GENOME-WIDEANALYSIS BY SNP ARRAY

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SNP ARRAY INT H E D I AG N O S I S O F I N T E L L E C T U A L D I S A B I L I T Y A N DC O N G E N I T A L A B N O R M A L I T I E S

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Conventional and molecular cytogenetics (FISH) . . . . . . . . . . . . . . . . 4

1. Karyotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

2. Fluorescence in situ hybridisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Emerging technology: CGH/SNP Array for Genome-wideexploration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1. Comparative Genomic Hybridisation Array: CGH Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

2. Single Nucleotide Polymorphism Array: SNP Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

SNP Array validation and interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

1. Pathological CNVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

2. CNVs assumed as Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

3. Unclassified LOH & CNVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Clinical cases resolved by SNP Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

1. Application of SNP Array in the diagnosis of mental retardation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

2. Application of SNP Array in the diagnosis of infertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20

3. Application of SNP Array to prenatal diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

4. Targeted applications of the SNP Array . . . . . . . . . . . . . . . . . . . . . . .24

5. Detection of Supernumerary Chromosomal Markers (SCMs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26

6. Benefits of SNP array in POC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28

Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Indications for SNP Array applications toclinical genetic testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Sample requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

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Contacts

AuthorSaïd EL MOUATASSIM, PhD

Molecular Genetic department, Biomnis LyonSaïd El Mouatassim, PhDGrégory Egea, MD

Phone number: +33 (0)4 72 80 23 65Fax number: +33 (0)4 72 80 23 66

Biomnis International DivisionInternational Division17/19 avenue Tony GarnierBP 732269357 Lyon cedex 07FrancePhone number: +33 (0)4 72 80 57 42Fax number: +33 (0)4 72 80 73 56E-mail: international@biomnis.comWebsite: www.biomnis.com/international

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Introduction

The emergence of DNA microarray (CGH Array/SNPArray) has revolutionised conventional cytogeneticdiagnostics. This new technique analyses the wholegenome with a higher resolution than that observedwith classical karyotyping. Single nucleotidepolymorphism (SNP) Array can detect and provide adetailed characterisation of the cryptic chromosomalanomalies implicated in mental retardation andcongenital abnormality (CA), which are not detectedby conventional methods. With the resolutionadvantage and due to the fact that this technologydoes not require a significant quantity of DNA, thescope of application of SNP Array is widened toinclude prenatal diagnostics. This review presentsclinical cases that confirm the advantages of SNPArray in cytogenetic laboratory practice and definesthe indications for the prescription of SNP Arraytesting.

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Figure 1. Karyotype of a child with Down's syndrome(47,XY,+21)

Conventional and molecular cytogenetics (FISH)

1. Karyotyping

Karyotype analysis studies the number and structureof chromosomes (Figure 1). It gives an overallperception of the chromosome rearrangements withinthe whole human genome. Karyotyping isrecommended when confronted with certain well-established clinical indications such as commonaneuploidies (trisomy 21, 13 and 18), polymal-formativesyndromes seen in newborn babies or during ultrasound examination, repetitive miscarriages, sterility,intellectual disability with dysmorphological featuresand intra-uterine growth retardation. The diagnosis ofa chromosomal abnormality is crucial as it means thatprecise genetic counselling can be given. Duringpregnancy, the identification of an unbalanced foetalchromosomal abnormality is important for monitoringpatient welfare.

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Figure 2. The percentage of the detected anomalies byconventional cytogenetics for ID.

Karyotype limits

In certain cases, the pathologist can be confrontedwith chromosomal abnormalities that are difficult tocharacterise, or with cases where the clinical etiologystrongly suggests the presence of a chromosomalabnormality but the karyotype is normal. In thesesituations, the karyotype reaches its limitation due toits low resolution. The most well known example isintellectual disability (ID). Despite the prevalence of ID(2-3%) and its impact on the individual and their family,karyotyping can only detect 5-10% of the patientssuffering from ID (Figure 2). It could stem fromchromosomal imbalances where the size is lower thanthe resolution of the karyotype (5-10 Mb).

One of the common applications of FISH is in thediagnosis of chromosomal microrearrangements,which are usually specific microdeletions ormicroduplications syndromes including DiGeorgesyndrome (22q11.2), Prader-Willi syndrome and

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Figure 3. FISH results showing hybridisation of centromericprobes on interphase nuclei (A), painting (B), locus specific(C) or telomeric probes on metaphase (D).

