"familial" versus "sporadic" intellectual disability: contribution of dna copy...
TRANSCRIPT
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Designing a Comprehensive Stepwise Diagnostic Approach to Iranian Patients with Mental
Retardation, by Determining the Molecular Profile of DNA Copy Number Variations
Tehran University of Medical SciencesDepartment of Medical Genetics
Presented for PhD Degree
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Presented by:Maryam Rafati
Supervised by:SR Ghaffari MSc MD PhD
Tehran University of Medical SciencesDepartment of Medical Genetics
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Advisors:ZT Keihanidoust MD, MR Eshraghian PhD
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Main Goal
Determining the genetic causes of “Familial MR”
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Main Questions
Is there any differences between “Sporadic” and “Familial” MR regarding their underlying genetic causes?
Should a different diagnostic approach be adopted for “Familial” rather than “Sporadic” MR?
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Outline
Introduction Materials and method Results Discussion
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Introduction
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Higher Cerebral Development Dysfunction
Mental retardation
Cerebral palsy Abnormal motor actions and postural mechanisms Non-progressive abnormalities of the developing brain limited, stereotypic, and uncoordinated voluntary movements
Autism A behaviorally defined syndrome characterized by
▪ Atypical social interaction▪ Disordered verbal and nonverbal communication▪ Restricted areas of interest▪ Limited imaginative play▪ A need for sameness
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Mental retardation
Mental retardation is a serious and lifelong disability that places heavy demands on society and the health system
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American Association on Mental Retardation, 1992
“ mental retardation is not something you have, like blue eyes or a bad heart, nor is it something you are, like short or thin. It is not a medical disorder or a mental disorder… mental retardation reflects the “ fit “ between the capabilities of individuals and the structure and expectations of their environment. “
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Definition
MR is accepted as having three components:
1) Significantly abnormal intellectual performance, generally determined by a test of intelligence
2) Onset during development before the age of 183) Impairment of the ability to adapt to the environment
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Global developmental delay
Reserved for children five years of age or younger
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Global developmental delay
Global developmental delay (DD) describes significant delay in two or more of the following areas:
Cognition Speech/language Gross/fine motor skills Social/personal skills Daily living
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Prevalence
Prevalence: 1% - 3%
Mild MR occurring 7-10 times more frequently than moderate or severe MR. Mild MR: 29.8/1000 Mod-severe MR: 3.8/1000
In Iranian population: 1.8 – 2.7%
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Why diagnosis?
Estimating the recurrence risk in future pregnancies Prenatal diagnosis Minimizing the number of diagnostic procedures Short-term and long-term prognosis Treatment options
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Recurrence risk
Variable depending on the etiology
From very low ( the same as normal population) to 50% and even in rare situations to 75 -100%
Irrespective of etiology, empiric risk: 8.4%
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Recurrence Risks for Severe MR
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Study Brothers Sisters All SibsMale index caseHerbst and Baird (1982)
1 in 12 1 in 33 1 in 18
Bundey et al. (1985)
1 in 10 1 in 20 1 in 13
Female index caseHerbst and Baird (1982)
1 in 22 1 in 17 1 in 19
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Classification
Severity:
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• (IQ : 50-70)Mild
• (IQ : 35-50)Moderate
• (IQ : 20-35) Severe
• (IQ < 20)Profound
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Classification
Pedigree analysis:
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Sporadic
Familial
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Etiology
Genetic
Non-genetic
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Prenatal and perinatal events
Infections
Environmental factors
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Genetic causes of sporadic MR
