This document describes a study conducted at the Medical Genetics Center in Iasi, Romania to diagnose the genetic causes of mental retardation in patients. Researchers developed a clinical protocol involving tests like karyotyping, fragile X testing, and MLPA to efficiently screen 200 patients. Results identified numerous chromosomal abnormalities, four specific monogenic disorders, and subtelomeric rearrangements in some patients. The De Vries score was found useful for case selection. MLPA was reliable for subtelomeric screening and identifying complex chromosomal changes. The protocol provides an effective approach but could be optimized further.

1. DIAGNOSIS AND MANAGEMENT OF PATIENTS
WITH MENTAL RETARDATION AND
MULTIPLE CONGENITAL ANOMALIES – IASI
MEDICAL GENETICS CENTER’S EXPERIENCE
Rusu Cristina (1), Volosciuc M. (1), Braha E. (1),
Butnariu L. (1), Panzaru M. (1), Popescu R. (1), Caba L
(1), Ivanov Iuliu (2), Gorduza Vlad (2), Covic Mircea (1)
(1) Medical Genetics Centre, “Sf. Maria” Children’s Hospital, Iasi, Romania;
(2) Immunology and Genetics Lab, “Sf. Spiridon” Hospital, Iasi, Romania

2. MENTAL RETARDATION – DEFINITIONS
 Mental retardation (MR) = QI < 70;
 Prevalence: 3%; Mild MR - 7-10 x more frequent than
moderate/ severe MR;
 Evaluation supposes gathering information from specialists
(educator, psychologist, physician) + parents;
 Classification:
◦ Mild (IQ 50-70) – 85%;
◦ Moderate (IQ 35-50) – 10%;
◦ Severe (IQ 20-35);
◦ Profound (IQ <20);
13/07/09 2

3. MENTAL RETARDATION – DETERMINISM
 Variable according to MR severity;
 Mild MR:
o Environment – 75-90% (social factors !);
o Genetic factors – 10-25%;
 Severe MR:
o Environment – 50%;
o Genetic factors – 50% (monogenic/ multifactorial disorders/
chromosomal abnormalities).
13/07/09 3

4. MODERATE / SEVERE M.R. – GENETIC CAUSES
 CHROMOSOMAL ABNORMALITIES:
◦ Numerical: trisomy 21, 13, 18, Klinefelter → karyotype;
◦ Structural:
 Deletions/ duplications: 4p-, 5p- → karyotype;
 Microdeleions/ duplications: Sdr Williams, Prader Willi,
Angelman → FISH;
 Subtelomeric rearrangements → MLPA;
 MONOGENIC DISORDERS:
◦ X-linked mental retardation: Fragile X syndrome → DNA test
(PCR, Southern blot);
◦ Metabolic disorders: Lesch Nyhan, Menkes → biochemical test.
◦ POLYGENIC CAUSES
◦ MITOCHONDRIAL DISORDERS
13/07/09 4

5. MATERIAL AND METHOD
 We have designed, optimized and applied an investigation protocol
for mentally retarded individuals; the protocol:
• Covers most genetic causes of moderate/ severe MR;
• Adapted to our lab (equipments/ cost of the reagents);
 We have taken 200 mentally retarded patients examined in Iasi
Medical Genetics Center and analyzed the efficiency of the protocol:
• If De Vries score (proposed for subtelomeric rearrangements’
identification) could be used broadly for chromosomal abnormalities
detection;
• If the results provided by screening methods (antiFMRP test and MLPA)
are concordant with diagnostic tests (long range PCR and FISH);
• If the methods chosen are reliable;

6. PROTOCOL OPTIMIZATION
 Use selection scores, so that the percent of positive tests
will be good enough;
 Choose the cheapest, less dangerous method that provides
good results;
 Use a sequence of reactions: first screening tests and then
diagnostic tests, so that the abnormality will be identified
precisely in the end;
 Reducing the cost by reducing the amount of reagents used;

7. CLINICAL PROTOCOL
 PERSONAL HISTORY (pre/intra/postnatal → exclude MR produced by
the environment);
 Extended FAMILY HISTORY → pedigree;
 ANTHROPOMETRIC MEASUREMENTS (Ht, Wt, HC, others);
 Detailed PHYSICAL EXAMINATION (+ photos);
 PSYCHOLOGIC EVALUATION;
EVALUATION
 DE VRIES SCORE for case selection;
 TEST the child with MR → anomalies identified → test the parents;
 GENETIC COUNSELLING (before test, followed by written consent of
the parents; new session when test results were available);
 Data recorded in a DATABASE specially designed;

8. DE VRIES SCORE (2001): 3/> points necessary
Criteria Score
Mental retardation family history
• Compatible with monogenic inheritance 1
• Incompatible with monogenic inheritance (including discordant phenotypes) 2
Prenatal onset growth retardation 2
Abnormal postnatal growth (1 point each, maximum 2 points)
• Microcephaly 1
• Short stature 1
• Macrocephaly 1
• Tall stature 1
2/> facial anomalies (mostly hypertelorism, nasal and auricular defects) 2
Extrafacial anomalies (1 point each, maximum 2 points), especially:
• Hand anomalies 1
• Heart defects 1
• Hypospadias +/- criptorchydism 1