2. Fluorescence in situ hybridisation (FISH): a targeted genome exploration.

FISH is based on the property of nucleic acids tofollow the denaturation and hybridisation processunder specific conditions of temperature, salinity andpH. A denatured probe (labelled single-strandedDNA) can specifically hybridise with its targetsequence. Hybridised probes are then highlighted byimmunodetection and detected using a fluorescentmicroscope. FISH can be performed on nuclei ormetaphases with a resolution of 150 kilo-bases.Several types of probes (centromeric, painting, locusspecific or telomeric; Figure 3) can be used to detecta chromosomal abnormality or confirm the presenceof a known syndrome.

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Figure 4. FISH detection of Microdeletional syndromes.(A) DiGeorge Syndrome (deletion 22q11.2), (B) Prader-Willisyndrome and Angelman syndrome (deletion 15q11q13),(C) Wolf-Hirschhorn syndrome (deletion 4p16).

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Angelman syndrome (15q11q13), Miller-Dieker (17q13.3)syndrome for the deletions, as well as Weideman-Beckwith syndrome (11p15) and Wolf-Hirschhornsyndrome (deletion 4p16) (Figure 4).

FISH limits

The known microdeletional syndromes and micro-rearrangements of the terminal regions are only asmall part of the pathologies that can be diagnosedby FISH. There remain numerous syndromes linkingID, CA and dysmorphia of unknown origin which couldbe caused by chromosomal microrear-rangementsthat are not diagnosed by FISH. This represents alimitation of the FISH technology. FISH does notexamine the whole genome and can therefore studyonly the targeted regions with the help of specificprobes.

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Diagram 1. Schematic representation of non-allelichomologous recombination.

Emerging technology: CGH/SNPArray for Genome-wide exploration

Human genome sequencing has allowed theunderstanding of microarrangement mechanismsinvolved in genesis of cryptic rearrangementimplicated in ID and CA. These "genomic disorders"are the result of non-allelic homologousrecombination (NAHR) between LCR sequences2 "LowCopy Repeat". LCR makes up the molecular base ofcryptic rearrangements whereby the directconsequence is DNA copy number variation. Thedeletions, insertions and duplications are qualified ascopy number variations (CNVs) or copy numberpolymorphisms (CNPs).

A CNV is defined as a segment of DNA of 1000 basesor more which is present in a variable number ofcopies in comparison to standard DNA. CNVs caninfluence the gene expression by the deregulation ofthe genes or their regulator sequences, by thecreation of fusion genes, or by directly altering thegene copy number.

Duplication

Deletion

CNVs can cause congenital diseases involvingmicroduplications or microdeletions. All of the currentscientific data reinforces the idea that the applicationof karyotyping or FISH remains insufficient for thediagnosis of the micro-rearrangements involved in IDand CA. Low karyotyping resolution (5-10 Mb) and thetargeted analysis of FISH represent a significantrestriction for ID and CA diagnosis. However, DNAmicroarrays have proved their utility in the diagnosisof ID and CA. They detect genomic disorders in 22.6%of patients reported as normal by karyotypinganalysis3.In this review, we present the clinical interest of SNPArray use (HumanCytoSNP-12 BeadChip) in clinicalgenetic testing including ID, CA or infertility; in prenataldiagnosis and targeted identification of chromosomalmarkers not identified by karyotyping and thedetection of the loss of heterozygosity (LOH)implicated in certain syndromes.

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Figure 5. Schematic representation of the CGH Arraytechnology.

1. Comparative Genomic Hybridisation Array:CGH Array

CGH Array provides a pangenomic analysis of thehuman genome with a better resolution than that usedin karyotyping analysis4,5. Currently, CGH Array isbased on the competitive hybridisation of the DNA ofthe patient being tested (labelled by a greenfluorochrome) and a normal DNA reference (labelledwith a red fluorochrome) on a significant number ofhuman DNA sequences (oligonucleotides spreadacross the whole human genome). After hybri-disation, the fluorescence intensities of eacholigonucleotide on the array is calculated using ascanner (Agilent®) and the fluorescence ratio iscalculated to determine the copy number of eachDNA marker tested. This calculated ratio detects thecopy number variation (CNV) between the testedpatient's DNA in comparison with the normal DNAreference. A CNV corresponding to a loss in geneticmaterial (deletion) in the patients will be representedby a decreased fluorescent ratio whereas the CNVscorresponding to a gain in genetic material (dupli-cation) show a raised fluorescent ratio (figure 5).

