Chromosome Instability Syndromes

Hailey Fancher CLGT 5503 Oct 27, 2011 !

What are chromosome Instability Syndromes?

Chromosome instability syndromes are rare genetic disorders caused by mutations in DNA damage repair genes which result in DNA damage repair defects They exhibit a high level of spontaneous and induced chromosome abnormalities Each syndrome has its own spectrum of clinical features but they all share increased hypersensitivity to DNA damaging agents and risk of malignancies.

DNA Damage Repair

DNA damage occurs frequently
> 20,000 DNA lesions received by a cell each day.

Genomic replication and transcription can be blocked if damage is not repaired correctly

Causes of DNA Damage

http://www.nature.com/scitable/content/dna-damage-repair-mechanisms-and-consequences-14459141

DNA Damage Response

Sensors and Detection
DNA Damage

Tolerance Apoptosis
If DNA r

Transcriptional Response
epair fa

Checkpoint Activation

ils

DNA Repair
http://www.prettyshady.com/2010/up/magnifying-glass.gif http://www.e-nox.net/images/illustrations/dna_damage_full.jpg

DNA Damage Repair

Bypass DNA Adducts when replicating Damaged and Mispaired Bases
Nucleotide excision repair Base excision repair Mismatch repair

Double Strand Breaks

Translesion DNA Synthesis

Nonhomologous Homologous end joining Recombination

Ataxia Telangiectasia (AT)

Also known as Louis Barr syndrome Autosomal recessive disorder that results from mutations in the ATM gene Carriers may have an increased rate of breast cancer Prevalence rate ~ 1:40,000 -1:100,000 live births in the USA ~.2-1.0% of the general population are obligate carriers

ATM gene
150kb in length and has 66 exons Located on chromosome 11q22.3 Encodes a serine-protien kinase There are known to be more then 500 unique disease causing mutations in the ATM gene. No mutation hot spots Many ethnic populations have specic ATM founder mutations Most North Americans with AT are compound heterozygous Majority of mutations result in loss of ATM protein

Mechanism
ATM kinase responds to DNA damage mainly DNA double strand breaks, changes in chromatid structure and damage caused by ionizing radiation.

http://mcr.aacrjournals.org/content/6/4/517/F2.medium.gif

Common Clinical Features

Progressive cerebellar ataxia Oculomotor apraxia Choreoathetosis Telangiectasias on the whites of the eyes and the skin Frequent infections due to immunodeciency Hypersensitivity to ionizing radiation Hypersensitivity to chemotherapy Increased risk of cancer (mainly leukemia and lymphoma)
http://www.websters-online-dictionary.org/images/wiki/wikipedia/commons/thumb/1/19/Ataxia-telangiectasia2.gif/ 250px-Ataxia-telangiectasia2.gif

Life Expectancy
Most individuals survive to an age > 25 years old and some even survive into their 50s. The most common cause of death is bronchiectasis The second most common cause of death is cancer.

Cytogenetic Features
Higher then normal rates of spontaneous chromosome aberrations such as chromosome breaks, acentric fragments, dicentric chromosomes structural rearrangements, and anuploidy Majority show a 4-20x greater than normal frequency of induced chromosome aberrations when lymphocytes are irradiated during the G2 phase of the cell cycle.

Cytogenetic Features
Most common chromosome break points are 7p14, 7q35, 14q12, 2p11, 2p12, 22q11-q12 Signature t(7:14)
http://atlasgeneticsoncology.org/Kprones/ataxia.html

Molecular Genetics
Around 90% of ATM sequence variants can be detected by sequence analysis Targeted mutation analysis is available for some of the more common mutations found in specic ethnic populations such as the c.103C>T mutation seen in North African Jewish populations Duplication/deletion analysis can be used to detect large genomic deletions or duplications If there is sufcient family history, linkage analysis can also be used.

Testing Strategy
Testing Strategy
Proband Preform immunoblot assay of ATM protein level Preform molecular genetic testing of ATM to identify diseasecausing mutations Perform molecular genetic testing if the disease-causing mutations in the family has been previously identified Perform molecular genetic testing if the disease-causing mutations in the family has been previously identified

Carrier Prenatal

Bloom Syndrome
Very rare autosomal recessive disorder caused by a mutation in the BLM gene As of 2009 only 265 people have ever been registered as having Bsyn Frequency in general pop. is unknown Founder effect is present in the eastern European Ashkenazi Jewish population
! 1:48000 live births. ! 1% obligate carriers

BLM Gene
Located on chromosome 15q26.1 Encodes Bloom syndrome protein. 14 reported nonpathogenic variants > 60 known pathogenic mutations Most of the pathogenic mutations result in loss of Bsyn protein or a nonfunctional BSyn protein.

http://weill.cornell.edu/bsr/genetics/

Mechanism
BLM gene encodes a RecQ helicase BLM increases the accessibility of the DNA lesion BLM also acts as an effector in the DNA damage signaling cascade It is believed that BLM plays a key role in the maintenance of genomic stability during DNA repair, recombination and replication An abnormally high rate of recombination and mutation is seen when the BSyn protein is not present or nonfunctional

http://www.genomeintegrity.com/content/1/1/14/gure/F1!

