Topics

1. Topics • Interpretation of pedigrees • Autosomal dominant (including trinucleotide repeats), recessive, X linked, mitochondrial, imprinting (risk assessment questions). • Knowledge about genetics of common adult monogenic disorders. • When is testing appropriate. • presymptomatic testing (HD, children etc) • Cancer genetics: breast, bowel cancer, rare genetic tumour syndromes. • How genetic tests are performed and their interpretation. • Basic science knowledge questions.

2. Mechanisms of Genetic Diseases • Dominant • Recessive • X linked • Trinucleotide repeat diseases • Mitochondrial Inheritance • imprinting • Complex Disease

3. Dominant Conditions • Neurofibromatosis (NF2). • Adult polycystic kidneys (ADPKD) • BRCA, HNPCC, MEN • Von Hippel Lindau. • Tuberous Sclerosis. • Marfan syndrome, EDS, OI • HMSN (CMT 1a, HNPP). • Hypertrophic Cardiomyopathy.

4. Dominant Pedigrees • Vertical Transmission • Are all offspring affected? • Does father to son transmission occur • Are new mutations common? • Interpretation of pedigrees complicated by somatic mosaicism, reduced penetrance and variable expressivity.

5. Mechanisms of Dominant Diseases 1. Dominant Negative Abnormal protein disrupts function of other proteins Occurs when a gene forms part of a complex Common with disorders affecting CT E.G. Marfan syndrome - FBN1 15q21 Collagen disorders - EDS, OI

6. Mechanisms of Dominant Diseases 2. First Hit Mutations A somatic second hit required to develop the condition Affected individuals inherit a predisposition Mechanism of many familial cancer syndromes E.G. Retinoblastoma, MEN, HNPCC, LFM

7. Carcinogenesis Initiating events: Tumour Suppresser Genes Accelerating events: Oncogenes First Mutation  Second Mutation  Third Mutation  …

8. Familial Bowel Cancer • Hereditary Non-Polyposis Colorectal Cancer HNPCC: 2-5% of familial CRC Modified Amsterdam Criteria At least 3 relatives with cancer: Colorectal, Endometrial, Small bowel, Ureter / Renal pelvis. Histology Confirmed. One the 1° relative of the other 2 (2 generations with CRC) One or more cancers diagnosed before age 50 • Familial Adenomatous Polyposis: 2% • Other Rare Syndromes:Peutz-Jeagar, Hyperplastic Polyposisis, Cowden syn.

9. Bowel Cancer in HNPCC Amsterdam Criteria Predominantly Right Sided, Early onset  Mucinous and Poorly differentiated types Synchronous and Metachronous tumours

10. Gene Mutations in HNPCC

12. Question 2000 1 3 • A 45-year-old man develops stage C cancer of the caecum. There is a strong family history of bowel cancer in the absence of polyps and there is no history of colitis. • The most likely underlying inherited genetic abnormality is in: • A) the ras gene. • B) the DNA mismatch repair (MMR) gene. • C) the p-glycoprotein (MDRI) gene. • D) the adenomatous polyposis coli (APC) gene. • E) the deleted in colon cancer (DCC) gene.

13. Question 1999 1 13 • A 35-year-old man presents with rectal bleeding. He describes the blood as being mixed with the stool. He is otherwise well. His brother was diagnosed with bowel cancer at the age of 38. • At colonoscopy he was found to have two exophytic tumours, one at the hepatic flexure, the other in the distal transverse colon. The remainder of the colon was normal. Biopsies of both areas showed adenocarcinoma. • The most likely genetic disorder in this family is: • A) familial adenomatous polyposis coli (FAP). • B) hereditary non-polyposis colorectal cancer (HNPCC). • C) Gardner's syndrome. • D) Peutz-Jeghers syndrome. • E) a p53 gene mutation.

14. Question 2003 2 18 • Hereditary non-polyposis colon cancer (HNPCC or Lynch syndrome) is associated with a number of extracolonic malignancies. • Which one of the following extra-colonic malignancies is most strongly associated with this diagnosis? • A) Melanoma. • B) Sarcoma. • C) Leukaemia. • D) Endometrial cancer. • E) Renal cell cancer.

15. Question 2001 2 12 • A 40-year-old man had profuse colonic polyposis diagnosed 15 years ago. A clinical diagnosis of familial adenomatous polyposis (FAP) had been made. He had a total colectomy. There is no family history of polyposis or colorectal cancer. • Mutation studies fail to identify a pathogenic mutation in the adenomatous polyposis coli (APC) gene in a blood sample. • The normal DNA result is best explained by: • A) the correct clinical diagnosis being juvenile polyposis. • B) the mutation occurring in a non-coding region of the APC gene. • C) gonadal mosaicism of the APC gene mutation. • D) the causative mutation being in another gene. • E) the APC gene mutation occurring only in cells derived from adenomatous polyps.