Microarray scanning

Test DNA Reference DNA

Test Reference Test Reference Test Reference

Deletion Normal Duplication

Microarray

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2. Single Nucleotide Polymorphism Array:SNP Array

In comparison to CGH Array, SNP Array determinesthe CNV and the LOH (loss of genetic material of oneof the two parents). In Biomnis, we use SNP Arraytechnology (Illumina®). The HumanCyto-SNP-12BeadChip used in our laboratory offers a whole-genome scanning panel. It includes 300 000 markersgenome-wide tag SNP and markers targeting allregions of known cytogenetic disease. This includesdense coverage of approximately 250 genomicregions commonly screened by cytogeneticlaboratories, including subtelomeric regions, peri-centromeric regions, sex chromosomes and targetedcoverage in approximately 400 additionnal disease-related genes (www.illumina.com).

HumanCytoSNP-12 BeadChip detects CNVs and LOHrelevant to many types of genomic variationsincluding duplications, deletions, LOH and mosaicism.SNP Array offers CNV analysis using the intensity ofthe markers and the genotype using B allele frequency value (BAF).

SNP Array is based on the whole genomicamplification, tagging and hybridisation on the Arrayslides. The BeadChips are then scanned using aniScan Reader (Illumina®) and the data analysis isperformed using GenomeStudio and CaryoStudio(Illumina®). The BAF is the value between 0 and 1 andrepresents the proportion contribution of one SNPallele (B) to the total copy number. A BAF value of 0.5indicates a heterozygous genotype (AB), whereas 0and 1 indicates a homo-zygous genotype (AA, BB,respectively). In the case of a deletion, present in allcells, the deleted region will show homozygosity-bands at 0 and 1 (AA, BB genotypes) and loss of theBAF value at 0,5 (Loss of AB heterozygous genotype).Whereas, a region of single-copy-number gain, in allcells will, in addition to the two bands of homozygousSNPs at BAF = 0 (AAA) and BAF = 1 (BBB), also showtwo additional bands: one at BAF = 0,33 with SNPs

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having genotype AAB and one at BAF = 0,67 withSNPs having genotype ABB (figure 6 and 7). Thefollowing resolutions are generally applied: Loss ≥ 150Kb, gain ≥ 200 Kb and LOH ≥ 5 Mb.

Genomic DNA (200-400 ng)

PCR-Free Whole-Genome Amplification

Fragment DNA

Figure 6 A. Schematic representation of the SNP Arraytechnique (Illumina®). DNA isolated from peripheral blood oramniotic fluid is amplified, fragmented and hybridised onthe SNP Array.

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Figure 6 B. C. The slides are scanned using iScan scanner(Illumina®) and the results are analysed using GenomeStudio and Caryostudio Software. FISH or quantitative real-time PCR are used to confirm any abnormal findings eitherat the time of initial testing or upon receipt of parentalsamples, depending on the abnormality, while methylation-specific.

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Figure 7. SNP Array results using HumanCytoSNP-12BeadChip (Illumina®) showing complex genomic changeson chromosome 8p. [arr8p23.3.p23.1(221411-6914226)x1,8p23.1.p21.3(12583059-22995348)x3]

Detection of a complex genomic changes inpatient with ID

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SNP Array validation and interpretation

UCSC (University of California, Santa Cruz) buit Hg19is generally used to analyse data. Copy-number-variation (CNVs) are systematically checked in thepublic databases DGV (Database of GenomicVariants), Chop database and literature. Otherdatabases are also used such as DECIPHER(Database of Chromosomal Imbalance and Phenotypein Humans using Ensemble Resources) and OMIM(Online Mendelian Inheritance in Man). The finalresults follow the International Standards forCytogenomic Array Consortium nomenclature (ISCA2009).

1. Pathological CNVs

The most important criterion to classify CNVs aspathological is its association with a known abnormalphenotype. CNVs with a direct association to anabnormal phenotype or known syndrome areassumed to be pathological. CNVs associated with aknown increasing risk of an abnormal phenotype isalso assumed to be pathological.