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Genetic causes of sporadic MR
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33%
3%9%
1%
55%
Chromosomal abnormalitiesFragile-X syndromeX-linked MRInborn errors of metabolismDe novo dominant mutations
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Genetic causes of sporadic MR
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9%
24%
3%9%1%
55%
Microscopic aberrationsSubmicroscopic aberrationsFragile-X syndromeX-linked MRInborn errors of metabolismDe novo dominant mutations
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Submicroscopic Cromosomal Abnormalities
Subtelomeric Rearrangements• 0.5-15%• Unselected patients: 5%
Common Microdeletion and Microduplication (CMMSs) Syndromes• 5.8-9.5%
Genomic Copy Number Variations (CNVs)• 10-17%
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Diagnostic approach to sporadic MR
Guidelines based on the assessment of1. Chromosomal abnormalities
▪ Microscopic▪ Submicroscopic
2. Fragile-X syndrome
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Diagnostic approach to MR
2005 (Van Karnebeek et al.) For all the patients:
1. Family history, neurologic and dysmorphologic exam2. Karyotype3. Assessment of Fragile-X syndrome
For highly selected patients:1. Investigation of Subtelomeric aberrations (FISH) 2. Investigation of common microdeletion and
microduplication syndromes (CMMSs)
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Diagnostic approach to MR
Many studies during 2005-2010
1. Karyotype2. Assessment of Fragile-X syndrome3. Assessment of DNA copy number differences
(Array-CGH, MLPA, …)
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Diagnostic approach to MR
2009 (Hochstenbach et al.), retrospective investigation of 36325 patients karyotype
2010 (Miller et al.), analysis of array testing on 21698 patients
Conclusion:chromosomal microarray is a first-tier clinical
diagnostic test
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Diagnostic Methods
Karyotype Assessment of fragile-X syndrome FISH MLPA Array-based techniques
Array-CGH SNP Array
Exome sequencing Next-generation sequencing
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Karyotype
The first technique for studying chromosomal abnormalities
Advantages: Genomic Detection of balanced abnormalities
Disadvantages: Low resolution (3-5 Mb) Labor intensive
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FISH
The first molecular cytogenetic technique
Advantages: Higher resolution
Disadvantages: Limited tergets
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MLPA
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MLPA
Advantages: Investigation of 40-50 genomic regions in a single reaction Quantitative results (carrier detection) Detection of duplications High-throughput Universal primers Relatively inexpensive
Disadvantages: Inability to detect
▪ Balanced abnormalities▪ Low-level mosaicism▪ Novel DNA copy number differences
Limited genomic targets
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Array-CGH
BAC-array CGH Oligonucleotide
array-CGH
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SNP array
Advantages: Very high resolution
(>1000000 probes) Detection of LOH
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Next-generation sequencing
Promising technique in detecting novel genetic changes (CNVs, single gene disorders)
Technique of choice in near future
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Materials and Methods
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Overview of the study design
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Sporadic Patients
Clinical screening
Laboratory investigati
ons
Familial patients
Clinical screening
QuestionnaireScoring
Laboratory investigatio
ns of selected families
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Sporadic patients
Inclusion criteria: Developmental delay (DD)or Mental retardation (MR)or Multiple congenital anomalies (MCA)
Exclusion criteria: Known inborn errors of metabolism Documented non-genetic causes of DD/MR
▪ Neonatal uncontrolled icter▪ Perinatal events
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Familial patients
Inclusion criteria: The presence of at least two individuals in the first
degree relatives affected with DD/MR/MCA
Exclusion criteria: Families with known genetic causes
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UNIQUE SAMPLE SET
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Sampling
Place: Imam Khomeini Hospital
Duration: 1386 (Aban) -1390 (Mehr)
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Screening phase
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Completing the questionnaire
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Data extraction
