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					Chromosomal Genetics and Pathology (Dr. Teshima‟s material)

cytogenetic techniques – chromosome test
     need to culture living cells
     eg. venous blood in heparin tube, stimulate T cell growth with PHA, grow in medium
       plus FCS for 72 hours
     eg. amniotic fluid – grow in medium plus FCS for about 7 days
     arrest cells in metaphase (colcemid – inhibits mitotic spindle )
     fix cells with methanol/acetic acid
     stain with trypsin-Giemsa to produce characteristic „G-banding‟ patterns

general chromosome info
    diploid cell: 6 billion base pairs, 46 chromosomes
    nucleosome = DNA wound around histone proteins
    basic chromosome fibre = long chain of nucleosomes
    metaphase – chromosomes packed together very tightly (1/10,000 normal size) to ensure
       proper segretation during cell division
    non-metaphase cell cycle – chromosomes unpack to allow for DNA transcription and
       replication
    centromere = place where mitotic spindles attach, 171 bp tandem repeats (a.k.a. alpha
       satellites), can create create DNA probes specific for each chromosome‟s centromere (ie.
       for FISH)
       http://www.nature.com/nsu/011011/011011-1.html
       http://www.path.queensu.ca/present/harrison/michener/sld001.htm
    telomere = chromosome ends, tandem repeats of TTAGGG, synthesized by telomerase,
       function to stabilize chromosome structure
    subtelomeric repeats = each chromosome arm has characteristic sequences here, that can
       be probed with FISH
    classify according to location of centromere: metacentric = centromere in the middle,
       submetacentric = centromere closer to one end, acrocentric = centromere very close to
       one end (only satellites and telomeres, no important genes, on short arm)
    acrocentric chromosomes: 13, 14, 15, 21, 22
    FISH = fluorescence in-situ hybridization
           o direct – DNA probe tagged with fluorescent marker (eg. FITC = fluorescein
                isothiocyanate) hybridizes with particular chromosomal locus
           o indirect – DNA probes attached to a hapten (eg. biotin or digoxigenin) hybridize,
                then fluorescently tagged markers (eg. avadin or anti-digoxigenin) are used to
                detect the probes
           o FISH probes - specific to locus, centromere, or sub-telomere region… group of
                probes can 'paint' an entire specific chromosome

meiosis
 meiosis I separates homologous pairs, meiosis II separates sister chromatids
 non-disjunction can occur at either I or II
   genetic recombination (crossing over) necessary for orienting homologous chromosomes to
    opposite poles for proper segregation, and for producing new gene combinations

aneuploidy
 live births can occur with trisomy 13 (Patau‟s Syndrome), 18 (Edward‟s Syndrome), 21
   (Down‟s Syndrome)
 most trisomies and chromosomal abnormalities are spontaneously aborted
 risk for Down‟s increases with maternal age above 30 (exponential increase)
 origins of the extra chrom. 21: 87% maternal… of those, 75% meiosis I
 oocytes undergo recombination in 3-5mo fetal life and then remain arrested in meiosis I until
   ovulation
 by contrast, sperm are continuously produced and undergo both meiosis I and II in ~64 days
 oocytes – 20% aneuploid, sperm – 1-2% aneuploid
 2 step hypothesis to explain non-disjunction in female germ cells:
    step 1 – prior to birth, chromosomes undergo reduced recombination
    step 2 – after birth, environmental / intracellular conditions affect completion of meiosis
 possible genetic susceptibility – polymorphisms in MTRR and MTHFR genes linked to
   Down Syndrome… these genes are involved in folate metabolism, which is necessary for
   methylation, and centromere methylation is essential for normal chromosome segregation
 recurrence risk after liveborn with trisomy 21:
    <30 maternal age – 1% (due to possible parental gonadal mosaicism, or genetic
       predisposition for non-disjunction)
    >30 maternal age – 1% plus age-related risk