2. CNVs assumed as Polymorphisms

Parental DNA, when available, is tested by anothertechnique such as FISH or Real time PCR, to identifyinherited CNVs. Rare CNVs associated with anincreased risk or abnormal phenotype, inherited froma healthy parent, is assumed to be benign. If a similarCNV was found in more than 3 individuals, the CNV isqualified as a polymorphism.

3. Unclassified LOH & CNVs

LOH is of unknown clinical relevance, except in casesof uniparental disomy of inherited regions. De novoCNVs or LOH of unknown (absence of disease-relatedgene) or uncertain clinical importance are defined asunclassified variants.

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Clinical cases resolved by SNP Array

1. Application of SNP Array in the diagnosis ofintellectual disability

Case 1: Detection of the Wolf-Hirschhornsyndrome.

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Figure 8 A. SNP Array results of an infant, with intellectualdisability. This result shows the presence of a deletion 4p16.3of 8,7 Mb, [arr4p16.3p16.2p16.1(38,283-8,757,013)x1-8.7Mb]responsible for the Wolf-Hirschhorn syndrome.

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Figure 8 B. FISH results confirming the presence of theWolf-Hirschhorn syndrome (Red spot) on the chromosome4 (Green spots).

Deletion 4p16.3

Wolf-Hirschhorn syndrome is the result of achromosomal deletion at 4p16. This syndrome ischaracterised by growth retardation, musclehypotonia and a developmental retardation withmental retardation.

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Case 2: Detection of Prader-Willi and Angelman syndrome

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Figure 9 A. SNP Arrays results shows the presence of LOHon chromosome 15q11.2 harbouring the Prader-Willi andAngelman syndrome region.

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Figure 9 B. Confirmation of PWS by PCR and fragmentanalysis using STR markers specific for chromosome 15.

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Prader-Willi (PWS) is a genetic disorder involvingseveral genes on chromosome 15q11-13. Thischromosomal region is influenced by“imprinting” which refers to expression of genesfrom one parent’s chromosome with silencing ofthe other parent’s chromosome. In the case ofPWS, the paternal region is active, so thatdeletion or failure of inheritance of the paternalregion causes the syndrome. PWS ischaracterised by mental retardation, hypo-gonadism, hypotonia, obesity, characteristicfacial appearance. PWS is a result of paternal 15qdeletion or maternal 15q uniparental disomy.

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2. Application of SNP Array in the diagnosis of infertility

Case 3: Detection of genomic changes inpatient with premature ovarian failure pre-senting a normal Karyotype

Patients with premature ovarian failure (POF)show partial deletions on chromosome X and X-autosome translocations. Seventy per cent ofthe deletions in the terminal end of chromo-some X are responsible for POF6. The criticalregions are located between Xq13.3 and Xq26-q27 containing POF1 (Xq21.3-q27) and POF2(Xq13.3-q21.1), necessary for ovarian deve-lopment. POF is also linked to a FMR-1 genepermutation involved in fragile X syndromefamilial transmission. Studies have shown that20% of permutated women have POF7,8. In aroutine laboratory practice, the translocations ofchromosome X and the screening for the FMR-1gene is systematically performed for patientswith POF. However, the karyotype analysis, dueto its low resolution, gives normal results in somecases presenting cryptic chromosomalabnormalities (Figure 10).

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Figure 10 A. Karyotype results showing normal karyotype46,XX in a woman with Premature Ovarian Failure.

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Duplication Deletion

Figure 10 B. SNP Array results of the same patient withPOF showing the presence of duplication on chromosome2 and a deletion on chromosome X. This result wasconfirmed by FISH (data not shown).

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Figure 11 A. Prenatal diagnosis of a fœtus with a karyotypesuspecting an anomaly of chromosome 21.

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3. Application of SNP Array for prenatal diagnosis

Case 4: Detection of genomic changes infetus with ultrasound findings

Prenatal diagnosis requires fast and sensitivetests to manage abnormal pregnancies. Prenataltesting is commonly performed usingkaryotyping and FISH analysis. In certain cases,the pathologist is confronted with ultrasoundresults which strongly suggest chromosomaldisorders (polymalformative signs, growthretardation, nuchal translucency, etc.), whereasthe karyotype is normal. This is certainly due tothe low resolution of the karyotype. In thesecases, the interest of detecting these genomicchanges by CGH Array or SNP Array becomesprimordial as these techniques provide apangenomic analysis with a better resolution9-17.The low quantity of DNA (50 ng) necessary toperform a SNP Array (without cell culture ofamniotic fluid), the better resolution of the arraysin comparison to a karyotype, the simultaneousdetection of CNVs as well as LOH andpolyploidy represent additional arguments forthe application of the SNP Arrays to prenataldiagnosis.

