blood enzymatic quantification.

NEWBORN URINE SAMPLE COLLECTION INSTRUCTIONS: • Place the WHATMAN 903 paper upon the neonate genitals and close the nappy and prick on the heel to obtain the blood. • Then, Teave the blood to dry and remove the paper soaked with urine ( If the neonate hasn’t urinated we will have to wait, it’s to be expected that newborns urinate after being pricked on the heel). • If the newborn has defecated, we have to wash them, replace the 903 paper and wait for them to urinate. • finally, leave the paper to dry.

paper card used to collect urine samples

ACKNOWLEDGEMENTS THANKS TO: -Dr.Chamoles neurochemistry laboratory: Dr. M. Blanco; Dr. A.B. Schenone; Dr. M. Szlago. -All the laboratory personnel from the metabolopathies laboratory (Santiago de Compostela) specially Beatriz Osorio, and all doctors that supply us with urine samples. -Biomarin. -Genzyme. -Shire Human Genetic Therapies.

-and you, for listening this presentation.

THANK YOU

Reykjavick (Iceland), June 2007