Drawing pedigree based on the questionnaires data Data entry to the Progeny
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ScoringPhase 1
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Scoring criteria
Number of affected individuals in siblings(example: 4 affected persons 1+2+3+4=10)
Affected parents: each of them 2 scores
Prenatal onset (sonographic positive findings)
Multiple organ involvements: each one 1
Non-consanguinity of parents: 1
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Laboratory phase
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Genetic counseling
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Drawing pedigreePast medical history
PrenatalPerinatalpostnatal
Recording past medical history in detail
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Pedigree drawn real time
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Pedigree drawn by progeny
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Informed consent signed before sampling by patients and their guardians
Informed concent
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Physical examination
Measurement of height and head circumference
Neurologic exam Dysmorphologic exam
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Photography
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Example: accessory nipples in 3 affected individuals in a family
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Sampling
Heparinized Blood EDTA Blood
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Designing patients database using Progeny software
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Stepwise approach to MR
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Karyotype
Assessment of Fragile-X syndrome
Assessment of subtelomeric rearrangements (MLPA)
Assessment of CMMSs (MLPA)
Scoring
Array-CGH
SNP Array Rafati M
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Karyotype
G-Banding: Lymphocyte culture Harvest Slide preparation Giemsa staining
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Fragile-X syndrome
PCR screening: Determining CGG repeat expansion of FMR1 gene
Triplet-primed PCR: Determining pre-mutations and full mutations
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MLPA optimization
Step 1: Detection of common chromosomal aneuploidies
Step 2: Assessment of deletions and duplications of F8 gene in
severe hamophilia A patients
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Subtelomeric rearrangements
Techniques: MLPA
▪ Using two alternative probe mixes▪ Family study to rule out polymorphisms
FISH (in case of positive findings)
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CMMSs
Technique:
MLPA Using 1 screening and 2 confirmatory probe mixes
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Investigated Syndromes:
Cri du Chat syndrome MECP2 / Xq28 duplication
1p36 deletion syndrome
DiGeorge syndrome 22q Rubinstein-Taybi syndrome
2p16 microdeletion
DiGeorge region 2 Smith-Magenis syndrome 3q29 microdeletion
Langer-Giedion syndrome Sotos syndrome 5q35.3 9q22.3 microdeletion
Miller-Dieker syndrome WAGR syndrome 15q24 deletion syndrome
NF1 microdeletion syndrome
Williams syndrome 17q21 microdeletion
Prader-Willi / Angelman Wolf-Hirschhorn 4p16.3 22q13 / Phelan-Mcdermid
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Scoring Phase 2
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Scoring criteria
Number of affected individuals1+2+3+…
Non-consanguinity +3
Recurrent abortion +2
Pedigree analysis (Dominant) +3
Multiple congenital anomalies +3
Severity of MR Moderate +2Severe
+3
Affected parents (each of them) +2
Dysmorphism+2
Negative scores
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Number of normal siblings -1-2-3…
Consanguinity-3
Pedigree analysis (Recessive) -3
Pedigree analysis (X-linked) -3
•Positive scores
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Selection
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array-CGHRafati M
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Array-based techniques
Array-CGH Agilent 44K EmArray Cyto6000
SNP Array Illumina humanOmni 1-Quad 1134514 markers Resolution: 6 kbp
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Results
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Screening phase
Number of families Number of patients
Sporadic patients 279 279
Familial patients
• First step 551 1260
• Second step (questionnaire) 355 647
Total 830 1539
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Laboratory phase
Number of families Number of patients
Sporadic 55 55
Familial 116 371
Total 171 426
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Screening phasesporadic patients
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Screening phasesporadic patients