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Figure 11 B. The SNP Array results confirm the presence ofa deletion in chromosome 21q of 4.6 Mb[arr21q22.3(42323005-46923252)x1] associated withholoprosencephaly phenotype.

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Figure 12 A. Prenatal diagnostic showing an inversion onchromosome 12 (46,XX,inv(12)(p13.1q24.1).

4. Targeted applications of the SNP Array

Case 5: "Balanced" rearrangements with anabnormal phenotype

Certain patients are carriers of translocations,insertions and inversions etc.), with no loss orgain in genetic material appearing on thestandard karyotype. The same patients whenanalysed by SNP Array show the presence ofduplication or deletion in the initial rearran-gement, not detected by the karyotype. This isimportant when the karyotyping of the f�tusshows a seemingly balanced de novo trans-location. Figure 12 shows a case of a f�tus withcryptic deletion, detected only by SNP Array,following an inversion of chromosome 12resulting in severe mental retardation, delayeddevelopment, small head and craniofacialabnormalities.

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Figure 12 B. SNP Array results of the same sample showinga microdeletion of 5.2 Mb at the break point of theinversion on chromosome 12[arr12p12.1p11.23(21416873-26670081)x1].

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Figure 13 A. Prenatal karyotyping with a non identifiedmarker.

5. Detection of Supernumerary ChromosomalMarkers (SCMs)

Case 6: Prenatal identification of chromo-some marker

SCMs are defined as additional chromosomal ofcomplex or abnormal structure. SCMs can bedetected by karyotyping but their origin isusually difficult to identify. The structure of theSCMs is variable: derivatives (der), inversionduplication (inv dup), ring (r), isochromosome (i),minute chromosomes (min). SCMs are correlatedwith known clinical syndromes (Pallister-Killiansyndrome and Cat-eye syndrome). Within thescope of prenatal investigations, the frequencyof SCMs is estimated at 0.075%. They can besuspected during the ultrasound and/or whenthe mother’s age is relatively old. In any case, itremains important to identify the origin and thestructure of the SCMs and the presence orabsence of euchromatin.

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Figure 13 B. The application of the SNP Array technologyconfirmed the origin of the marker. This marker is a resultof a duplication of the chromosome 16q24.

Figure 13 C. This data was confirmed by FISH.Mos46,XX[20]/47,XX,+mar.arr 16q24(82796939-83328805)x3

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6. Benefits of SNP array in Products of Conception(POC)

Case 7: First trimester miscarriage causedby fetal triploïdy

Fetal Death (FD) occurs in approximately 15% ofclinically identified pregnancies. Cytogeneticabnormalities are present in 50% ofspontaneous miscarriages (FD < 22 weeks ofamenorrhea (WA) and in 6-13% of stillbirths (FD >22 WA). Samples originating from theseoccurrences can be analysed by karyotype,CGH array or SNP array.The advantage of SNP array over karyotypingcannot be refuted: the failure rate does notsurpass 1% compared to the 40% failure rate ofthe karyotype (Figure 14A). In additional, theresolution is 50 times more defined in SNP array,which in turn increases the sensitivity andtherefore the number of pathogenicabnormalities detected (Figure 14A).The SNP array also has an advantage over CGHarray : it enables the detection of triploidies,which represent close to 5% of FD < 22 WA,which is impossible in CGH array. Figure 14B shows the case of a POC sampleanalysed by SNP array: fetal triploïdy resulted ina first trimester miscarriage.

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Figure 14A : Comparison of karyotype and SNP arrayperformance in products of conception analysis.

Figure 14B: SNP array result shows a triploïdy, here is theexample of chromosome 1, all chromosomes have acomparable profile. Log R ratio has not deviated due tonormalisation, triploïdy is visible thanks to genotypingdata (B Allele Freq).