9. INVESTIGATION PROTOCOL
 KARYOTYPE (→ numerical & structural chromosomal abnormalities);
 BARR TEST – for cases that associate abnormal sexual development;
 Fragile X screening and diagnosis – done for cases with speech delay/
autism (early signs of Fragile X); positive & negative samples included;
◦ ANTI-FMRP TEST (immunohistochemical test done on hair root) →
screening;
◦ Long range PCR (identifies premutations and complete mutations) →
diagnostic;
 MLPA (2 separate kits P036 and P070) → subtelomeric rearrangements (if
the results in both kits are abnormal) and polymorphisms (if the result is
abnormal in a single kit);
 FISH → microdeletions / confirm subtelomeric rearrangements;
 DNA stored for subsequent tests (after parental consent);

10. RESULTS
 53% of patients (106) have been selected for genetic testing; the rest
were due to social causes/ perinatal events/ fetopathies etc;
 4 patients had specific monogenic disorders and a DNA sample has
been taken for further confirmation;
 15 patients had a Barr test → 3 cases confirmed;
 99 patients had a karyotype → 43 cases identified; many of them
were Down syndrome;
 7 patients had a FISH test for microdeletion → 1 case confirmed;
 55 patients had a MLPA test → 4 cases identified; FISH
confirmation is in due course;
 8 patients had FraX testing → all normal;
 Structural chromosomal abnormalities identified by karyotype
have been confirmed by MLPA.

11. TEST EFFICIENCY
100%
90%
80%
70%
60%
50% Abnormal
Normal
40%
30%
20%
10%
0%
Barr Kar FISH MLPA FraX

14. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
01-001.2 SCNN1D
01-001.0 TNFRSF18
01-247.1 SH3BP5L
02-000.3 ACP1
02-000.3 ACP1
02-241.2 CAPN10
03-000.3 CHL1
03-000.3 CHL1
03-198.8 BDH1
04-000.5 GPI7
04-000.3 ZNF141
04-191.1 FRG1
05-000.3 PDCD6
05-000.3 LOC133957
05-180.6 GNB2L1
06-000.3 IRF4
06-000.3 IRF4
06-170.7 PSMB1
07-000.9 CENTA1
07-000.9 UNC84A
07-158.6 VIPR2
08-000.4 FBXO25
08-000.4 FBXO25
08-144.6 ZC3H3
09-000.9 DMRT1
09-000.4 DOCK8
09-139.8 EHMT1
10-000.5 DIP2C
10-000.2 ZMYND11
10-135.0 PAOX
11-000.2 RIC8A
11-000.2 BET1L
11-133.6 hCAP-D3
12-000.12 SLC6A12
12-000.3 JARID1A
12-132.2 ZNF10
Mapview
13-019.2 PSPC1
Mapview
13-019.2 PSPC1
13-112.8 F7
14-019.9 CCNB1IP1
14-019.9 PARP2
po36-88.D04_08121819RL^CEQsCSV.csv
14-105.0 MTA1
15-021.5 NDN 15-021.4 MKRN3
15-099.3 ALDH1A3
P070.88.G01_09032408H9^CEQsCSV.csv
16-000.4 DECR2 16-000.04 POLR3K
16-088.6 GAS8
17-000.1 RPH3AL 17-000.1 RPH3AL
17-078.4 TBCD
18-000.2 THOC1 18-000.2 USP14
18-075.9 C18orf22
19-000.2 PPAP2C 19-000.5 CDC34
19-063.8 CHMP2A
20-000.2 ZCCHC3 20-000.3 SOX12
20-062.2 OPRL1
21-014.7 STCH 21-014.5 RBM11
21-046.9 HRMT1L1
DUPLICATION 9p
22-016.0 IL17R
22-016.6 BID
22-049.6 RABL2B
X-000.5 SHOX
X/Y-000.5 SHOX (PAR region)
X/Y-154.8 SYBL1 (PAR region)

16. Ratio
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
01-001.0 TNFRSF18
02-000.3 ACP1
03-000.3 CHL1
04-000.3 ZNF141
5-000.3 LOC133957
06-000.3 IRF4
07-000.9 UNC84A
08-000.4 FBXO25
09-000.4 DOCK8
10-000.2 ZMYND11
11-000.2 BET1L
12-000.3 JARID1A
13-019.2 PSPC1
14-019.9 PARP2
15-021.5 NDN
P070.111.H01_09032408HA^CEQsCSV.csv
16-000.4 DECR2
17-000.1 RPH3AL
18-000.2 THOC1
19-000.2 PPAP2C
20-000.2 ZCCHC3
21-014.7 STCH
22-016.0 IL17R
dup(21)(q22.3;q11.2)
X-000.5 SHOX

17. CONCLUSIONS
 De Vries score - useful for case selection (both chromosomal
abnormalities and subtelomeric rearrangements);
 MLPA - reliable, fast and unexpensive method that can be used
as a screening method for subtelomeric rearrangements;
 MLPA may be used as a complementary method to identify marker
chromosomes/ complex chromosomal abnormalities;
 The protocol could be further optimized by extending MLPA for
other applications;
 Cases with normal results/ polymorphisms - reevaluated in order to
get a final diagnosis;
 Cases identified - followed in order to prevent complications;
clinical description of subtelomeric rearrangements
is an ongoing process.

18. 13/07/09 18