prenatal diagnosis, case discussion
TRANSCRIPT
1
Prenatal diagnosisCase Discussion
92th Seminar in Clinical Genetics, Tehran, Iran
17 December 2015
SR Ghaffari, MSc MD PhDMR Rafati, MD PhD
12/17/2015
2
The First Family
12/17/2015
3
Mutation versus Polymorphism?!
12/17/2015
4
Family pedigree
12/17/2015
5
Previous Investigations
Variants detected in the affected individual (Wilson disease):ATP7B Gene c.1919C>T Homozygous Exonic (known mutation) c.3903+6C>T Homozygous Intronic
Variants investigated in his spouse (not affected):ATP7B Gene c.1919C>T Not Detected c.3903+6C>T Heterozygous
12/17/2015
6
A known non-pathogenic variantc.3903+6C>T
12/17/2015
7
New investigations
The pathogenic mutation is confirmed in the patient
12/17/2015
8
New investigations
The pathogenic mutation is not detected in the patient’s spouse
12/17/2015
9
Conclusion
The pregnancies of this family are not at increased risk of Wilson disease
Prenatal diagnosis of Wilson disease is not recommended
12/17/2015
10
The Second Family
12/17/2015
11
Cryptic Chromosome Abnormality
12/17/2015
12
Family pedigree
12/17/2015
13
Results
Based on pedigree analysis
Hereditary chromosome abnormality is highly suspected
Investigations of the affected daughter (6Y):
Karyotype: 46XXMLPA using Subtelomeric probemixes
Partial trisomy of 4qPartial monosomy of 5p
Chr pos Length (nt) Ratio
01p 130 Normal (0.94)01q 306 Normal (1.03)02p 137 Normal (0.92)02q 314 Normal (0.92)03p 144 Normal (0.95)3q 322 Normal (1.05)04p 151 Normal (1.03)4q 330 Gain (1.51)05p 158 LOH (0.5)05q 338 Normal (0.95)06p 165 Normal (0.96)06q 346 Normal (1.02)07p 172 Normal (1.1)07q 354 Normal (1)08p 179 Normal (0.95)08q 362 Normal (0.99)09p 186 Normal (1.04)09q 370 Normal (1)10p 194 Normal (0.96)10q 378 Normal (1.08)11p 202 Normal (0.87)11q 386 Normal (1.09)12p 208 Normal (0.91)12q 394 Normal (0.93)13p 218 Normal (0.97)13q 402 Normal (1.14)14p 226 Normal (0.93)14q 410 Normal (1.21)15p 234 Normal (0.94)15q 418 Normal (1)16p 242 Normal (0.91)16q 426 Normal (1.04)17p 250 Normal (1.04)17q 434 Normal (0.97)18p 258 Normal (0.96)18q 442 Normal (1.07)19p 266 Normal (0.93)19q 450 Normal (1.03)20p 274 Normal (0.94)20q 458 Normal (1.22)21p 282 Normal (1.06)21q 466 Normal (1.13)22p 290 Normal (1.03)22q 474 Normal (1.12)X/Yp 298 Normal (1.07)X/Yq 482 Normal (1.15)
12/17/2015
14
Metaphase FISH
Metaphase FISH confirmed Partial trisomy of 4q and Partial monosomy of 5p in the patient
Investigation of the parents: Father: normal Mother: subtelomeric translocation between 4qter and
5pter
12/17/2015
15
Chr pos Length (nt) Ratio Ratio
01p 130 Normal (0.94) Normal (0.9)01q 306 Normal (1.03) Normal (1.06)02p 137 Normal (0.92) Normal (0.92)02q 314 Normal (0.92) Normal (0.99)03p 144 Normal (0.95) Normal (0.92)3q 322 Normal (1.05) Normal (1.07)04p 151 Normal (1.03) Normal (0.95)4q 330 Gain (1.51) Normal (1)05p 158 LOH (0.5) Normal (0.9)05q 338 Normal (0.95) Normal (1.02)06p 165 Normal (0.96) Normal (0.87)06q 346 Normal (1.02) Normal (1.02)07p 172 Normal (1.1) Normal (0.89)07q 354 Normal (1) Normal (1.14)08p 179 Normal (0.95) Normal (0.93)08q 362 Normal (0.99) Normal (0.94)09p 186 Normal (1.04) Normal (0.95)09q 370 Normal (1) Normal (1.08)10p 194 Normal (0.96) Normal (0.97)10q 378 Normal (1.08) Normal (1.02)11p 202 Normal (0.87) Normal (0.89)11q 386 Normal (1.09) Normal (1.08)12p 208 Normal (0.91) Normal (0.95)12q 394 Normal (0.93) Normal (1)13p 218 Normal (0.97) Normal (1.01)13q 402 Normal (1.14) Normal (1.05)14p 226 Normal (0.93) Normal (0.95)14q 410 Normal (1.21) Normal (1.05)15p 234 Normal (0.94) Normal (1)15q 418 Normal (1) Normal (1.04)16p 242 Normal (0.91) Normal (0.92)16q 426 Normal (1.04) Normal (1.03)17p 250 Normal (1.04) Normal (0.96)17q 434 Normal (0.97) Normal (1.04)
Prenatal diagnosis using MLPA Technique
Prenatal diagnosis:Fetus was unaffected
Control Positive
Fetal Sample
12/17/2015
16
The Third Family
12/17/2015
17
Prenatal Diagnosis of Tyrosinemia Type 1 Using Next Generation Sequencing Technique
12/17/2015
18
Family pedigree
12/17/2015
19
Family History
II-I: presented at emergency department with agitation and restlessness at 1 month of age and died a few days later without definite diagnosis.