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Highlights

Karyotyping provides an overall analysis of the humangenome but the resolution of the test remains lowcompared to new techniques based onoligonucleotide arrays (CGH Array and SNP Array).These new technologies enhance the geneticetiological diagnosis of MR and CA (Figures 15 and 16).SNP Arrays provide a genome-wide study of allimbalanced genomic anomalies with a resolutionclose to 100 kb. The advantages of SNP Arrays arenumerous:

Overall analysis of the genome;

Highlighting genomic changes that are not detectedby karyotyping;

Identification and characterisation of the loss of genomic material in the case of structural anomalieswith an apparently balanced karyotype;

Detection of the loss of heterozygosity, generallyseen in cases of uniparental disomy;

Identification and characterisation of the super-nume-rary chromosome markers seen in the karyotype;

SNP Array does not require prior cell culture. Theadvantage of working directly with amniotic fluidavoids culture selections, and a decrease in the turnaround time from 10-15 days to only 1 week without parental confirmation.

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Figure 15. Overview of distribution of various etiologicalcauses of developmental delay and mental retardation1-2.

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Figure 16. Advantages of SNP Array.

32

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33

American Col lege of Medical Genet ics hadrecommended replacing karyotyping with ChromosomalMicroarrays as “First-Line” Postnatal Test. Earlier thisyear, ISCA International Standards for Cyto-genomicArrays Consortium was recommended using arrays as“first-tier” tests to assess individuals with unexplainedDevelopment Delay (DD) and Intellectual Disability,Autism Spectrum Disorders (ASD) or MultipleCongenital Anomalies (MCA). ISCA ConsensusStatement. AJHG, 201018, International Guidelines19,20

Indications for SNP Array applicationsto clinical genetic testing

HumanCytoSNP-12 BeadChip is indicated for:

Unexplained developmental delay or intellectual disability.

Dysmorphic features or congenital anomalies

Autism spectrum disorders, seizures or a clinicalpresentation suggestive of a Chromosomal syndrome.

Patients with a normal chromosome analysis resultand abnormal phenotype.

Patients with suspected UPD.

For unbalanced rearrangements, SNP Array can beused to size the deletion or duplication, or identifythe genes involved and their content.

For apparently balanced rearrangements and anabnormal phenotype, SNP Array can be used totest for cryptic deletions/duplications at the break-points.

Infertility and sexual disorder development.

>

>

>

>

>

>

>

>

34

References

1. Rauch A, Hoyer J, Guth S, et al. (2006): Diagnostic yieldof various genetic approaches in patients withunexplained developmental delay or mentalretardation. Am J Hum Genet, (140A): 2063–2074.

2. Stankiewicz P, Lupski JR (2002): Genome architechture,rearrangements and genomic disorders. Trends Genet,18:74-82.

3. Gijsbers AC, Lew JY, Bosch CA, et al. (2009): Anewdiagnostic work flow for patients with mental retardationand/or multiple congenital abnormalities. Eur, J HumGenet, 17(11):1394-402.

4. Pinkel D, Segraves R, Sudar D, et al. (1998): Highresolution analysis of DNA copy number variation usingcomparative genomic hybridization to microarrays. NatGenet. (20):207-211.

5. De Vries, BA Pfundt R, Leisink M, et al. (2005):Diagnostic Genome Profiling in Mental Retardation. AmJ Hum Genet; 77(4): 606–616.

6. Devi A, Benn PA. X-chromosome abnormalities inwomen with premature ovarian failure. J Reprod Med1999 ; 44 : 321-4.

7. Sherman SL (2000). Premature ovarian failure in thefragile X syndrome. Am J Med Genet; 97 : 189-94.

8. Murray A, Webb J, Crimley S, et al. (1998): Studies ofFRAXA and FRAXE in women with premature ovarianfailure. J Med Genet; 35 : 637-40.

9. Shaffer LG, Coppinger J, Alliman S, et al. (2008):Comparison of microarray-based detection rates forcytogenetic abnormalities in prenatal and neonatalspecimens. Prenat Diagn; 28: 789–795.

10. Rickman L, Fiegler H, Carter NP, et al. (2005): Prenataldiagnosis by Array-CGH. Eur J Med Genet; 48: 232–240.

11. Rickman L, Fiegler H, Shaw-Smith C et al. (2006):Prenatal detection of unbalanced chromosomalrearrangements by Array CGH. J Med Genet; 43: 353–361.

