PRENATAL TESTING FOR GENETIC MUTATION CARRIER PARENTS

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PRENATAL TESTING FOR GENETIC MUTATION CARRIER
PARENTS

• Dr Ritika Bajaj
• MBBS, MS (OBGYN), Fellowship in Maternal Fetal
  Medicine (AIIMS, New Delhi)
• Consultant Fetal Medicine
• Jindal IVF Sant Memorial Hospital, Chandigarh

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IMPACT OF GENETIC DISEASE

• CHILDHOOD
• Genetic disorders account for-
• 50 of childhood blindness
• 50 of childhood deafness
• 50 of all cases of severe learning difficulties

• SPONTANEOUS MISCARRIAGES
• 40-50 of all recognized 1st trimester pregnancy
  loss have a chromosomal abnormality

• NEWBORN INFANTS
• 2-3 of neonates have at least one major
  congenital anomaly
• 50 of these are caused exclusively or partially
  by genetic factors
• Incidence of chromosomal abnormalities in
  neonates- 1 in 200
• Incidence of single gene disorders in neonates- 1
  in 100

As infectious causes of perinatal mortality
decline, the relative contribution of genetic
causes in this subgroup has increased
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PRESENTING COMPLAINTS IN CLINICAL PRACTISE

• Infertility- Karyotype
• Recurrent pregnancy loss- Karyotype
• Abnormal ultrasound findings in fetus during
  pregnancy
• History of stillbirth/ unexplained death of
  previous child during infancy
• History of intellectual disability/ developmental
  delay in previous child/ close relative
• History of genetic disorder in family (mental
  handicap/ physical handicap/ malformation/
  lethality)

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COMMON QUESTIONS THAT BRING COUPLES TO FETAL
MEDICINE SPECIALIST/ CLINICAL GENETICIST

• Why did it happen?
• Can it happen again?
• What can be done in next pregnancy?

• Genetic diagnosis
• Recurrence risk
• Prenatal testing

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TYPES OF GENETIC DISORDERS

• CLASSIC CHROMOSOME
• DISORDERS
• Full aneuploidies (Trisomy/ Monosomy)
• Partial aneuploidies (Deletions/ Duplications of
  sufficient size that they can be seen on
  Microscope)
• Phenotype practically always abnormal

• MICRODELETIONS/ MICRODUPLICATIONS/ COPY NUMBER
  VARIANTS
• Very small imbalances
• Not detected on Standard Karyotype
• May not always lead to clinical abnormality

• MENDELIAN DISORDERS
• Mutation at a single genetic locus
• Autosomal Dominant/ Autosomal Recessive
• X-linked dominant/ X-linked Recessive/ Y-linked

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APPROACH TO A COUPLE WITH HISTORY OF GENETIC
DISEASE

• 3 generation family history (PEDIGREE), Ascertain
  the pattern of inheritance
• Try to make a clinical diagnosis (Role of
  clinical geneticist)
• GENETIC TESTING OF THE INDEX CASE BASED ON
  PRESUMPTIVE CLINICAL DIAGNOSIS
• Battery of genetic tests available, different
  tests give different information
• Which genetic test to use- Depends upon the
  presumptive clinical diagnosis
• NO SINGLE GENETIC TEST TO DIAGNOSE ALL GENETIC
  CONDITIONS
• Many genetic tests have a Turn Around Time of 4-6
  weeks
• Ideally Molecular Genetic testing should be done
  in the preconception period or at first ANC visit
  itself
• Prenatal testing possible if exact molecular
  diagnosis is available
• Majority of genetic disorders do not have a cure
• Current focus-
• Prevention of genetic disorder (PGT)
• Timely prenatal diagnosis

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TOOLS FOR PRENATAL DIAGNOSIS