Robertsonian translocation
 2 acrocentric chromosomes (D =13, 14, 15; G = 21, 22) joined at the centromere region
 of all infants with Down Syndrome, 5% have a Robertsonian translocation… of these,
    >50% are der(D;21), especially der(14;21)… half of these are inherited
    <50% are der(G;21), especially der(21;21)… 95% of these are de novo
 when they join, the short arms are lost… but these contain only repetitive rRNA sequences so
   the loss isn‟t deleterious
 balanced carrier (eg. mom) – 45, XX, der(14;21)(q10;q10)
 affected child – 46, XY, der(14;21)(q10;q10), +21
 carriers can produce: normal gametes, balanced gametes, unbalanced gametes
 carriers for diff. Robertsonian translocations have different risks of affected child
    rob(13;14) – risk <1%
    rob(14;21) – risk 2% if paternal carrier, 10% if maternal carrier
    rob(21;21) – risk 100%
 uniparental disomy (can be the result of a „resolved‟ Robertsonian trisomy)
    eg. rob(13;14)  UPD 14 or rob(D or G;15)  UPD 15
    associated with adverse clinical outcomes as well (ie. deleterious recessive genes)
prenatal diagnosis for aneuploid syndromes
 indications – increased risk
    advanced maternal age
    previous child with trisomy 21
    Robertsonian carrier
    1st trimestre: increased nuchal translucency, increased hCG, decreased PAPP-A
    positive MSS (16wks) – measures alpha-fetoprotein, hCG, and unconjugated estriol…
       predicts risk of Down syndrome, trisomy 18, open neural tube defect
 options for sampling
    CVS – 10wks, 1% risk of miscarriage
    amniocentesis – 15-16wks, 0.5% risk of miscarriage
 options for testing
    karyotyping – labour intensive, need lg. sample, 2 week wait
    interphase FISH – less work, smaller sample, 2 day wait
        guidelines(?): risk >5%, major anomaly on US, potential for intervention
        positive – sufficient for management, negative – should karyotype

reciprocal translocations
 eg. baby karytype abnormality found: 46, XX, 5q+ (extra-long 5q arm)
    mom‟s karyotype tests normal: 46, XX
    dad‟s karyotype tests abnormal: 46, XY, t(5;14)(q35;q24)
    thus baby‟s karytype is actually: 46, XX, der(5)t(5;14)(q35;q24)pat
 reciprocal translocation carriers
     1/500 of couples (2-5% of those with multiple miscarriages)
     empirical risk of chrom. unbalanced offspring only 10%
     alternate segregation: both normal or both abnormal chromosomes, balanced gametes
     adjacent-1 segregation: one normal and one abnormal chromosome, unbalanced gamete
     adjacent-2 segregation: both normal and abnormal of same chromosome (rare)
     3:1 (ie. non-disjunction) or 4:0 (rare)
 t(11;22) translocation
     most common constitutional translocation in humans, >>100 noted in literature
     t(11;22)(q23;q11), due to low-copy repeats in 11q23 and similar repeats in 22q11.2
     risk of affected offspring <5%
     affected offspring must have extra der(22), via meiotic non-disjunction
 affects of translocation breakpoints
     through gene – disrupts function
     outside gene – disrupts chromatin structure or regulatory elements
     useful in identification of disease genes
 diagnosis
     cryptic rearrangements identified by FISH subtelomeric testing
     eg. detection of subtelomeric rearrangements in idiopathic mental retardation
     HSC introduced TEL-FISH in Apr 2002 but v. expensive ($750cdn)
      clinical checklist includes: normal G-banding and devel. delay or IQ<70, plus some of:
       family hx of MR, prenatal onset growth retardation, postnatal growth abnormalities, >2
       facial dysmorphic features, >1 non-facial dysmorphic feature or congenital abnormality


genome-wide FISH: multiplex (M-FISH) and spectral karyotyping (SKY)
    pool of human chromosome painting probes, each labelled with a different fluor
      combination (5 fluorocromes -> 31 different combinations, only 24 diff. chromosomes)
    hybridized to metaphase chromosomes
    each chromosome has a unique emission spectrum, that can be detected with fluorescent
      microscope some special equipment
    computer program displays chromosomes digitally using easy-to-distinguish colours
    great for finding translocations in clinical genetics, leukemias, solid tumors

genomic disorders
 diseases caused by rearrangements involving one to several megabases of a chromosomal
   region
 eg. microdeletion syndromes
 results in gene dosage imbalance
 can cause congenital malformation, mental retardation… large impact on human health

microdeletion syndromes
 deletion of small part of a chromosome, at a specific location
 loss of contiguous genes resulting in clinical features of syndrome
 eg. William syndrome (http://www.williams-syndrome.org/facts.htm)
    deletion at 7q11.23 (diagnosis with elastin-specific FISH)
    involves elastin and other genes
    supravalvular aortic stenosis and other features
 possible mechanism of origin – misalignment of repeats during recombination resulting in
   deletion or duplication
 eg. chrom. 17p11.2
    deletion causes Smith Magenis syndrome
    duplication causes „duplication syndrome‟
 eg. peripheral myelin protein (PMP22) gene
    deletion causes Hereditary Neuropathy with Liability to Pressure Palsies (HNPP) -
       episodes of numbness and muscle weakness
    duplication causes Charcot Marie Tooth disease type 1A(CMT1A) - slow progressive
       atrophy of distal limb muscles
 many microduplication syndromes remain unidentified – usually milder phenotype