Diagnosis Number of patients Diagnostic method
Chromosomal abnormalities Numerical 23 karyotype
Structural 9 karyotype
• Mosaicism 4 karyotype
Syndromes DiGeorge Syndrome 2 FISH
Miller-Dieker Syndrome 1 Clinical and paraclinical findings
Rubinstein-Taybi Syndrome 1 Clinical and paraclinical findings
Kabuki Syndrome 1 Clinical and paraclinical findings
Hallermann-Streiff Syndrome 1 Clinical and paraclinical findings
Laurence-Moon-Bardet-Biedl Syndrome
1 Clinical and paraclinical findings
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Continued
Single gene disorders Phenylketonuria (PKU) 3 Measurement of Phenylalanine level
Methylmalonic Acidemia (MMA) 1 MS/MS
Aminoacids HPLC Maple Syrup Urine Disease
(MSUD)2
GM2 Gangliosidosis (Tay-Sachs Disease)
2 Enzyme activity assessment of Hexosaminidase A
GM2 Gangliosidosis (Sandhoff Disease)
1 assessment of Hexosaminidase A and B Enzyme activity
Niemenn-Pick Disease 1 Mucopolysaccharidosis type I
(MPS1)1
Krabbe Disease 1 Whole gene sequencing
Non-ketotic Hyperglycinemia (Glycin Encephalopathy)
1 Whole gene sequencing
Metachromatic Leukodystrophy 1 assessment of arylsulfatase Enzyme activity
Saposine-B Deficiency 1 Whole gene sequencing
Total 582/15/2012
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Screening PhaseFamilial Patients
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Number of affected individuals in the first degree relatives
2 3 4 5 6 8 12
49.40%
30.20%
10.60%
4.70%
1.70%0.40%
0.00%
36.30% 36.30%
17.50%
5.00%
2.00%2.00%
0.98%
Screening phase
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Laboratory Phase Findings
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MLPA optimization
STEP 1: Prenatal diagnosis of chromosomal aneuploidies As a stand-alone test:
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74 amniotic fluid samples
5 Trisomy 21
1 XXY
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MLPA optimization
STEP 2:
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347 Previously Investigated Severe
Hemophilia A Patients
10 Patients No PCR amplification in
some exons
Confirmation of deletionsDetermining the deletion
extensionCarrier detection
1 patient with no mutation detected
Duplication of exon
24
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MLPA
Optimization
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Sporadic MRGenetic test Abnormal finding Number (%)Karyotype Trisomy 21 4 (7.2%)
Partial deletion of 9p telomeric region
1 (1.8%)
Fragile-X syndrome CGG repeat expansion 1 (1.8%)Subtelomeric rearrangements
Duplication of 2p telomeric region
1 (1.8%)
CMMSs Williams syndrome 1 (1.8%)Cat Eye syndrome 1 (1.8%)
Cytogenetically visible chromosomal abnormalities
5 (9%)
Cryptic chromosomal abnormalities
3 (5.4%)
Fragile-X syndrome 1 (1.8%)Total 9 (16.3%)/55
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Sporadic Williams syndrome
Summary of clinical findings: Global Developmental delay Mental retardation Facial dysmorphic features
▪ Micrognathia ▪ Midface hypoplasia▪ Wide mouth▪ Prominent upper and lower lips▪ Short philtrum▪ Full nose tip▪ Periorbital fullness
Non-facial dysmorphic features▪ Additional skin creases of the fingers
Personality▪ Outgoing overfriendly personality
Investigations with normal results Karyotype Visual status
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Dysmorphic features
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MLPA analysis
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Cat Eye syndrome
Summary of clinical findings: Developmental delay Hypotonia Facial dysmorphic features
▪ Dolichocephaly ▪ Cleft palate▪ Low-set ears▪ External ears deformity
Failure to thrive (FTT) Congenital Heart Disease (CHD)
Large ASD with left to right shunt Bilateral inguinal hernia
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MLPA analysis
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Patient resultsInvestigated genes using 4
MLPA probemixes Genes Genomic regions
Gain P070 IL17RA
Cat eye syndrome region
- SLC25A18Gain P036 BID
- MICAL3- USP18- LCR22-A
Gain P064 CLTCL1
commonly-
deleted DiGeorge region
DiGeorge region
- HIRAGain P064 CDC45Gain P245 / P064 CLDN5Gain P245 GP1BB
- TBX1- TXNRD2- DGCR8
Gain P064 ARVCFLCR22-B
- ZNF74Gain P064 KLHL22
- MED15LCR22-C
Normal P245 / P064 SNAP29
Not Investigated
- LZTR1- LCR22-D- HIC2- PPIL2- TOP3B
LCR22-E- RTDR1- GNAZ- RTDR1- RAB36
LCR22-F- SMARCB1
LCR22-G- SNRPD3
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Laboratory PhaseFamilial Patients
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Karyotype
All normal Inversion of chromosome 2 (normal variation)
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Fragile-X Syndrome
Description Number (%)
Diagnosed families 8 (6.9%)
