prenatal diagnosis, case discussion

1. SR Ghaffari, MSc MD PhD
MR Rafati, MD PhD
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5.  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
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6. c.3903+6C>T
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7. New
investigations
The pathogenic
mutation is confirmed
in the patient
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8. New
investigations
The pathogenic
mutation is not
detected in the
patient’s spouse
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9.  The pregnancies of this family are not at increased risk of
Wilson disease
 Prenatal diagnosis ofWilson disease is not recommended
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13. Results
Based on pedigree analysis
Hereditary chromosome
abnormality is highly suspected
Investigations of the affected
daughter (6Y):
Karyotype: 46XX
MLPA using Subtelomeric
probemixes
Partial trisomy of 4q
Partial 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)
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14.  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
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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)
Prenatal diagnosis using MLPA
Technique
Prenatal diagnosis:
Fetus was unaffected
Control
Positive
Fetal
Sample
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19.  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 out
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20.  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.
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21.  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
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22.  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 twoTurkish patients with
Tyrosinemia type I
 Further investigations showed the same heterozygous
mutation in both phenotypically normal parents
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23. SequenceAnalysis
A: Father
B: Mother
C: Fetus
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24.  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
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27.  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.
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30.  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.
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34.  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.
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35. Family Members
ABCA4
c.6005+1delG
Phenotype
(Patient) Homozygous Retinitis Pigmentosa
(Spouse) Not Detected Normal
 Detected mutation in the patient: c.6005+1delG in ABCA4
Gene
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36. Sanger
Verification
The pathogenic
mutation is confirmed
in the patient
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37. Carrier testing
The pathogenic
mutation is not
detected in the
patient’s spouse
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38.  The pregnancies of this family are not at increased risk of
Retinitis pigmentosa
 Prenatal diagnosis of retinitis pigmentosa is not
recommended
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42. Mutation
detection
The pathogenic mutation is
confirmed in the patient with
Wolfram syndrome
Detected mutation:
WFS1: c.1362_1377del16
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43.  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
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44. Mutation
detection
The pathogenic mutation is
detected in the patient with
Wilson disease
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47.  The couples are both carrier of the detected mutation in
ATP7B gene (carrier ofWilson disease)
 Genetic investigation ofWilson disease is indicated in their
children for early diagnosis and treatment if necessary.
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