• INVASIVE TESTING
• CVS (Chorionic Villus Sampling)
• AMNIOCENTESIS

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AMNIOCENTESIS Process of withdrawing amniotic
fluid from uterine cavity for diagnostic/
therapeutic purposes Optimal gestation
16-17wks Early amniocentesis (lt15wks)
Increased fetal loss, complication rates, culture
failure COMPLICATIONS- DRY TAP Fetal
membranes tent over needle tip. More common in
early amniocentesis (Incomplete fusion of amnion,
chorion decidua parietalis) FETAL LOSS 1 in
300 to 1 in 500. Can occur upto 4 wks after
procedure BLOODY TAP lt 1 cases. Blood almost
always of maternal origin. Usually does not
affect amniotic cell growth
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Amniocentesis done at 19 wks Patient had history
of bleeding PV off on USG- Subchorionic
hematoma RBCs in pellet after centrifugation
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• CVS
• OPTIMAL GESTATION After 11 weeks (Once NT, NB
  Scan is available)
• Advantage of CVS over amniocentesis
• DNA analysis can usually be carried out directly
  on villi obviating the need and delay of a cell
  culture as required after amniocentesis 
• Yield of cells and DNA from CVS much greater than
  20ml of amniotic fluid
• Provides a shift towards earlier diagnosis and
  option of termination at an earlier gestation
• COMPLICATIONS
• Fetal loss Comparable to amniocentesis in
  experienced hands
• LIMITATIONS-
• Mosaicism 1-2 samples

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CHROMOSOMAL ABNORMALITYBalanced Chromosomal
Translocation In Either Parent
CASE NO. 1
Karyotype- 46,XX,t(810)(p21q22) Balanced
reciprocal translocation involving breakage at
specific breakpoints on ch- 8 10 and exchange
of fragments
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REPRODUCTIVE IMPLICATIONS OF BALANCED RECIPROCAL
TRANSLOCATION IN EITHER PARENT

• Behavior of Balanced reciprocal translocation at
  meiosis-
• Problems arise at meiosis because chromosomes
  involved in translocation cannot pair normally to
  form Bivalents
• They form a cluster known as Pachytene
  Quadrivalent
• Each chromosome aligns with homologous material
  in quadrivalent

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Pattern of Segregation Segregating Chromosomes Chromosome Constitution in Gamete
22 22 22
Alternate AD Normal
BC Balanced translocation
Adjacent-1 (non-homologous centromeres segregate together AC or BD Unbalanced (combination of partial monosomy and partial trisomy in zygote)
Adjacent-2 (homologous centromeres segregate together) AB Or CD Unbalanced (combination of partial monosomy and partial trisomy in zygote)
31 31 31
3 chromosomes A B C A B D A C D B C D Unbalanced, leading to trisomy in zygote
1 chromosome A B C D Unbalanced, leading to Monosomy in zygote
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RISKS IN RECIPROCAL TRANSLOCATION

• When counseling a carrier of balanced
  translocation, risk of birth of an abnormal baby
  depends upon the particular rearrangement
• This risk is usually somewhere between 1 and 10

Karyotype of child- 46,XX,der(8)t(810)(p21g22)ma
t Additional material on short arm of ch- 8 due
to maternal balanced translocation
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CASE NO. 2

• Achieved normal milestones was attending
  regular school till 13 yrs of age
• At 13 yrs of age-
• Eating difficulty with mild drooling
• Gait disturbances
• Tremors
• Speech difficulty
• Dystonia
• Ceruloplasmin levels, 24hr urinary copper
  excretion, Slit lamp examination of eyes, MRI
  findings s/o of Wilsons disease
• When couple presented to us at 12 wks POG, eldest
  child was already on treatment for Wilson/s
  disease
• However, NO Genetic evaluation had been done

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DILEMMAS IN THIS CASE

• Couple was from a remote area and had not brought
  the affected child
• Already 12 wks of gestation no genetic
  diagnosis available
• In view of clinical diagnosis of Wilsons
  disease, 25 recurrence risk in each pregnancy

• Role of Clinical geneticist very important
• Sample of affected child collected from home in
  this case as complete evaluation and clinical
  diagnosis was available and logistic issues
• Couple counselled that prenatal diagnosis can be
  provided only if genetic testing of previous
  child reveals pathogenic variation in ATP7B gene
• Coordination with lab in view of urgency of
  genetic diagnosis in prenatal testing