Clinical Features
Pre and postnatal growth retardation Dolichocephaly Facial sun sensitivity and skin lesions Feeding problems Immunodeciency Increased risk of cancer Hypersensitivity to radiation and chemotherapy Fertility issue Thumb hypoplasia

http://weill.cornell.edu/bsr/clinical_description/ http://humgen677s11.weebly.com/

Life Expectancy
Average death age of 26 years. The most common cause of death is cancer

Cytogenetic Features
have higher then normal rates of chromatid gaps, breaks and rearrangements 1-2% of cultured BSyn lymphocytes show a quadriadial chromosome conguration. In individuals with BSyn sister chromatid exchanges (SCE) are seen at a rate of 40-100 per metaphase whereas they are seen at a rate of less than 10 per metaphases in normal individuals.

http://weill.cornell.edu/bsr/lab_diagnosis/

Molecular Genetic Testing

Can be used to conrm a clinical diagnosis of BSyn or to determine the type of mutation Targeted Mutation analysis is available only for the most common mutations including the eastern European Ashkenazi Jewish population c.2207_2212delinsTAGATTC BLM mutation The rest of the BLM mutations can be identied by sequence analysis of the whole BLM coding region.

Testing Strategy
Testing Strategy
Proband Preform SCE assay on lymphocytes Preform SCE assay on skin fibroblasts if blood is normal or inconclusive and mosaicism is suspected. Preform molecular testing to detect homozygosity for a disease causing mutation at BLM or compound heterozygosity for two different disease causing mutations Perform molecular genetic testing if the disease-causing mutations in the family has been previously identified or if Ashkenazi Jewish can test for the common mutation Preform SCE assay on fetal cells Perform molecular genetic testing if the disease-causing mutations in the family has been previously identified

Carrier

Prenatal (for at risk pregnancies)

Fanconi Anemia
Multi-gene disorder that results from a mutation in one of the 15 genes associated with FA The genes are each responsible for their own complementation group. The prevalence rate ~1:360,000 live births Carrier frequency ~1:300. Founder mutations are present in the Ashkenazi Jewish population
Carrier frequency of ~1:89

Complementatio n Group

Gene Symbol

Chromosom e Locus

Portion of FA Attributable to Mutations in This Gene 60%-70 ~2% ~14%

Role of Normal Gene Product A phosphoprotein that is a component of the FA core complex A component of the FA core complex A component of the FA core complex Regulates homologous recombination repair and has function in stabilization of stalled replication forks and regulation of cytokinesis Forms protein complex with FANCI Directly binds to FANCD2. Acts as a flexible adaptor protein required for the assembly of the FA core comple x An phosphoprotein that is a component of the FA core comple x An phosphoprotien that forms a protein complex with FANCD2 An DNA-dependent ATPase and a 5'-to-3' DNA helicase that binds directly to BRCA1 An E3 ubiquitinprotein ligase that is a component of the FA core comple x An component of the FA core complex and is phosphorylated in response to DNA damage

Common Mutations c.1115_111 8del c.3788_379 0del

FA Associated Genes
The FA associated genes are autosomal recessive except for FANCB, which has a Xlinked recessive The majority of carriers are considered to be asymptomatic except for FANCD1/BRCA2 heterozygotes whom may have an increased risk of breast, ovarian and other cancers.

FA-A FA-B FA-C

FANCA FANCB FANCC

16q24.3 Xp22.3 9q22.3

c.345+4A> T c.1642C>T c.67delG

FA-D1

BR C A 2

13q12.3

~3%

FA-D2 FA-E

FANCD2 FANCE

3p25.3 6p22-p21

~3% ~3%

FA-F

FANCF

11p15

~2%

FA-G

FANCG

9p13

~10%

c.307+1G> C c.9252A>G c.1480+1G >C

FA-I

FANCI

15q25-q26

~1%

FA-J

BR I P 1

17q22

~2%

FA-L

FANCL

2p16.1

~0.2%

FA-M

FANCM

14q21.3

~0.2%

FA-N FA-O

PALB2 RAD51C

16p12 17q22

~0.7% ~0.2%

Regulates localization and stability of BRCA2 protei n Component of several protein complexes involved in homologous recombinati o n Involved in resolution of homologous recombination intermediat e s

