Arda Lembet_Array CGH in Prenatal Diagnosis.ppt [Uyumluluk Modu]

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Array CGH 

( Comparative Genomic Hybridization ) in 

Prenatal Diagnosis 

Arda Lembet,MD 

Associate Professor 

Obstetrics and Gynecology 

Total

• Postnatal genetic evaluation 

–Dysmorphic feautures 

–Cognitive difficulties 

• Prenatal conditions 

–Structural anomalies 

–Stillborn infants

Basic introduction to arrays 

• Metaphase karyotyping: 

–Aneuploidy,deletions,duplications,rearrangements 

–5-10 megabases 

• Microarray analysis: 

–Submicroscopic deletions, duplications 

–100 times smaller than ( kilobase range )



– Copy number varitions ( CNV ): 

•Regions of DNA larger than a kilobase ( 1000 

basepair ) 

•Autosomes each DNA has 2 copies 

•Deletions, duplications, trisomies 

•Benign CNVs 

•Disease causing CNVs 

•CNVs with unknown significance 

•1 Mb

The impact of human copy number variation 

on a new era of genetic testing 

• Copy number variations ( CNV ): 

–Normally found only once on each chromosome 

–29,133 CNVs in Database of Genomic Variants 

–12 % of the human genome is CNV 

–CNVs contribute 0.12% -7.3 % of g. variability 

–41% of all CNVs overlap with known genes 

– www.1000genoems.org 

–NAHR , 10 -4 per generation

The impact of human copy number variation 

on a new era of genetic testing 

• Copy number variations ( CNV ): 

• Single nucleotide polmorphism arrays 

– Loss of one allele 

– Loss of heterozygosity ( LOH ) 

– Uniparental disomy 

– Non – paternity 

– Consanguinity



–Comparative genomic hybridization 

•Method: DNA comparison 

•Deletions and duplications 

•Point mutaions, balanced translocations, inversions can 

not be detected 

•Microarray analysis technique

Array coverage: Density / backbone 

• Probes on arrays: 

– Oligonucleotides : 30-50 base pair ( bp ) 

– Bacterial artificial chromosome ( BAC ) : 

• 150-750 bp 

– Single nucleotide polymorphisms ( SNP ) 

– Targeted array 

– Whole genome arrays

Comparison of the types of chromosomal abnormalities 

that can be detected by karyotype and array CGH

Resolution and detection rate of arrays

Construction of a prenatal microarray 

• NICHD :Summer 2011, Prenatal vs. postnatal 

• 5-18 % of children with multipl anomalies 

and /or developmental delay and a normal 

karyotype will have a disease causing CNV 

• Low density targeted arrays vs / whole 

genome arrays

Current indications for microarray 

analysis in prenatal diagnosis 

• Evaluation of ultrasound structural 

anomalies 

• Delineating marker chromosomes 

• Reciprocal balanced translocations 

• Evaluation of stillborn infants



• Evaluation of ultrasound structural anomalies : 

–Fetal samples for standart indications , 

– 5-6 % clinically significant CNV , 

–When structural anomaly present microarray analysis will 

have a detection rate of 1-3 % beyond that of karyotype. 

–What is the value if no ultrasound abnomality 

–Expert opinion



• Evaluation of ultrasound structural anomalies : 

–Cardiac, CNS, skeletal, urogenital, renal 

–Increased NT 

–IUGR

• 50 fetuses with major structural anomaly 

–Cardiac,CNS,skeletal,urogenital,growth disorder,GI 

• Mean GA : 24.5 weeks 

• 4 / 50 fetuses had abnormal array 

• 1 clinically significant 2% 

• 3 inherited benign variants 6%

• 300 samples AS – CVS 

• AMA, US abnormality, family history, p. concern 

• 58 ( 19.3 % ) CNVs 

–40 (13.3 %) Benign 

–15 ( 5 % ) Pathological 

–3 ( 1 % ) Uncertain clinical significance 

–7 (2.3 %) a CGH contributed new information 

–2 ( 1 % ) w/o a CGH no abnormality would have 

been detected

• NT > 99 th, CVS- normal G band, 

• 100 fetuses f/u 22 months 

• HR-CGH and MLPA 

• 80 liveborn 

–3 ( 4 % ) had syndromes ( MR,sotos syndrome ) 

–18 % adverse pregnancy outcome overall 

–CGH and MLPA did not detect any chromosome abn.with 

syndromes

Identification of submicroscopic chromosomal aberrations in 

fetuses with increased NT and an apparently normal karyotype 

Leung et al,UOG March 2011 

• NT > 3.5 , aCGH 44 K oligonucleotide , validation 

• CNV 6 / 48 ( 12.5 % ) , 1-7.8 Mb 

• 5 / 48 ( 9.5 % ) clinical significant – validated 

• 4 / 48 ( 8.3% ), 1p36 excluded ( normal v ) 

–Pathogenic CNV 20 % -US abnormality 

5.3% -No US abnormality 

• Abnormal array, median NT 4.35 mm




anomalies 






• Delineating marker chromosomes. 

–Nonsatellited markers:15% abnormal phenotype 

–Satellited marker risk: 11 % 

–Acrocentric chromosome 

–Heterochromatin vs euchromatin




anomalies 







anomalies 






• Evaluation of stillborn infants : 

–5% of structurally normal stillborn fetuses 

–35-40 % of structurally abnormal or macerated 

•Will have an abnormal karyotype 

–Quality of the karyotype

Other potential benefits of microarray 

• Turnaround time: 

–1-2 weeks for a conventional karyotype 

• No need for cell culture 

–Sample sites: DNA extraction sites …. 