12. Sahoo T, Cheung SW, Ward P, et al. (2006): Prenataldiagnosis of chromosomal abnorma-lities using array-

35

based comparative genomic hybridization. J MedGenet; 43: 719–727.

13. Friedman JM (2009): High resolution array genomichybridization in prenatal diagnosis. Prenat Diagn; 29:20–28.

14. Kleeman L, Bianchi D, Shaffer LG, et al. (2009): Use ofarray comparative genomic hybridization for prenataldiagnosis of f�tuses with sonographic anomalies andnormal metaphase Karyotype. Prenat Diagn; 29: 1213–1217.

15. Van den Veyver IB, Patel A, Shaw CA, et al. (2009):Clinical use of array comparative genomic hybridization(aCGH) for prenatal diagnosis in 300 cases. PrenatDiagn; 29: 29–39.

16. Srebniak M, Boter M, Oudesluijs G, et al. (2011):Application of SNP Array for rapid prenatal diagnosis:implementation, genetic counselling and diagnosticflow. Eur J Hum Genet.; 19(12):1230-7.

17. Fiorentino F, Caiazzo F, Napolitano S, et al. (2011):Introducing array comparative genomic hybridizationinto routine prenatal diagnosis practice: a prospectivestudy on over 1000 consecutive clinical cases. PrenatDiagn; 31(13):1270-82.

18. ISCA Consensus Statement: Building momentum: Arraysas first-line? AJHG, May 2010

19 : de Leeuw N, Dijkhuizen T, Hehir-Kwa J Y, et al. (2012):Diagnostic interpretation of array data using publicdatabases and internet sources. Hum Mut; 33, 930–940.

20. ACMG standards and Guidelines, Genet Med, 2011 &2013

21. Levy B, Sigurjonsson S, Pettersen B, et al. (2014):Genomic Imbalance in Products of Conception: Single-Nucleotide Polymorphism Chromosomal MicroarrayAnalysis. Obstet Gynecol; 124, 202–209.

36

TypeSpecimen

Requirements

SpecimenCollection

and Shipping

WholeBlood

5 ml EDTA wholeblood *

Store and transportsample at room temperature

*For paediatric samples, please also sendEDTA samples of the parents

Amnioticfluid

5 ml of amniotic fluidStore and transportsample at room temperature

POC

Please also includematernal EDTA sam-ple to exclude conta-mination

Store and transportsample at room temperature

TAT: 10 days

Signed genetic consent form and detailed clinicalinformations are essential

Sample requirements

37

Notes

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Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Conventional and molecular cytogenetics (FISH) . . . . . . . . . . . . . . . . 4

1. Karyotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

2. Fluorescence in situ hybridisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Emerging technology: CGH/SNP Array for Genome-wideexploration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1. Comparative Genomic Hybridisation Array: CGH Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

2. Single Nucleotide Polymorphism Array: SNP Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

SNP Array validation and interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

1. Pathological CNVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

2. CNVs assumed as Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

3. Unclassified LOH & CNVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Clinical cases resolved by SNP Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

1. Application of SNP Array in the diagnosis of mental retardation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

2. Application of SNP Array in the diagnosis of infertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20

3. Application of SNP Array to prenatal diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

4. Targeted applications of the SNP Array . . . . . . . . . . . . . . . . . . . . . . .24

5. Detection of Supernumerary Chromosomal Markers (SCMs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26

6. Benefits of SNP array in POC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28

Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Indications for SNP Array applications toclinical genetic testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Sample requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

39

Contacts

AuthorSaïd EL MOUATASSIM, PhD

Molecular Genetic department, Biomnis LyonSaïd El Mouatassim, PhDGrégory Egea, MD

Phone number: +33 (0)4 72 80 23 65Fax number: +33 (0)4 72 80 23 66

Biomnis International DivisionInternational Division17/19 avenue Tony GarnierBP 732269357 Lyon cedex 07FrancePhone number: +33 (0)4 72 80 57 42Fax number: +33 (0)4 72 80 73 56E-mail: international@biomnis.comWebsite: www.biomnis.com/international

GENOME-WIDEANALYSIS BY SNP ARRAY

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SNP ARRAY INT H E D I AG N O S I S O F I N T E L L E C T U A L D I S A B I L I T Y A N DC O N G E N I T A L A B N O R M A L I T I E S