ASCP Molecular

indications for cytogenetic chromosome analysis

tissues suitable for karyotype

-Indications

• developmental problems- delay, FTT, dysmorphic features, congenital malformations, ambiguous genitalia, MR
• Stillbirth or neonatal death
• infertility or recurrent spontaneous abortions
• amenorrhea
• advanced maternal age
• family history or chromosome abnormality
• neoplasms

Tissues usable:

-PB lymphocytes (sodium heparin- green top)

-BM cells (sodium heparin- green top)

-skin biopsies

-fetal cells (Amnio/CVS)

-tumor cells

ASCP Molecular

Constitutional conditions d/t structural chromosome abnormalities

-Beckwith-Wiedemann

-Cri-du-Chat

-DiGeorge/VCF

-Miller-Dieker

-Pallister-Killian

-Rb (germline)

-Russell-Silver

-Smith-Magenis

-WAGR

-Williams

-Wolf-Hirchhorn

Constitutional conditions

-Beckwith-Wiedemann: Paternal UPD 11p15 (IGF2)

        --gigantism, macroglossia, omphalocele, hemihypertrophy, neonatal hyperglycemia, Wilm's tumor

-Cri-du-Chat: Del 5p

        --Meowing cry, low birth wt, FTT, microceph, facies

-DiGeorge/VCF: Del 22q11

        --cong heart defects (conotruncal), palate abn, immunodef and hypocalcemia

-Miller-Dieker: del 17p13 (LIS)

        --microceph, lissencephaly, sz, MR

-Pallister-Killian: + isochrom 12p

        --diaphragmatic hernia, hypotonia, short limbs, imperforate anus, talipes, facies

-Retinoblastoma (w/ birth defects): Del 13q14 (Rb)

        --Retinoblastoma (uni or bilat), osteosarcoma, craniofacial

-Russell-Silver: maternal UPD Chr 7

        --Pre and post natal growth retardation, skeletal asymmetry, triangle shaped face

-Smith-Magenis: Del 17p11

       --FTT, dysmorphic, hoarse voice, hearing loss, self-destructive behavior, sleep abn

-WAGR: Del 11p13 (PAX6 &WT1)

       --Wilm's tumor, Aniridia, Genitourinary malformations, MR

-Williams: Del 7q11 (ELN, elastin)

       --Aortic stenosis, small stature, hypercalcemia, outgoing personality

-Wolf-Hirchhorn: Del 4p

      --Pre and post natal GR, severe hypotonia, microceph, cleft lip/palate, chx facies with prominent forehead

ASCP Molecular

B-ALL with recurrent cytogenetic abnormalities and prognosis associated with each

B-ALL

-t(12;21)(p13;q22):  TEL-AML1 (ETV6-RUNX1), found in children; GOOD prog

-t(1;19)(q23;p13):   E2A-PBX1; child>adult; improved sensitivity to intense chemo

-t(v;11q23):  MLL gene, multiple translocation partners; found in infants <1yr; POOR prog

-t(9;22)(q34;q11):  BCR-ABL1 (usually minor breakpoint= p190 protein, esp children), adults 50/50: p230/p190;  found in adults>children; POOR prog

-t(5;14)(q31;q32): B-ALL with eosinophilia; IGH-IL3; rare, uncertain prog

**Hyperdiploid: 51-65 chr, usually +X, 4, 6, 10, 14,17,18; +21 in almost all; common in children; GOOD prog

-Hypodiploid: <46 chr; near haploid= children only; happens in adults and kids; POOR prog

ASCP Molecular

Common cytogenetics in CLL

CLL

-Del 13q14- in 50%, GOOD prog if only abn

-Del 11q22 (ATM)- POOR prog

-Del 17p13 (p53)- POOR prog

-tris 12 (+12)- uncertain prog, in 20%

-Mutated- somatic hypermutation of IGH variable= lower risk of progression

-Unmutated- germline IGH variable= higher risk of progression

ASCP Molecular

Cytogenetics in MM

MM

-Hyperdiploid: significantly better survival; +3, 5, 7, 9, 11, 15, 19, 21; typically don't have structural chr abn

-Hypodiploid: aggressive dz; complex structural rearrangements

    --14q32 translocations

    --monosomy 13 or del 13q14

ASCP Molecular

Cytogenetics in MDS

MDS

-Good risk:

    --Normal karyotype

    --Isolated del(5q)

    --Isolated del(20q)

    -- -Y

-Poor risk:

    --Complex abn (≥3)

    -- -7 or del(7q)

-intermediate risk:

    --anything else

ASCP Molecular

Cytogenetics in CML

CML

**t(9;22)(q34;q11): BCR-ABL, major breakpoint= p210

   some have µ brkpt= p230

   -Ph chr = derivative 22

Use Quant RT-PCR to assess response to imatinib (Gleevec)

   -Imatinib competes with ATP for binding the BCR-ABL tyr kinase

T315I point mutation= a/w resistance to imatinib and other tyr kin inh

-chr abn a/w progression= +8, iso(17q), and +der22 (Ph chr)

ASCP Molecular

AML with recurrent cytogenetic abn

1- with balanced transloc/inv

complete the translocation, what genes involved and prog

-t(8;21)

AML with recurrent cytogenetic abn

1- balanced transloc/inv

-t(8;21)(q22;q22): RUNX1-RUNXT1 (formerly AML1/ETO); favorable prog

ASCP Molecular

AML with recurrent cytogenetic abn

1- with balanced transloc/inv

-inv(16)

AML with recurrent cytogenetic abn

-inv(16)(p13q22) or t(16;16)(p13;q22)

- CBFB-MYH11

-favorable prog

-AML with eosinophilia

ASCP Molecular

AML with recurrent cytogenetic abn

1- with balanced transloc/inv

-t(15;17)

AML with recurrent cytogenetic abnormalities

-t(15;17)((q22;q12): PML-RARA

formerly APML (M3)

Favorable prog, with ATRA tx

ASCP Molecular

AML with recurrent cytogenetic abn

1- with balanced transloc/inv

-t(9;11)

AML with recurrent cytogenetic abn

-t(9;11)(p22;q23): MLLT3-MLL (11q23)

-intermediate prog, but better than all others with 11q23 (MLL) abn

ASCP Molecular

AML with recurrent cytogenetic abn

1- with balanced transloc/inv

-t(6;9)

AML with recurrent cytogenetic abn

-t(6;9)(p23;q34)

  -DEK-NUP214

POOR prog

ASCP Molecular

AML with recurrent cytogenetic abn

1- with balanced transloc/inv

-inv(3)

AML with recurrent cytogenetic abn

-inv(3)(q21q26) or t(3;3)(q21;q26)

-RPN-EVI1

-POOR prog

ASCP Molecular

AML with recurrent cytogenetic abn

1- with balanced transloc/inv

-t(1;22)

AML with recurrent cytogenetic abn

t(1;22)(p13;q13)

-megakaryoblastic

RBM15-MKL1

prev poor, but now responds well to intensive chemo

ASCP Molecular

AML with recurrent cytogenetic abn

what two variant APML translocations are a/w ATRA resistance?

-t(11;17)(q23;q21)- 11q23- ZBTB16

and

-t(17;17)(q13;q21)- 17q13- STAT5

ASCP Molecular

AML with recurrent cytogenetic abn

2- with gene mutations

there are 3 (2 are provisionary)

AML with recurrent cytogenetic abn

-with gene mutations

-FLT3: tyrosine kinase receptor

   --FLT3-ITD mutations (juxtamembrane domain)- adverse outcomes

   --FLT3-TKD (tyr kin domain)- uncertain prog

-NPM1- aberrant cytoplasmic expression of nucleophosmin (NPM) d/t mutation in nuclear homing

   --favorable prog in nl karyotype and absent FLT3

-CEBPA- favorable prog in nl karyotype

ASCP Molecular

Cytogenetics in mature B-cell neoplasms

-Follicular

-MALT

-Splenic marginal zone

-DLBCL

-Burkitt

-Mantle cell

Mature B-cell neoplasms

-Follicular: t(14;18)(q32;q21)= IGH-BCL2

-MALT- by location

   --lung/gastric: t(11;18) - API2-MALT1

   --ocular/adnexa/salivary: t(14;18) - IGH-MALT1

   --thyroid/ocular adnexa/skin: tris 3

-Splenic marginal zone: del(7)(q22-32)