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Amniocentesis done at 164 weeks of gestation
after pathogenic mutation identified in ATP7B gene
FETUS UNAFFECTED
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Clinical suspicion of Osteopetrosis Baby died
at 7.5 months of age Genetic testing of baby
could not be done DNA of baby not available
CASE NO. 3
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ROLE OF TARGETED CARRIER SCREENING OF PARENTS
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• Because clinical evaluation of previous child had
  been done, clinical diagnosis (Osteopetrosis)
  made by Geneticist was available
• Based on clinical diagnosis, Targeted Carrier
  screening was able to identify mutation in
  parents
• Both parents heterozygous for c.674GgtA mutation
  in exon 3 of TCIRG1 gene
• 60 cases of infantile osteopetrosis are due to
  mutations in TCIRG1 gene
• Because pathogenic mutation was already
  identified when couple presented to us, CVS was
  done for prenatal diagnosis

CVS- FETUS UNAFFECTED
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CASE NO. 4

• Both parents Beta Thalassemia Minor
• WIFE-
• Hb- 11gm, HbA2- 4.9
• HUSBAND
• Hb- 13.4gm, HbA2- 4.8
• Late presentation- 186 wks
• Counselled regarding 25 risk of Thalassemia
  major
• Amniocentesis done
• Trio sequencing done to save time
• Pathogenic variant identified in parents and
  fetal DNA evaluated for the same mutation

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• On an average, 10 of Indians are carriers for
  Beta Thalassemia gene mutation
• HPLC/ Hb electrophoresis should be offered to all
  women who come for pre-conceptional counselling
  or at the First ANC visit
• Hb should not be used as a criteria for offering
  Hb electrophoresis/ HPLC as B Thalassemia
  carriers can have Hb in the range of 10-11 gm
• In patients who present late for prenatal
  diagnosis- We counsel the parents and take a
  written consent explaining the need for legal
  route if report becomes available after legal
  limit for MTP and fetus is found to be affected

FETUS- BETA THALASSEMIA MAJOR
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CASE NO. 5

• FTLSCS, B Wt.- 3.0 kg
• Age-appropriate milestones till 6 months of age
• H/O Recurrent LRTI
• Abdominal distension, Hepatomegaly, jaundice at 6
  months of age
• Hepatorenal failure
• Died at 7 months of age
• H/0 5 abortions at 3 months POG
• Cause for abortions- Not identified

Died at 7 months Age d/t Hepatic failure
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• Genetic evaluation of child had been done
• Variant c.82GgtT identified in SLC25A20 gene
• Variant was classified as VOUS
• (VOUS- Variant Of Uncertain Significance)
• SLC25A20 gene is associated with
  Carnitine-acylcarnitine translocase deficiency
• Carnitine-acylcarnitine translocase deficiency is
  AR metabolic disorder of long-chain fatty acid
  oxidation
• Clinical features- Neurologic abnormalities,
  hypotonia, Cardiomyopathy, skeletal muscle
  damage, LIVER DYSFUNCTION

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VOUS (VARIANTS OF UNCERTAIN SIGNIFICANCE)

• ACMG recommends a 5-tier system of classification
  of Variants
• Pathogenic (2) Likely pathogenic (3) Uncertain
  significance (4) Likely benign (5) Benign
• Counseling for VOUS should preferably be
  undertaken by Clinical geneticists
• VARIANT REANALYSIS
• Knowledge of variants is continuously evolving so
  an unreported variant or a VOUS at the time of
  initial Exome sequencing may be labelled
  pathogenic in future
• Testing additional family members may result in
  re-classification of variants
• Testing should be ordered only by specialists who
  are comfortable with the interpretation and
  explanation of results
• Information should include options for
  reproductive decision making, pregnancy and
  perinatal management