–Also potential benefit for stillbirth

Limitations of microarray 

•Balanced translocations will not be detected. 

•Standart microarray will not identify 

polyploidies . 

•Low-level mosaicism : 

•Unknown clinical significance: ISCA-NIH

Current guidelines 

• American College Medical Genetics: 

– Multiple anomalies not spesific to a well delineated genetic 

syndrome, nonsyndromic developmental delay and 

intellectual disability and autism spectrum disorders. 

– Children with growth retardation, speech delay..etc


• American College of Obstetrics and 

Gynecologists ( ACOG ) 

–First line test for prenatal evaluation of chromosomal 

abnormalities remains unknown and conventinal 

karyotyping remains the primary cytogenetic tool.


• American College of Obstetrics and Gynecologists ( ACOG ) 

– However, targeted arrays in combination with genetic counseling can be 

offered in the setting of an abnormal ultrasound finding and a normal 

karyotype result. 

– It can also be offered in cases of fetal demise with congeital anomalies 

when conventional karyotype is unobtainable. 

– Pretest and posttest genetic counseling

Future perspectives 

•Acceptable cost 

–Array CGH: 442 £ Karyotype: 117 £ 

–FISH :214 £ MLPA: 245 £ 

• Fetal cells from various sites 

–ECC, fetal cells, cell-free fetal DNA 

• Single nucleotide changes 

• Parental origin of del / dup 

• Spesific point mutation analysis

Asphyxiating Thoracic Dystrophy: 

A case report 

31 yo 

G3P2, alive 0. 

Referred due to shortness of long bones. 

Gestational age : 14 w 4 d. 

No consanguinity. 

Family history unremarkable

OB HISTORY 

• G 1 was terminated at 24 th weeks. 

-USG examination: 

• Shortness of long bones.( predominatly humerus 

and femur. BPD: 24 wk, FL: 17 wk, HL: 18 wk) 

• Bowing of humerus and femur 

• Narrow thorax 

-A/S: normal karyotype

Autopsy: 

•Rhizomelia 

•Brachydactyly 

•Narrow thorax 

•Clinodactyly at 5 th fingers

-Radiographic findings: 

• Bowing at femur and humerus 

• Metaphyseal irregularities at all tubuler bones 

• Abnormalities at iliac bones. ( square-shaped iliac 

bone) 

• Short ribs 

• Handlebar clavicles 

• Diagnosis: ATD / Short limb polydactyly related 

syndromes

•2nd pregnancy was terminated at 17 th 

week . 

•-USG: 

• Shortness of long bones ( FL

-G3 Current pregnancy USG findings: 

• Shortness of long bones ( FL, HL< 5 % ) 

• Bell-shaped thorax 

• The other organs and systems were 

unremarkable. 

Karyotype analysis was normal.

•Molecular karyotype analysis ( cytogenetics 

whole genome 2.7 M Array) 

•Deletion at ACVR1 and ACVR1C genes 

located at 2q23-24 region) 

–Fibrodysplasia ossificans progressiva

ATD ( Jeune syndrome) 

• AR skeletal dysplasia, 1/100,000-1/130,000 live 

births 

Clinical and radiological findings: 

• Bell-shaped / long narrow thorax 

• Varying degree of rhizomelia 

• Short hands, polydactyly 

• Pelvic abnormalities ( trident acetabulum, 

hypoplastic ileum) 

• Renal, hepatic, retinal, pancreatic abnormalities

•Most cases of ATD are sporadic, prenatal 

diagnosis is very difficult in low-risk 

women without a previous history 

•Clinical outcome is poor, early prenatal 

diagnosis is important. 70% of ATD cases 

die in early childhood from pulmonary 

hypoplasia and respiratory distress 

Chen et al. Prenatal Diagn 2003; 23

•ATD is known to be genetically 

heterogeneous. 

•The molecular basis of ATD is unknown, 

with few clues to the location of genes 

likely to contribute to pathogenesis.( Morgan et al. J 

Med Genet 2003.)

•To map a locus for ATD, we performed a 

genome wide linkage search

RESULTS 

•15q13 region: heterozygote deletion 

Morgan et al identified homozygosity for 

mutations in a specific locus to 

chromosome 15q13 in five consangineous 

families.

• DYNCH2H1 mutations on chromosom 11q. , 

heterozygote deletion. 

In a consanguineous family and in 2 isolated cases 

with short rib-polydactyly syndrome type III 

(263510), Merrill et al. (2009) identified 

homozygosity or compound heterozygosity for 

mutations in the DYNC2H1 gene. The 

abnormalities in short rib-polydactyly syndrome 

are primarily related to the effect on the skeleton, 

reflecting an essential role for DYNC2H1 in cilia 

function in cartilage.

•We exclude IFT80 mutations. 

Mutations in the intraflagellar transport 80 ( 

IFT80) gene have been identified in 3/ 39 

families , ascribing ATD to the ciliopathy 

group. 

. 

( Beasles et al. Nat. Genet. 2007).

CONCLUSION 

• We identified heterozygote mutations at 

chromosome 15q13 and 11q (DYNC2H1). 

• It is difficult to identify the exact responsibility of 

these mutations on the occurence of disease. 

• Whether the disease is the consequence of only 

one of these two mutations or their combined 

effect remains to be determined.

Arda Lembet_Array CGH in Prenatal Diagnosis.ppt [Uyumluluk Modu]

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