Seven cases of intellectual disability analysed by genomewide SNP analysis. Rodney J. Scott

Seven cases of intellectual disability analysed by genomewide SNP analysis Rodney J. Scott

Ability to interrogate Genomic Material ~1930 Crude analyses 2012 Highly precise analyses

A (very) brief history of Genetics.. After Mendel

Karyotyping ~ 1930s, lost and rediscovered 1950s Transposable elements (100 s - 1000s bp in length, spread far apart) Restriction Fragment Sites frequency ~ 1/1,000,000 bp - 1/2000 bp (allowed for the development of FISH) DNA Microsatellite Markers spaced ~ 50,000 bp across the entire genome Single Nucleotide Polymorphisms (SNPs) 1 every 300 400 bp!!!

Karyotyping

Fluorescent in situ hybridization (FISH)

Chromosome Genomic Hybridization (CGH)

Array CGH using overlapping fragments of DNA

Costs $ SNP arrays have developed very rapidly: Size: 50K 100K 320K 550K 1000K 5000K Year: 2002 2004 2005 2006 2007 2010 Time (years) Offer the advantage of very dense coverage with up to 5,000,000 data points across the genome (array CGH only 30,000) Fine assessment of small gains or losses Identification of uniparental disomy

CNV terminology Log R Ratio Based on normalised intensity data. A value of 0 means there are 2 alleles present (balanced). Sig <0 means there is a deletion. Sig >0 means there is a duplication. B-allele Frequency Has a value of 0, 0.5, or 1. 0 = AA genotype 0.5 = AB genotype 1 = BB genotype Normal Log R ratio and B-allele frequency BB AB AA

CNV terminology Log R Ratio Based on normalised intensity data. A value of 0 means there are 2 alleles present (balanced). Sig <0 means there is a deletion. Sig >0 means there is a duplication. B-allele Frequency Has a value of 0, 0.5, or 1. 0 = AA genotype 0.5 = AB genotype 1 = BB genotype 1 copy gain BBB ABB AAB AAA

CNV terminology Log R Ratio Based on normalised intensity data. A value of 0 means there are 2 alleles present (balanced). Sig <0 means there is a deletion. Sig >0 means there is a duplication. B-allele Frequency Has a value of 0, 0.5, or 1. 0 = AA genotype 0.5 = AB genotype 1 = BB genotype 1 copy loss B A

Software Packages partek Partek Incorporated KaryoStudio/GenomeStudio Illumina SNP and Variation Suite 7 Golden Helix Nexus BioDiscovery

Capability Overview partek KaryoStudio SVS 7 Nexus User Friendly No Yes Yes Yes Visualisation OK Poor OK Tops Tests for Association Quality of output? N/A OK OK OK Poor OK Good Support Very Poor OK Tops OK Analysis types CNV (inc LOH) and SNP CNV CNV (inc LOH) and SNP CNV (inc LOH), and integration of expression and mirna data

Genome-wide SNP analysis as a new tool for cytogenetic analysis.

Case 1 Male Normal height and weight at birth. Growth retardation at 13 years (133 cmm <1 st percentile, head circumference 53 cm). Severe generalized psychomotor delay Dysmorphic facial features (open mouthed expression, thin upper lip, prominent cupids bow, smooth phitrum, up-tilted nose, low set ears, down slanting palpebral fissures with mild hypertelorism and high arched palate) Ventricular septal defect (poor peripheral circulation, tapering fingers, hyperventilation) Poor sleep and hand wringing.

Case 2 Male Severe intellectual disability Dysmorphic features (deep set eyes, right ptosis, hypoplastic alanase, very curly hair) Atrial septal defect (ASD) Ventricular septal defect (VSD), hypoplastic aortic arch and left ventricle, Multiple arteriovenous malformation (AVMs), mild coarctation. Hypoplasia of cerebellar vermis. Postnatal short stature Microcephaly Gastric oesophageal reflux Poor feeding Pulmonary HT.

Case 3 Female Previously identified apparently balanced translocation, t(x,6)(q13;q22.2) Complex cardiac defect, (aortic arch abnormality with narrowed right-sided carotid artery and large VSD).