-DLBCL: BCL2 (18q21) rearrangement or BCL6 (3q27)

-Burkitt: mc= t(8;14)(q24;q32)= MYC-IGH

  -less common are other translocations of MYC (8q24): with IGKappa (2q11) or IGlambda (22q11)

-Mantle cell: t(11;14)(q13;q32)- CCND1-IGH; cyclinD1 (11q13)

ASCP Molecular

Cytogenetic abn in mature T-cell neoplasms

-T-cell prolymphocytic leukemia

-ALCL

Mature T-cell neoplasms

-T-cell Prolymph leukemia: inv(14)(q11;q32)

   --TCRalpha (TRA)-TCL1A or TCL1B (14q32)

or -t(14;14) same genes

   --also freq have abn chr 8 and del(11q23) (ATM)

-ALCL: t(2;5)(p23;q35)- ALK-NPM

less common- t(1;2) - TPM-ALK

   -ALK= 2p23

ASCP Molecular

genes involved in myeloid and lymphoid neoplasms with eosinophilia

Eosinophilia

-PDGFRA (4q12)

-PDGFRB (5q31-q33)

-FGFR1 (8p11)

ASCP Molecular

Cytogenetics of ST tumors

-alveolar rhabdomyosarcoma

Alveolar rhabdomyosarcoma

-t(2;13)(q35;q14)- PAX3-FKHR

   -less common: t(1;13)- PAX7-FKHR- better prog

ASCP Molecular

Cytogenetics of ST tumors

-Embryonal rhabdomyosarcoma

Embryonal Rhabdomyosarcoma

-Del(11p15)-

  -Beckwith-Wiedemann region

  -complex translocations

ASCP Molecular

Cytogenetics of ST tumors

-Ewing Sarcoma

Ewing Sarcoma

-t(11;22)(q24;q12)- EWS-FLI1

-other variants with 22q12 (EWS) in small %

ASCP Molecular

Cytogenetics of ST tumors

-Synovial sarcoma

Synovial sarcoma

-t(X;18)(p11;q11)- SYT (18q11)- with either SSX1, SSX2 or SSX4 at Xp11

ASCP Molecular

Cytogenetics of ST tumors

-Alveolar soft part sarcoma

-Alveolar soft part sarcoma

-der(17) or t(X;17)(p11;q25)- ASPSCR1- TFE3

ASCP Molecular

Cytogenetics of ST tumors

-Clear cell sarcoma

Clear cell sarcoma

-t(12;22)(q13;q12)- ATF1-EWS

ASCP Molecular

Cytogenetics of ST tumors

-Desmoplastic small round cell tumor

DSRCT

-t(11;22)(p13;q11)- WT1-EWS

ASCP Molecular

numerical chr abnormalities

-Turner syndrome

Turner syndrome

-1/5000 live births

-99% of 45,X conceptuses are lost

-1/2 of cases are monosomy X d/t paternal meiotic  (only contain maternal X)

-others are mosaics/variants, with loss of some X material

-short stature, gonadal dysgenesis, cystic hygroma/webbed neck, coarctation of aorta, renal anomalies

ASCP Molecular

numerical chr abnormalities

Down's syndrome

Down's syndrome

-1/800 live births (but still majority of conceptuses are lost)

-usually d/t maternal nondijunction in meiosis (a/w adv mat age)

-small % d/t transloc involving 21q; iso(21q) o tris 21q

-CHx facies, MR, hypotonia, cardiac defects, duodenal atresia, leukemia (AML- very high risk)

ASCP Molecular

numerical chr abnormalities

-Patau

-Edwards

Patau- trisomy 13, rare, 1:20K-25K live births; heart def, holoprosencephaly, rocker bottom feet, clenched fists, microphthalmia, polydactyly, severe MR, rarely survive 1mo

[image] [image]

Edwards- trisomy 18, 1/6K-8K live births

- heart def, FTT, low-set malformed ears, prominent occiput, receding jaw, clenched fist, rockerbottom feet, MR, few survive to 1 yr

[image] [image]

ASCP Molecular

Cytogenetics of hydatidiform moles

Hydatidiform moles

-Complete mole- 46,XX (90%)

   --d/t empty egg fertilized by one haploid sperm, then undergoing endoreduplication.  Only paternal genes present