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Evaluation by clinical geneticist Both parents
underwent testing for the variant identified in
SLC25A20 gene Sanger sequencing confirmed the
presence of variant in SLC25A20 gene in both
parents As parents were Heterozygous carriers
for SLC25A20 gene, VOUS variant was reclassified
as Likely pathogenic Genetic counselling done
regarding 25 risk of recurrence Parents opted
for prenatal testing by CVS
FETUS HETEROZYGOUS (UNAFFECTED)
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• Husband- Myotonic Dystrophy Type I
• Mild DM1 (Mild myotonia)
• People with mild DM1 may have fully active lives
  a normal or minimally shortened lifespan
• Parents counselled
• Myotonic Dystrophy is Autosomal Dominant Triplet
  CTG repeat disorder with 50 risk of transmission
  to children
• ANTICIPATION
• Fetus may inherit repeat lengths considerably
  longer than transmitting parent
• May lead to increasing disease severity
  decreasing age of onset in successive generations

CASE NO. 6
Myotonic Dystrophy Type I
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Molecular genetic diagnosis was available in
father Couple opted for prenatal testing CVS
done at 12 weeks
FETUS UNAFFECTED
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• 5th pregnancy- Unexplained IUD _at_ 28 wks
• Genetic testing of fetus done
• Fetus Heterozygous for mutation in CFTR BTD
  gene
• Genetic counselling done
• Need to rule out carrier status of parents as
  both diseases (Cystic Fibrosis Biotinidase
  deficiency) cause significant morbidity
• Both parents found carriers for mutation in BTD
  gene (Biotinidase deficiency)

CASE NO. 7
POG 12 wks
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• In Present pregnancy- CVS done for both-
• Fragile X syndrome
• Biotinidase defeciency
• TAKEAWAY-
• Whenever we find a genetic variant, we need to
  evaluate fully (even with very rare conditions)
• What is rare in literature or in western world
  may not be very rare in our settings

FETUS AFFECTED (HOMOZYGOUS FOR BTD GENE MUTATION)
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10 yrs old Male child with DMD (Duchenne
Muscular Dystrophy) GENTIC ANALYSIS- Deletion of
exon 48-50 in Dystrophin gene Molecular Genetic
testing of Mother- No Deletion or Duplication in
79 Exons of Dystrophin gene
CASE NO. 8
DMD
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• DMD pathogenic variant identified in affected
  Male is not detectable in Maternal leukocyte DNA
• It could be a de novo pathogenic variant
• It could be because of Maternal germline
  mosaicism
• What is Germline Mosaicism?
• Some of the mothers egg cells carry the
  Dystrophin gene mutation while other egg cells
  donot
• Likelihood of maternal germline mosaicism in DMD
  is 15 to 20
• Even when the mother is not a carrier, it is
  possible that she has Germline mosaicism for the
  mutation and is at risk of having a second son
  with DMD
• Prenatal testing should be offered to mother in
  all subsequent pregnancies

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CASE NO. 9
Present pregnancy- NT/ NB Scan- CRL- 54mm NT-
3.5mm (gt99th centile) Unossified NB
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FETUS- TRISOMY 18
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THANK YOU

• Prenatal testing in genetic mutation carrier
  parents is much more than CVS or Amniocentesis
• In fact, that might be the easiest part
• Knowledge of Clinical Genetics is the backbone of
  prenatal testing in Single Gene Disorders

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• PRACTICAL CONSIDERATIONS-
• R/O Down syndrome if either parent carries
  Robertsonian translocation involving ch- 21

Source of Robertsonian translocation Risk of having baby with Trisomy 21
Mother carries 13q21q or 14q21q translocation 10
Father carries 13q21q or 14q21q translocation 1 to 3
Either parent carries 21q21q translocation 100
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ROBERTSONIAN TRANSLOCATION IN EITHER PARENT
CASE NO. 2

• Robertsonian translocation- Breakage of two
  acrocentric chromosomes (13, 14, 15, 21 and 22)
  at or close to their centromeres with subsequent
  fusion of their long arms
• Short arms of each chromosome are lost (no
  clinical importance as they contain genes only
  for ribosomal RNA for which there are multiple
  copies on other acrocentric chromosomes)
• Total chromosome number is reduced to 45
• This is a functionally balanced rearrangement