II-2 was a 21-year old boy with normal growth and development.
II-3:
Failure to thrive Hepatosplenomegaly Rickets Increased level of tyrosine (375 μmol/L; reference range <145
μmol/L) The patient died at the age of 9 months before any molecular
genetic investigation carried out12/17/2015
20
PND
The family requested prenatal diagnosis of tyrosinemia while the mother was at 12 weeks of gestation
Following a comprehensive genetic counseling session in which the benefits and limitations of this approach were thoroughly discussed, the parents opted to do fetal sampling for genetic testing.
12/17/2015
21
Method
Chorionic villous sampling Maternal contamination was then ruled out Mutation analyses of three genes associated with
tyrosinemia including FAH, TAT and HPD using Next Generation Sequencing
12/17/2015
22
Results
A heterozygous mutation (c.709C>T) in FAH gene was detected in the fetus.
This was a nonsense mutation leading to a premature stop codon and a truncated protein (p.Arg237Ter) which had previously been reported in in two Turkish patients with Tyrosinemia type I
Further investigations showed the same heterozygous mutation in both phenotypically normal parents
12/17/2015
23
Sequence Analysis
A: Father
B: Mother
C: Fetus
12/17/2015
24
Conclusion
successful application of next generation sequencing in prenatal diagnosis of even well characterized genetic disorders, when The time is a limitation factor More than one (specially large) responsible genes
are involved A “founder” or a “previously detected” mutation
is not present
Hence, the conventional molecular genetic investigations can not be employed
12/17/2015
25
The Forth Family
12/17/2015
26
NGS experience in Iran
12/17/2015
27
Family history
The family were concerned about the recurrence of the hearing impairment in their children
Based on pedigree analysis, the autosomal recessive inheritance was proposed and therefore the recurrence risk of hearing impairment in the fetus was 1 in 32 (30-40 times more than normal population)
Genetic investigation of the affected individual using NGS was recommended.
12/17/2015
28
Family pedigree
12/17/2015
29
NGS Analysis
12/17/2015
30
NGS Results
A homozygous mutation (c.2644_2644delG) in COL9A1 gene was detected in the patient.
This was a nonsense mutation leading to a premature stop codon and a truncated protein (p.Val882fs).
Carrier screening of the detected mutation in at risk family members including the couple was then recommended.
12/17/2015
31
The Fifth Family
12/17/2015
32
Advantages of Genetic Investigations using NGS
12/17/2015
33
Family pedigree
12/17/2015
34
Family history
High recurrence risk based on pedigree analysis Using gamete donation had been offered in
previous genetic counseling sessions
2 times ART with egg donation: no pregnancy
Genetic investigation and mutation analysis in the patient using NGS was recommended.
12/17/2015
35
Family Members ABCA4c.6005+1delG Phenotype
(Patient) Homozygous Retinitis Pigmentosa
(Spouse) Not Detected Normal
NGS Results
Detected mutation in the patient: c.6005+1delG in ABCA4 Gene
12/17/2015
36
Sanger Verification
The pathogenic mutation is confirmed in the patient
12/17/2015
37
Carrier testing
The pathogenic mutation is not detected in the patient’s spouse
12/17/2015
38
Conclusion
The pregnancies of this family are not at increased risk of Retinitis pigmentosa
Prenatal diagnosis of retinitis pigmentosa is not recommended
12/17/2015
39
The sixth Family
12/17/2015
40
Prenatal Diagnosis of Wolfram SyndromePGD of Wolfram Syndrome
High Recurrence Risk of Wilson Disease
12/17/2015
41
Family pedigree
12/17/2015
42
Mutation detection
The pathogenic mutation is confirmed in the patient with Wolfram syndrome
Detected mutation:WFS1: c.1362_1377del16
12/17/2015
43
Prenatal diagnosis of Wolfram Syndrome
The fetus was unaffected
After 2 years, the family requested preimplantation genetic diagnosis (PGD) of Wolfram syndrome
Genetic investigation of Wilson disease in the affected individual of the family was also recommended
12/17/2015
44
Mutation detection
The pathogenic mutation is detected in the patient with Wilson disease
12/17/2015
45
Carrier detection
12/17/2015
46
Family pedigree, new
12/17/2015
47
Carrier detection
The couples are both carrier of the detected mutation in ATP7B gene (carrier of Wilson disease)
Genetic investigation of Wilson disease is indicated in their children for early diagnosis and treatment if necessary.
12/17/2015
48
Thank You
12/17/2015