Diagnosed patients 23
Males 21 (91.3%)
Females 2 (8.7%)
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Fragile-X syndrome
Control (unaffected) Control (affected) Contrlo (unaffected) MR-63 (No Band) MR-35 (No Band) Control (unaffected) Control negative (No DNA) Size marker (100-1000bp)
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1 2 3 4 5 6 7 8 1 2 3 4 5 6
•Control (affected)•MR-69-MO (Mother)•MR-69-S2M (Normal male)•MR-69-P2M (Affected male)•Size marker (100-1000bp)•Control negative (No DNA)
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Pedigree
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Subtelomeric Rearrangements
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Family ID MLPA probemix Results Type of aberrations
MR-92P036
P070
Partial monosomy of 13qter and partial trisomy of 9pter
Clinically significant pathogenic changes
MR-51P036
P070Xpter and Xqter Gain
MR-130P036
P0701pter Gain
Aberrations with unknown clinical significance
MR-130P036
P07012qter Gain
MR-1 P070 3pter loss
Genomic variants
MR-1 P070 4qter Gain
MR-61 P070 4qter Gain
MR-105 P070 4qter Gain
MR-109 P036 21q11.2 Gain
MR-116 P070 4qter loss (homozygous)Rafati M
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Unbalanced Subtelomeric Rearrangement
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Pedigree
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Family history
Non-consanguineous healthy parents History of one abortion at 12 weeks of gestation Two infantile deaths due to Multiple Congenital
Anomalies (MCA) One affected child with developmental delay/mental
retardation (DD/MR)
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Clinical findings
Second pregnancy:
Cleft lip/cleft palate Intestinal atresia Death at 2 months of age Failure to thrive (FTT)
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Clinical findings
Third pregnancy: (A 12-year old girl )
At birth: Wt: 2850 gr HC: 33 cm Microcephaly Poor sucking (feeding difficulties) Facial Dysmorphism:
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Facial and non-facial dysmorphic features
▪ Hypertelorism ▪ Ptosis ▪ Epicanthal fold▪ Low-set ears▪ Broad nasal bridge▪ Long forehead
▪ Broad distance between 1st and 2nd toes
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Clinical findings
Developmental delay/mental retardation Intractable seizure (onset: 3.5 y) Behavioral disorder Autistic features
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Previous genetic tests
Karyotype: 46,XX
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Family history
Fourth pregnancy: Prenatal:
▪ IUGR▪ Choanal atresia▪ Polyhydramnios▪ Intestinal obstruction
Prenatal diagnosis (Cordocentesis): karyotype: normal
Postnatal: Club foot Duodenal atresia Death at 3 months of age
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Assessment of subtelomeric rearrangements
Technique: MLPA Result:
• Deletion of 13q telomeric region
• Duplication of 9p telomeric region
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FISH analysis
Confirmatory test: FISH Result:
• Partial monosomy of 13qter
• Partial trisomy of 9pter
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9pter
13qter
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Investigation of the Parents
Technique: Metaphase FISH
Mother: normal
Father: balanced translocation of 13q and 9p telomeric regions
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9pter
13qter
Chr 9
Chr 13
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Assessment of CMMSs
A familiy with hereditary Williams syndrome Others: no deletion or duplication detected
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Hereditary Williams Syndrome
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Patient 1 (the proband)
Clinical findings: ▪ Developmental delay (most prominently in walking)▪ Mental retardation▪ Growth retardation
Dysmorphic features: ▪ Microcephaly▪ Micrognathia▪ Midface hypoplasia▪ Wide mouth▪ Prominent lower lip▪ Long philtrum▪ Strabismus
Personality: outgiong overfriendly personality
Echocardigraphy Pulmonary stenosis 2/15/2012
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Patient 2 (the father)
Summary of clinical and paraclinical findings At age of 37-year:
Ht: 165.5 cm (<5th centile) HC: 53.5 cm (<5th centile)
Dysmorphic features: Micrognathia Strabismus Periorbital fullness Long philtrum
No hypertension (blood pressure: 125/80 mmHg) Echocardiography:
Mild aortic stenosis Tricuspid valva regurgitation Mitral valave regurgitation Mitral valve prolapse
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MLPA analysis
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Williams syndrome critical region
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Hereditary Williams syndrome