microdeletion 22q11.2
 eg. 46, XX.ish del(22)(q11.2q11.2)(TUPLE1-)… 3Mb hemizygous deletion
 current candidate gene is TBX1 (transcription factor)
 problems occur in particular pharyngeal arches during development
 variable clinical presentation: neonatal death  isolated hypernasal speech
   conotruncal heart defects, face, cleft palate, thymic and parathyroid defects, hypocalcemia,
    developmental disability
   medical management: cardiology, immunology, endocrinology, renal ultrasound,
    hematology, speech therapy, child development
   DiGeorge in infancy, Velocardiofacial in children
   mostly sporadic, familial in 5-10%

olfactory receptor gene superfamily
 clusters of olfactory receptor (OR) genes are found on most human chromosomes, some have
    more than one cluster
 unequal recombination b/w OR clusters on chrom. 8 (short arm) results in three recurrent
    chromosomal rearrangements: inverted duplication (distinct phenotype), supranumery
    chromosome (minor anomalies), 8p23 interstitial deletion (heart defect)
 these recombination events are associated with a maternal polymorphism – heterozygosity
    for an 8p submicroscopic inversion (seen in 26% of people of European descent)
 so the inversion polymorphism confers susceptibility to unequal recombination events
 another example: polymorphic genomic duplication on chromosome 15 is a susceptibility
    factor for panic and phobic disorders

DNA microarray method using comparative genomic hybridization (CGH)
 ~1000 BACs with DNA from locations across the human genome (spaced 2-4Mb apart)
 DNA spotted onto glass slides to make a BAC array
 DNA from patient and from normal reference – hybridize to array and detect any differences

Chromosome 15 alterations – Prader-Willi and Angelman syndromes
 these syndromes are usually due to a 4Mb deletion of the PWS region, which contains ~12
   genes including SNRPN (small nuclear ribonucleoprotein polypeptide N)
 detect with FISH – probes: centromere, PML (control), SNRPN (locus of interest)
 deletion on maternal 15  Angelman syndrome
 deletion on paternal 15  Prader-Willi syndrome

genomic or gametic imprinting
 “A reversible process whereby a gamete-specific modification in the parental generation can
   sometimes lead to functional differences between maternal and paternal genomes in diploid
   cells of the offspring.”
 germline erasure of existing imprints
 acquisition of imprint by gamete according to sex of germline
 maintenance of imprint in 2n somatic cells
 translation of imprint in somatic cells into monoallelic gene expression
 involves allele-specific methylation (ie. SNRPN is differentially methylated depending on
   parent of origin, thus only expressed on paternally inherited 15)

methylation-specific PCR
 treat specimen with bisulfite to convert all unmethylated C‟s to U‟s
   so unmethylated and methylated versions now have different sequences in the 5‟ promoter
    region
   use specific PCR primers to produce products of characteristic size
     maternal (methylated) – 174 bp PCR product
     paternal (unmethylated) – 100 bp PCR product
     normal – both 174 and 100, PWS – 174 only, AS – 100 only
   can also use Southern blot method
     maternal – 4.3 kb fragment
     paternal – 0.9 kb fragment
     PWS – 4.3 only, AS – 0.9 only

uniparental disomy
 PWS – two maternal 15s… AS – two paternal 15s
 issue in prenatal testing
   CVS – 46, XY / 46, XY, +15
   Amniocentesis – 46, XY
   trisomic zygote  trisomic placenta, UPD fetus

other imprinting syndrome
 Beckwith-Weidemann syndrome
     overgrowth malformation syndrome – large tongue, omphalocele, ear creases, risk of
        tumours, etc.
     results from mutations or epigenetic events involving imprinted genes in 11p15

features of imprinted genes
 clustered in domains
 differential methylation (expression with hypomethylation)
 differential histone H4 and H3 acetylation (expression with acetylation)
 reduced mRNA, differential replication
 atypical non-coding RNAs – H19, IPW, antisense transcripts from non-coding strand
 imprinting centre determines methylation
     <5% of PW and AS due to abnormal methylation from disruption of IC
     deletion (can be inherited) or no deletion (sporadic)
 10% of AS due to UBE3A mutation

summary
PWS: paternal deletion or mutation, maternal UPD, imprinting, translocation
AS: maternal deletion or mutation, paternal UPD, imprinting, translocation

				
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