FA-P

SLX4

16p13.3

~0.2%

Mechanism
The FA associated genes all work together in the FA pathway Pathway activated in response to replicative stress caused by DNA damage from cross linking agents or reactive oxygen species. !
(Source: http://genesdev.cshlp.org/content/24/16/1680/F1.medium.gif)

Clinical Features
Physical abnormalities
Short stature Abnormal skin pigmentation Malformations of the thumbs, forearms, skeletal system, eyes, kidneys and urinary tract, ears, heart, gastrointestinal system, and central nervous system Hypogonadism developmental delay Thrombocytopenia or leucopenia Pancytopenia Neutropenia

Bone marrow failure

Adult-onset aplastic anemia Increased risk of cancer

http://img.medscape.com/pi/emed/ckb/pediatrics_general/954354-960401-2104.jpg

Cytogenetic Features
FA cells have a higher frequency of spontaneous chromosome breaks and exchanges. There may also be dicentric and ring chromosomes observed. When cultured FA cells are exposed to cross-linking agents (Mitomycin C or Diepoxybutance) multiple chromosome aberrations such as chromosome breaks/exchanges and quadriradial chromosomes are induced. FA can be diagnosed by the increased frequency of induced chromosome aberrations when compared to controls in chromosome breakage studies Carriers of FA cannot be detected using chromosome breakage assays.
http://atlasgeneticsoncology.org/Kprones/Images/BreaksFanconi.gif

Molecular Genetic Testing

Mutations in FA associated genes can be detected by sequence analysis Duplication/deletion analysis is also clinically available for some of the FA genes but not widely used The most common molecular test is the detection of the common Ashkenazi Jewish FANCC mutation (c.345+4A>T) by targeted mutation analysis

Testing Strategy
Testing Strategy
Proband Perform DEB or MMC chromosomal breakage assay on lymphocytes Perform DEB or MMC chromosomal breakage assay on skin fibroblasts if blood is normal or inconclusive and mosaicism is suspected. If cytogenetic testing has confirmed the diagnosis of FA, perform complementation analysis to identify the mutated gene and then perform sequence analysis of the appropriate gene to identify mutation. For Ethnic groups where founder effect is present targeted mutation analysis of FANCC or sequencing of specific genes can be preformed Perform molecular testing if the disease-causing mutations in the family has been previously identified Perform DEB or MMC chromosomal breakage assay on fetal cells from chorionic villus sampling or amniocentesis Perform molecular testing if the disease-causing mutations in the family has been previously identified

Carrier Prenatal (for at risk pregnancies)

Xeroderma Pigmentosum
A disorder that results from a mutation in one of the 9 genes associated with FA The genes are each responsible for their own complementation group except for ERCC1 and POLH. The prevalence ~1:000,000 live births in North America. XP has a higher prevalence rate in Japanese, North African, and Middle Eastern populations.
In Japan, prevalence rate ~1:22,000 live births

XP Associated Genes
The XP associated gene mutations are all autosomal recessive Carriers of XP associated gene mutations are clinically normal except for carriers of an XPC mutation who frequently have lower than normal levels of XPC mRNA.
Complementation Group A B C D E Gene Symbol XPA ERC C 3 XPC ERC C 2 DDB2 Chromosome Locus 9q22.3 2q21 3p25 19q13.2q13.3 11p12-p11 Portion of XP Attributable to Mutations in This Gene 25% Rare 25% 15% Rare Role of Normal Gene Product Has role in maintaining singlestranded regions during repair 3'- 5' DNA helicase and component of TFIIH basal transcription factor compl e x Has role in recognition of DNA damage and global genome repair 5'- 3' DNA helicase and component of TFIIH basal transcription factor complex Has role in the initial recognition of UV-induced DNA damage in non-transcribed portions of the genome DNA endonuclease 5' to DNA lesions DNA endonuclease 3' to DNA lesions error-prone DNA polymerase DNA excision repair protein

F G XP Varient

ERC C 4 ERC C 5 POLH ERCC1

16p13.3-p13.13 6% 13q33 6p21.1-p12 19q13.2q13.3 6% 21% Rare

Mechanism
Exposure to UV radiation causes damage to DNA by the production of diprimadine photoproducts such as cyclobutane dimers