  -- no fetal tissue, risk of progression to choriocarcinoma (2-3% or less, like 1% in some articles)

  --10-15% may become invasive moles--> gestastional trophoblastic disease

-Partial mole- 69,XXX or 69, XXY

   --haploid ovum fertilized by two sperm or one sperm the undergoes reduplication

   --fetal tissue is present

   --essentially low to no risk of progression to choriocarcinoma

   --may still advance to gestational trophoblastic disease, but rarely

ASCP Molecular

Autosomal dominant inheritance- characteristics in inheritance and types with genes involved

Autosomal dominant

-Phenotype present in every generation

-each child of an affected parent has 50% chance of inheriting

Males and females equally affected

-Isolated cases caused by new mutation

Types/Examples

-Achondroplasia- FGFR3

-Autosomal dominant polycystic kidney disease- PKD1 (polycystin 1) and PKD 2 (polycystin 2)

-Familial hypercholesterolemia- LDLR

-Hereditary spherocytosis- multiple RBC cytoskeletal proteins- esp ankyrin and spectrin

-Marfan syndrome- FBN1 (fibrillin 1)

ASCP Molecular

Autosomal recessive inheritance

-Inborn errors of metabolism:

- Carbamoylphosphatase synthetase I def

Carbamoylphosphatase synthetase I def

-urea cycle disorder

Gene defect= carbamoylphosphatase synthetase

ASCP Molecular

Autosomal recessive inheritance

-Inborn errors of metabolism:

Gaucher disease

Gaucher disease

Lysosomal storage dz

gene defect= Glucocerebrosidase

ASCP Molecular

Autosomal recessive inheritance

-Inborn errors of metabolism:

Medium-Chain Acyl CoA def (MCAD)

Medium-chain Acyl CoA def (MCAD)

-fatty acid oxidation defect

-gene defect= medium chain acyl CoA dehydrogenase

ASCP Molecular

Autosomal recessive inheritance

-Inborn errors of metabolism:

Maple syrup urine disease

Maple Syrup Urine Disease

Disorder of- Amino acid metabolism

Gene defect: Branched chain α-ketoacid dehydrogenase

ASCP Molecular

Autosomal recessive inheritance

-Inborn errors of metabolism:

Phenylketonuria

Phenylketonuria (PKU)

disorder of amino acid metabolism

gene defect= phenylalanine hydroxylase

ASCP Molecular

Autosomal recessive inheritance

-Inborn errors of metabolism:

Tay-Sachs disease

Tay-Sachs disease

lysosomal storage d/o

gene defect: hexosaminidase A

ASCP Molecular

AR disease: Hereditary Hemochromatosis

-AR dz, d/t mutation in HFE (6p21)

-inappropriately high absorption of iron from GI tract; excessive storage of iron in- liver, skin, pancreas, heart, joints, testes and -->end-organ failure

-HFE= HLA Class I molecule, expressed as heterodimer with β2-microglobulin, binds transferrin receptor

-N. Europeans- 1 in 300 affected

-Screening test: transferrin-iron saturation= high and/or serum ferritin- high

majority of mutations are missense- C282Y- mc; 2nd mc= H63D

ASCP Molecular

X-linked Recessive inheritance:

-characteristics and examples with gene defects

XLR inheritance

Phenoype MUCH more often in males

-heterozygous (carrier) females- usually unaffected

-*NOT transmitted directly from father to son.

BUT all daughters of affected male will be carriers

Examples:

-Fabry's dz: α-galactosidase A

-G6PD def: G6PD

-Hemophilia A: fact VIII

-Hemophilia B: fact IX

-Lesch-Nyhan syndrome: Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

-Menkes dz: Cu2+-transporting ATPase

ASCP Molecular

X-linked dominant inheritance

XLD inheritance

All daughters of affected male are affected and NO sons are affected.

-*phenotype in females >males

-phenotype in females much milder

-rare mode of inheritance, few disorders involve XLD

Examples:

-X-linked hypophosphatemic rickets

-Incontinentia pigmenti (lethal in males)

ASCP Molecular

What is MELAS?

