prenatal diagnosis, case discussion

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