Case 4 7 month old female conceived by intracytoplasmic sperm injection Born by caesarean section at 35 weeks due to decreased foetal movement. Severe hydrops fetalis (birth weight of 4000 grams due to oedema) and congenital chylothorax. Chromosome studies and karyotyping identified a complex, but apparently balanced, translocation involving chromosomes 1q25, 1q32, 2q23, 7q22 and 16q24. Both parents have normal chromosomes. At 7 months of age development was appropriate for corrected age (weight 90th centile, length and head circumference 75th centile). Gastro-oesophageal reflux and occasional oedema of legs, feet and sides of face. Facial appearance: flat nasal bridge, small nose with anteverted nares, wide mouth, and prominent epicanthic folds with stellate irides, suggestive of Williams Syndrome but MLPA analysis for William syndrome negative.

Cases 5 & 6 Female Dysmorphic features (hypotelorism and asymmetric nose), Single central incisor and short toes. Postnatal short stature Growth hormone deficiency. Male Mild developmental delay, Cataracts Optic atrophy Transposition of the great vessels Sensoneuronal (SN) deafness.

Case 7 Male with mild intellectual disability, Dysmorphic features (dysplastic ears, accessory nipple x 2 and camptodactyly of index finger), VSD Undescended testes Sensorineural hearing loss Postnatal short stature, treated with growth hormone & hypogonadotrophic hypogonadism.

Case 1 Two deletions were identified using SNP arrays: A 2.2 Mb deletion located on chromosome 1q44 (position: 240,761,719-242,994,271) A 1.2 Mb deletion on chromosome 13q31.1 (position: 82,281,240-83,456,591). The SNP array result of the 1q44 deletion was confirmed using FISH with BAC probe RP11-424N15 deleted. The deleted region 1q44 contains 4 genes with unknown function (MGC33370, PNAS-4, FAM36A and MGC12458), and ADSS (catalyses first step in conversion of IMP to AMP), HNRPU (RNA binding protein) and SMYD3 (histone methyl transferase). Region 13q31.1 contains no genes.

Case 2 LOH of entire chromosome 2 was identified in this patient using SNP arrays. Parental haplotype analysis was carried out using eleven microsatellite markers along the full length of chromosome 2. Six microsatellite markers (D2S319, D2S131, D2S390, D2S367, D2S380 and D2S326) were fully informative in this family and revealed a paternal isodysomy of chromosome 2 in the patient.

Maternal Proband Paternal Maternal Proband Paternal Chromosome 2 Microsatellite marker analysis D2S367 D2S380 Paternal isodisomy detected

Case 3 A duplication of approximately 4.5 Mb located on chromosome Xq21.1-21.31 (position: 83,722,520-88,224,080) was identified using SNP arrays. Region Xq21.1-21.31 contains four genes with unknown function and ZNF6 (a zinc finger protein of unknown function, located within a region which has been associated with mental retardation), CHM (membrane trafficking) and KLHL4 (unknown function, contains an actin binding domain).

Case 4 A deletion of approximately 1.7 Mb was identified on chromosome 7q22.2-22.3 (position: 103,396,976-105,403,796) using SNP arrays. FISH confirmed the deletion and its location at the breakpoint of the translocation between chromosome 2 and 7. The deletion of BAC clones RP11-612L03 and RP11-96B13 at 7q22. The deleted region at 7q22.2-22.3 contains eight genes with unknown function.

Cen 1 2 3 4 Tel 1 2 3 4 der 2 der 7 der 7 der 7 N 7 N 7 N 7

Case 5 An increase in copy number score (from 2 to 3,4,5 and 6) at numerous X chromosome loci were observed in this patient.

Cases 6 & 7 Case 6 and 7 No apparent chromosomal aberrations were detected in these two patients.

Conclusions Case 1: Deletion of 1q44 reported previously Case 2: Isodysomy of Ch2, 4 cases identified world wide. Paternal imprinting remains a possibility Cases 3 & 4: Previously identified translocations. Both were accompanied by genomic copy number changes at the break points. Previous technologies unable to reveal such changes. Case 5: 25 copy number changes identified on Ch X. 11/25 are likely to be polymorphisms

Conclusions ctd. Detection rate using the SNP array approach appears to be high (~71%) Could be due to ascertainment bias (all other assays were less sensitive). Parental testing required to confirm findins SNP array technology is superior to any other that is currently available Higher resolution arrays will yield more information (1M arrays are available)

Conclusions ctd. Microarray technology is cost effective (increased rate of anomaly detection) Cytogenetics merging with the power of Molecular Genetics When will karyotyping be replaced as the frontline cytogenetic request.