MELAS

-A disorder of mitochondrial inheritance

Stands for:

Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like episodes

-Stroke-like episodes before 40 yrs, seizures, abd/or dementia, ragged-red fibers, and lactic acidosis

Other types of mitochondrial inheritance d/o:

-Kearns-Sayre syndrome

-Leber Hereditary Optic Neuropathy

-Myoclonic Epilepsy with Ragged red fibers (MERRF)

-Progressive external ophthalmoplegia

ASCP Molecular

Inherited Cancer predisposition syndromes

-VHL

-Li-Fraumeni

-Retinoblastoma

-Cowden

Inherited cancer syndromes

-Von hippel Lindau (VHL)- 3p25: hemangioblastomas of brain/spinal cord/retina; renal cysts and Clear cell RCC, pheochromocytoma, endolymphatic sac tumors;  Also multiple brain venous angiomas

-Li-Fraumeni- 17p13- TP53: ST sarcoma, breast ca, leukemia, osteosarcoma, melanoma, colon, pancreas, adrenal and brain ca.  Multiple cancers early in life

-Retinoblastoma- 13q14- RB1: retinoblastoma (uni or bilat), pinealomas, osteosarc, ST sarc, melanomas

-Cowden syndrome- PTEN- 10q23: facial trichilemmomas, papillomas, mucosal lesions; Lhermitte-duclos dz (=cerebellar dysplastic gangliocytoma)

  -also get breast ca, thyroid ca (esp follicular), endometrial ca and macrocephaly

      --Other cancer syndromes with PTEN mutation (10q23)- Bannayan-Riley-Ruvulcaba syn and Proteus syn

ASCP Molecular

Syndromes a/w APC gene mutation

APC (5q21-22)

**FAP: classical- thousands of polyps, carpeted colon, by late teens/early 20's, 100% develop colon ca.

   -also a/w- cancer of ampulla of vater, bile duct, thyroid, small intestine

-Attenuated FAP: fewer polyps, later age of onset

-Gardner syndrome- Colonic adenomatous polyposis, osteomas, ST tumors (epidermoid, fibromas, desmoid tumors)

-Turcot synd- CRC, or CR adenomas + CNS tumors (medulloblastoma)

ASCP Molecular

Peutz-Jeghers syndrome

STK11 (19p13)

Hamartomatous GI polyps, small bowel polyposis, mucosal pigmentation, gonadal (sex cord) tumors; and a/w cancer of: colon, breast, pancreas, uterus, ovary, lung, testes

ASCP Molecular

HNPCC-

 Bethesda 2004 guidelines

Variant syndromes

HNPCC- CRC with MSI

-d/t mutations in DNA-repair genes: mc MLH1 and MSH2

(others- MSH6, PMS1, PMS2)

Bethesda 2004 criteria for patients to test tumors for MSI:

- CRC in individual <50y

-Synchronous/metachronous CRC or other HNPCC tumors (any age)

-CRC with MSI histology in tumor (<60y): tumor infiltrating lymphocytes, Crohn's-like response, mucinous/signet rign differentiation, medullary growth patter

-CRC dx'd in ≥1 1st-deg relative with and HNPCC related tumor  or 1 cancer dx'd <50y

-CRC dx'd in ≥2 1st or 2nd deg relatives of any age

HNPCC variant syndromes:

-Muir-Torre: sebaceous neoplasms skin and internal malignancies

-Turcot: CRC or adenomas with CNS (glioblastoma)

ASCP Molecular

MEN syndromes

Multiple Endocrine Neoplasia (MEN)

MEN type 1 (Wermer): MEN1 (menin) 11q13- Parathyroid, Pituitary (prolactinoma), pancreatic (well-diff endocrine neopl- gastrinoma, insulinoma, glucagonoma)

   -Plus- facial angiofibromas, collagenomas, lipomas, meningiomas, ependymomas, leiomyomas

MEN type 2- All RET, 10q11

-MEN type 2A (Sipple): Parathyroid adenoma, pheochromocytoma, medullary thyroid carcinoma

-MEN type 2B: Pheochromocytoma, medullary thyroid carcinoma, mucosal neuromas, ganglioneuromatosis (GI tract), enlarged lips, "marfanoid" habitus