Source: http://carcin.oxfordjournals.org/content/29/3/455/F1.large.jpg

http://www.ncbi.nlm.nih.gov/books/NBK21936/

Clinical Features
UV hypersensitivity (UVA,UVB,UVC)
Severe sunburn with blistering persistent erythema with minimal sun exposure (60% of affected) Marked freckle-like pigmentation of the face seen before age two years) Photophobia Keratitis

Eye abnormalities
Atrophy of the skin of the lids

A greatly increased risk of cancer

Greater than 2000x increased risk of Cutaneous melanoma with an average onset age of 22 years Greater than 10000x increased risk of Non-melanoma with an average onset age of 9 years

Neurologic manifestations
Acquired microcephaly Diminished or absent deep tendon stretch reexes Progressive sensorineural hearing loss Progressive cognitive impairment
http://www.dermaamin.com/site/images/clinical-pic/X/ xeroderma-pigmentosum/xeroderma -pigmentosum2.jpg

Cytogenetic Features
Individuals with XP do not show signicantly increased rates of spontaneous chromosome aberrations but UV exposure induces higher then normal levels of sister cromatid exchange and chromatid aberrations. Chromosomal breakage studies on cells exposed to UV radiation can be preformed but this is not an established method of XP diagnosis.

Molecular Genetic Testing

Diagnosis of XP can be conrmed by Sequence analysis but only for XPA, XPC, ERCC2, ERCC5, and DDB2 Mutations in the remaining XP associated genes can be detected by labs that offer custom sequence analysis Diagnosis of XP is often based on clinical features due to the lack of availability of clinical molecular testing for XP mutations.

Testing Strategy

Testing Strategy
Proband Carrier Prenatal (for at risk pregnancies) Clinical Diagnosis and then perform sequence analysis if available for conformation Perform sequence analysis if available and the disease-causing mutations in the family has been previously identified Perform sequence analysis if available and the disease-causing mutations in the family has been previously identified

Diepoxybutane Breakage Assay for Postnatal Diagnosis of FA

DEB is a highly reactive cross-linking agent and a potent carcinogen Safety precautions
personal protection equipment class II biological safety cabinet DEB should be disposed of as hazardous chemical waste

Preferred speciman: Peripheral blood

Summery of Procedure
Peripheral blood is cultured and a day later DEB is added to the culture The cells are treated with the DEB for approximately 2 cell cycles (48-72 hours) Cultures are harvested then G-banded or solid stained The resulting metaphases are then analyzed The Canadian College of Medical Geneticists recommends that 50 Solid stained patient and control metaphases be analyzed for chromosome breakage.

Complications
Interpretation of the results can be complicated by mosaicism resulting in two populations of lymphocytes observed
One showing increased sensitivity to DEB One showing normal levels of chromosomal breakage

Normal cellular phenotype is a result of:

gene conversion events back mutations compensatory deletions/insertions
May produce a false normal DEB test result. If a clinical suspicion of FA remains after having a normal result DEB test result the test can be repeated using an alternative cell type, such as skin broblasts.

Case Study:

Blooms Syndrome in a 12-year-old Iranian Girl

Tayebi N, Khodael H. 2008

A 12-year-old girl was sent for genetic testing because of her short stature Probands signs and symptoms:
Recurrent urinary, respiratory and gastrointestinal infections Long narrow face with a predominant nose Malar and mandibular hypoplasia Sun sensitive erythema on buttery area of the face Hyper and hypo pigmentations on trunk and limbs Talengectasia spots on some areas of the trunk Generalized excessive hairiness Bone age was 2 years decient of her chronological age

Case Study:

Blooms Syndrome in a 12-year-old Iranian Girl

Tayebi N, Khodael H. 2008
Probands parents were rst cousins Proband is the second child born All other members of her immediate family were healthy Two of her maternal cousins had similar but more severe signs and symptoms Her pedigree shows autosomal recessive inheritance.

Case Study:

Blooms Syndrome in a 12-year-old Iranian Girl

Tayebi N, Khodael H. 2008

50 metaphases GTG-banded metaphases were analyzed and found to be abnormal Structural chromosome abnormalities observed in four of the observed cells:
46, XX; chrb(1) (q42) 46, XX; chrb(X) (q21) 46,XX; chrb(7) (p22) 46, XX; chrb(9)(q13)

SCE analysis showed a mean of 80 SCE per cell compared to a mean of 7 per cell for the control.

Case Study:
Final diagnosis:

Blooms Syndrome in a 12-year-old Iranian Girl

Tayebi N, Khodael H. 2008

46,XX(46)/46,XX;chrb(1)/46,XX;chrb(X)/ 46,XX;chrb(7)/46,XX;chrb(9).

These ndings support a clinical diagnosis of Bloom Syndome.

QUESTIONS??