Reference Number of Reported Families (Cases)
Method of WBS Ascertainment
Molecular Confirmation
TechniqueMorris et al. (1993) 3 (6) Clinical manifestations Quantitative southern
analysis (Ewart et al (1993) confirmed the deletion in two of the
families]Sadler et al. (1993) 1 (2) Clinical manifestations -
Ounap et al. (1999) 1 (2) Clinical manifestations FISH1 (just the son was investigated, the mother
was not available)
Pankau et al. (2001) 2 (4) Clinical manifestations FISH (both of the patients)
Metcalfe et al. (2005) 1 (2) Clinical manifestations FISH (both of the patients)
The present study 1(2) Screening for cryptic chromosomal abnormalities
MLPA2 ((both of the patients))
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Findings of array-based techniques
Clinically significant findings: Related to familial MR: 0 Unrelated to familial MR: 1
▪ Charcot-Marie-Tooth
Benign copy number changes: 3 families
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Familial MR and CMT
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Array Result
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Array result
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case a CGH ISCN nomenclature # probesSize
loss(bp) Size gaine (bp) Clinical target
MR-82 arr chg 6P25.3 (204528-284802)X1 6 80274 Benign CNC
arr chg 8P23.1 (7040596-8117330)X1 6 1076762 Benign CNC
arr chg 10q26.3 (135104029-135227522)X3 6 123,494<500Kb, CYP2E1 gene, SYCE1 gene
arr chg 16p11.2-11.1 (34339543-34584850)X3 5 245,308Benign CNC
arr chg 17p12 (14052497-15382791)X3 38 1,330,295
CNC with significance pathogenic effect,
COX10, HS3ST3B1, PMP22 Genes
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Family study
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Loss of heterozygosity (LOH)
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1 p31.3 p31.1 q12 q32.1 q41 q43 q44
Base Position 25,624,620 50,471,950 75,319,280 100,166,610 125,013,940 149,861,270 174,708,600 199,555,930 224,403,260 249,250,590
Cytogenetic Band p3 p3p3p3p3 p… p p32.p3 p31.1 p2p2pp21pp21p13 p1 p p1 q12 q21q2q21 q q23 q2q2 q…q31q3q31q32.1 q3q3 q41 q q43 q44Sequence (+) No sequence data file found for this chromosome.
LOC643837LOC643837LOC643837LOC643837LOC643837LOC643837AK091100FAM41C KIAA1922
IGSF21
BC040588BC131526CAMK2N1CAMK2N1CAMK2N1MUL1 ARCH
ZBTB8OSC1orf94
RRAGCRRAGCRRAGCRRAGCMYCBP
AKR1A1
ELAVL4DMRTA2
TMEM48
YIPF1YIPF1YIPF1YIPF1
NFIA
AK3L1
SGIP1SGIP1SGIP1SGIP1
SGIP1
PIN1LBC041341
IFI44L
KIAA0786LPHN2LPHN2LPHN2
LPHN2
U80773HFM1
CDC7
PTBP2PTBLPPTBP2
PTBP2
DPYD
LPPR4
BC043293
PRMT6
BC053344RBM15RBM15RBM15
BC069739
SLC16A1 CR936796FLJ00310DQ579288DQ586768BC053679BC071797BC029473DQ596563
TAGLN2
CS1SLAMF7SLAMF7
F11R
F11RF11R
FCER1G
FMO3
TNFSF18
TNFSF18TNFSF4
RFWD2
PAPPA2PAPPA2SEC16B
RGL1
FAM5C
FAM5CDBCCR1L
RGS18
RGS21RGS21
KCNT2
KCNT2
LRRN2
LRRN2SLC26A9
FAM72AFAM72A
CD46sytXIV
C1orf133
TGFB2
TGFB2SLC30A10
DUSP10DUSP10
DQ584993
TLR5
PARP1
SIPA1L2
SIPA1L2
BC016972RYR2RYR2
LOC339535
FMN2
FHOR2M7
OR2M7
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Pedigree
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Discussion
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Outline
Summary of results
Comparison of “Familial” MR with “Sporadic” MR
Comparison of the current study with the previous investigations of “Familial” MR
Diagnostic algorithms
Suggestions
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Sporadic MR
0.00%4.00%8.00%
12.00%16.00%
Sporadic MR (current study)Sporadic MR (previous stud-ies)
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Summary of results (familial MR)
0%1%2%3%4%5%6%7%
Familial MR (current study)
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“Familial” versus “Sporadic” MR
371 Patients from 116 Families
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Karyotype
Sporadic MR: 9.5% Present study: 0% (371
patients from 116 families)
Sporadic MR
Familial MR
0.00%
2.00%
4.00%
6.00%
8.00%
10.00%
Cytogenetically vis-ible abnormalities
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Fragile-X syndrome
Sporadic MR: 2-5% (mean: 3%)
Present study: 7% (371 patients from 116 families)
Sporadic MR
Familial MR0%1%2%3%4%5%6%7%
Fragile-X syndrome
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Subtelomeric aberrations
Sporadic MR: 0.5-9% (mean: 5%)
Present study: 0.9% (248 patients from 108 families)
Sporadic MR
Familial MR0%
1%
2%
3%
4%
5%
Subtelomeric Rearrangements
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Previous subtelomeric investigations
Reports in support of the importance of “positive family history” (most of the studies)
Reports with conflicting data
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Positive family history, as a predicting factor?