-Familial Medullary Thyroid carcinoma

ASCP Molecular

Neurofibromatosis

Neurofibromatosis

-NF1: gene= NF1- neurofibromin-1, 17q11.2

   --Cafe-au-lait spots, axillary and inguinal freckling, dermal neurofibromas, plexiform neurofibromas, iris Lisch nodules

-NF2: gene=NF2 or Merlin; 22q12.2

  --bilateral acoustic schwannomas; multiple meningiomas, cataracts; ependymomas

ASCP Molecular

Tuberous Sclerosis

Tuberous sclerosis

variable disease, caused by either:

-TSC1- hamartin- 9q34

-TSC2- tuberin- 16p13

Skin: hypomelanotic macules, facial angiofibromas, Shagreen patches, fibrous facial plaques, ungual fibromas

Brain: SEGA/subependymal glial nodules, cortical tubers, seizures, MR/DD

Kidney: angiomyolipomas, cysts

Heart: rhabdomyomas, arrhythmias

ASCP Molecular

Examples of chromosome breakage syndromes

Chromosome breakage syndromes

-Bloom synd- BLM, DNA helicase, get many cancers

-Fanconi anemia- many genes, most FACA; hypersensitive to DNA crosslinking agents; AML, Hepatocellular tumors

-Ataxia-telangiectasia- ATM (11q23), cerebellar ataxia, telangiectasias, hypoplastic thymus; leukemias, lymphoma, and solid tumors; hypersensitive to xrays

-Xeroderma pigmentosum- sensitive to UV light; get BCC and SCC

-Werner synd- WRN, DNA helicase; premature greying/aging, sarcomas

ASCP Molecular

BRCA1 and BRCA2

BRCA1 and 2

-loss of function germline mutation inherited in BRCA1 (17q21) or BRCA2 (13q12-13)

-Hereditary Breast and Ovarian cancer

-Proteins- involved in DNA repair

-Autosomal dominant inheritance- acquire 2nd hit in tumor

-Female carriers- 85% risk of breast cancer by age 70

-BRCA1: higher grade tumors, medullary fx, triple (-): ER-, PR-, her2Neu unamplified; ovary- most serous adenocarcinoma

ASCP Molecular

List examples of trinucleotide repeat d/o's

Trinucleotide repeat d/o

-Fragile X syndrome

-Friedreich ataxia

-Huntington disease

-Myotonic dystrophy

-Spinal and Bulbar muscular atrophy (Kennedy dz)

-Spinocerebellar ataxias

ASCP Molecular

Huntington's disease

HD, 4p16

Autosomal dominant

-atrophy of caudate and putamen

-due to expansion of CAG repeat in exon 1 of HD

*Normal: ≤26

Intermediate: 27-35

Incomplete penetrance: 36-39

Full penetrance: ≥40

-show anticipation; passed by paternal repeat expansion

Test: determine repeat size by PCR

ASCP Molecular

Hardy-Weinberg Law

hardy-Weinberg law

At a genetic locus with 2 alleles of frequency p and q:

 p² + 2pq + q² = 1

p²= freq of homozygous pp

2pq= freq of heterozygous

q²= freq of qq homozygotes

Example:

genotype in 100 subjects for 2 alleles: T and t and get: 81 TT, 18 Tt, and 1 tt.  Find frequency of T and t alleles

p= freq of T= 81/100 + (1/2)(18/100)= 0.9

q= freq of t= 1/100 + (1/2)(18/100)= 0.1

Example:

rare autosomal recessive d/o detected at 1 in 40,000. WHat is freq of heterozygous carriers in general population?

q= freq of rare allele

q²= 1/40,000 thus q= 1/200

p=1-q= ~1 with rare alleles, p approaches 1)

Freq of carriers= 2pq= 2 * (1) * (1/200)= 1/100

ASCP Molecular

basic chx and application for:

-Transcription mediated amplification

- Branched DNA

-Hybrid capture

-TMA: target is ribosomal RNA, uses RNA polymerase and Reverse transcriptase

   --used for: C. trachomatis, N. gonorrhoeae, M. tuberculosis

-Branched DNA: target DNA or RNA; not amplifying product, just signal

    --used for: HBV DNA, HCV RNA or HIV RNA

-Hybrind Capture: target DNA; useing RNA probe; antibodies detect hybrid RNA-DNA

    --used for: HPV, CMV, C. trachomatis, N. gonorrhoeae