Positive family history is the main single predicting factor of subtelomeric rearrangements
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Conflicting data in the previous studies
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The current study
Our results could be compared with the prevalence of hereditary subtelomeric aberrations (0-2%)
The present study finding:Severity of MR, dysmorphism and multiple congenital
anomalies are more important than positive family history by alone
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CMMSs
Sporadic MR: 5.8-9% Present study: 0.9% (245
patients from 107 families)
More coverage of common syndromes in this study (18 CMMSs)
Sporadic MR
Familial MR0%
1%
2%
3%
4%
5%
CMMSs
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CMMSs
Mechanisms: De novo: misalignment during recombination due to LCRs Hereditary:
▪ Balanced translocation in the parents▪ Autosomal dominant transmissions
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Array-based techniques
Sporadic MR: 10-17%
The findings were not explanatory for “familial” mental retardation
Sporadic MR
Familial MR
0%2%4%6%8%
10%12%14%16%18%
Genomic CNVs
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Interpretation
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“Familial” versus “Sporadic” MR
Karyo
type
Fragil
e-X Sy
ndrom
e
Subte
lomeri
c cha
nges
CMMSs
Other g
enom
ic CNCs
048
1216
Familial MRSporadic MR
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Familial versus Sporadic MR
Sporadic MR
Familial MR0.00%5.00%
10.00%15.00%20.00%25.00%30.00%35.00%40.00%
Chromosomal Abnormali-tiesFragile-X syndrome
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Interpretation
2011, Jehee et al.
Karyotype + MLPA investigations of subtelomeric rearrangements and CMMSs = detection of 76.5% of all chromosomal abnormalities detected by whole genomic array screening
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Conclusion
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No pathogenic
CNV is expected in
the remained families
More coverage on CMMSs
KaryotypeMLPA,
subtelomeric aberrations
MLPA, CMMSs75.6% No pathogenic
change in array-CGH study of highly selected
patients
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Low contribution of chromosomal abnormalities to “Familial” MR
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Autosomal dominant single gene disorders
2009-2011, Hamdan et al. Investigation of 197 synaptic genes (glutamate receptor, …)
in 95 patients: 11 new mutations found
2011, Nature, Vissere et al. Exome sequencing of 10 patients with sporadic MR: 6
pathogenic mutations found (60%) More than all of the previous investigations
New paradigm of de novo dominant mutations in MR
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New classification
Sporadic MR Familial
MR
0.00%10.00%20.00%30.00%40.00%50.00%60.00%70.00%80.00%90.00%
Chromosomal Abnormali-tiesFragile-X syndromeSingle gene disorders
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Comparison with the previous studies
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Previous investigations of Familial MR
Few published studies Studies on Iranian population
Autosomal recessive MR (2011, Najmabadi et al.) X-linked MR
A. R. Pouya et al. Fragile X syndrome screening of families with consanguineous and non-consanguineous parents in the Iranian population. European Journal of Medical Genetics (2009), doi:10.1016/j.ejmg.2009.03.014
Najmabadi H et al. Homozygosity mapping in consanguineous families reveals extreme heterogeneity of non-syndromic autosomal recessive mental retardation and identifies 8 novel gene loci. Hum Genet. 2007 Mar;121(1):43-8. Epub 2006 Nov 21
Kahrizi K et al. An autosomal recessive syndrome of severe mental retardation, cataract, coloboma and kyphosis maps to the pericentromeric region of chromosome 4. Eur J Hum Genet. 2009 Jan;17(1):125-8. Epub 2008 Sep 10.2/15/2012
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Parents Relationship
55%
15%
6%
24%
First CousinSecond CousinFar relatedNon-con-san-guineous
76%
24%
Parents Rela-tionship
ConsanguineousNon-consanguineous
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Pedigree analysis
78%
5%17%
Pedigree analysisAutosomal Recessive Established X-linkedPutative X-linked
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Sample Set Characteristics
40%
60%
Selected families
ConsanguineousNon-consanguineous
39%
49%
6%2% 4%
Screening phaseNon-consanguinousFirst CousinFirst Cousin, Once removedSecond CousinFar related
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Pedigree analysisthe present study
66%
24%
10%
Chromosomal or Autosomal Dominant
Autosomal Recessive
X-linked
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Comparison of studied sample sets
Autosomal Recessive X-Linked
Autosomal Dominant
0%10%20%30%40%50%60%70%80%
The present studyNajmabadi et al.
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Diagnostic Algorithms
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Sporadic MR
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Karyotype
Numerical or Structural Chromosomal Abnormality Normal
Assessment of Fragile-X syndrome
Fragile-X syndrome No CGG repeat expansion
Availability of array-based techniques + cost
Aavailable
array-CGH orSNP array
Pathogenic Copy Number
Variation
No pathogenic
change
Exome Sequencing (de novo dominant mutations)
New Suggestion
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Karyotype
Numerical or Structural Chromosomal Abnormality Normal
Assessment of Fragile-X syndrome
Fragile-X syndrome No CGG repeat expansion
Availability of array-based techniques + cost
Aavailable
array-CGH orSNP array
Pathogenic Copy Number
Variation
No pathogenic
change
Exome Sequencing (de novo dominant mutations)
Not Available
MLPA (Subtelomeric Rearrangements + CMMSs)
Subtelomeric Aberration
CMMSs
No pathogenic
change
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Familial MR
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Pedigree
Analysis
Autosomal Recessive
Assessment of Known
single gene disorders
Mutation Detected
No Mutation Detected
Exome Sequencing
Next-generation Sequencing
X-linked
Assessment of Fragile-X Syndrome
Fragile-X
Syndrome
No CGG repeat
Expansion
Assessment of Known XLMR
Mutation Detected
No Mutation Detected
Exome Sequencing
Next-generation Sequencing
Autosomal Dominant
DysmorphismMultiple Congenital
AnomaliesRecurrent Abortion/Infertility
Yes
Karyotype
Numerical or Structural
AbnormalitiesNormal
Availability of array-based techniques +
cost
Aavailable
array-CGH or
SNP array
Pathogenic Copy Number
VariationNo pathogenic
change
Exome Sequencing (de novo dominant
mutations)
Not Available
MLPA (Subtelomeric Rearrangeme
nts + CMMSs)
Subtelomeric Aberration
CMMSs
No pathogenic change
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Pedigree
Analysis
Autosomal Recessive
Assessment of Known single gene disorders
Mutation Detected
No Mutation Detected
Exome Sequencing
Next-generation Sequencing
X-linked
Assessment of Fragile-X Syndrome
Fragile-X
Syndrome
No CGG repeat
Expansion
Assessment of Known XLMR
Mutation Detected
No Mutation Detected
Exome Sequencing
Next-generation Sequencing
Autosomal Dominant
DysmorphismMultiple Congenital
AnomaliesRecurrent Abortion/Infertility
Yes
Karyotype
Numerical or Structural
AbnormalitiesNormal
Availability of array-based techniques +
cost
Aavailable
array-CGH or
SNP array
Pathogenic Copy Number
VariationNo pathogenic
change
Exome Sequencing (de novo dominant
mutations)
Not Available
MLPA (Subtelomeric Rearrangeme
nts + CMMSs)
Subtelomeric Aberration
CMMSs
No pathogenic change
No
Exome Sequencing
Next-generation sequencing
Unknown
Assessment of Fragile-X
syndrome
Fragile-X
syndrome
No CGG repeat
expansion
New Suggestion
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List
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Acknowledgements
Comprehensive Genetic Center, Imam Khomeini Hospital
Department of Medical Genetics, TUMS Hope Generation Foundation Tehran Welfare Organization Gene clinic
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Thanks for Your Attention
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