16. Question 1999 2 57 • Which one of the following is least likely to be associated with familial colon cancer? • A)Mutations in the adenomatous polyposis coli gene. • B)Mutations in DNA repair genes. • C)Chromosomal translocations. • D)Microsatellite instability. • E)Loss of heterozygosity for tumour suppressor genes.

17. Overview of Familial Contribution to Breast Cancer • 5 % breast cancer due to known genetic factor • 80% BRCA 1 & 2 • Others • ATM • E-cadherin1 • TP53 • CHEK2 BRCA1 & 2 16% 80% Genetic & other

18. Age of Onset Am J Hum Genet. May 2003; 72(5): 1117–1130.

19. risk in general population Menarche before 12 yrs Menopause after 55 yrs First live birth after 30yrs Nulliparity Obesity Alcohol use Previous biopsies BRCA carriers Breast feeding for one year  OC for 3yrs after 30  BRCA1 OC prolonged starting before 25  Parity in BRCA2  HRT post-oophorectomy incidence of breast ca Non-genetic Risk Factors

20. Indicators of a Genetic Cause • Family History • 3 generations, maternal and paternal sides • Age of diagnosis: pre-menopausal, particularly under 40 yrs • Male breast cancer • Bilateral breast cancer • Ovarian and breast cancer in the same individual or family line • Ethnicity

21. Question 2004 2 88 A 35-year-old mother with breast cancer reports that her aunt had developed breast cancer at 48 years of age. She is keen to clarify the risk of her young daughter developing breast cancer. A blood sample from the mother is submitted for mutation analysis of the BRCA1 and BRCA2 genes, but no mutation is found. What impact does the mutation analysis have on the estimate of the daughter’s risk of developing breast cancer? A. The maternal studies place the daughter at low risk of developing breast cancer. B. The maternal studies do not clarify the daughter’s risk. C. The maternal studies place the daughter at high risk of developing breast cancer. D. The daughter’s risk cannot be clarified without studies of her father’s BRCA1 and BRCA2 genes. E. The daughter’s risk cannot be clarified without studies of her own BRCA1 and BRCA2 genes.

22. Question 2001 2 28 • A 35-year-old woman seeks your advice about her risk of developing breast cancer. Which one of the following would place her at greatest risk of developing breast cancer? • A) Menarche less than 12 years. • B) Birth of first child after the age of 25. • C) Oral contraceptive use for more than 10 years. • D) Sister and aunt diagnosed with breast cancer. • E) Excision of a benign breast lump.

23. There are a number of questions addressing the Knudson two hit hypothesis.

24. Question 2002 1 58 • A man is admitted to hospital for surgical treatment of bilateral vestibular schwannomas (i.e. acoustic neuromas). He has a family history of the disorder and a familial mutation in the NF2 gene has been identified. At operation, samples of blood and tumour tissue are collected for DNA studies. A Southern blot of these samples (with a normal control sample for comparison) is probed with a DNA fragment derived from the NF2 gene. The result is shown in the figure below. • What is the most likely interpretation of this result? • A)The mutant NF2 allele has been lost from the patient’s blood. • B)The mutant NF2 allele has been lost from the patient’s tumour. • C)The mutant NF2 allele has been duplicated in the patient’s tumour. • D)The normal NF2 allele has been lost from the patient’s blood. • E)The normal NF2 allele has been lost from the patient’s tumour.

25. Mechanisms of Dominant Diseases 3. Haploinsufficiency Product of both alleles required for normal function Occurs in systems with minimal functional reserve Can also have effects from over expression. Can you think of an example?

26. Recessive Pedigrees • Horizontal Transmission • Carrier state exists • Are new mutations common? • Recurrence risk for siblings = ?% •  consanguinity.

27. Carrier Risk in Extended CF Pedigree I:1 I:2 Grandparent II:1 II:2 II:3 II:4 II:5 Parent Uncle (Aunt) III:1 III:2 III:3 III:4 III:5 III:6 III:7 Sibling Affected First Cousin IV:1 IV:2 Nephew (Niece) V:1 750

28. Carrier Risk in Extended CF Pedigree I:1 I:2 Grandparent 1/2 1/2 II:1 II:2 II:3 II:4 II:5 Parent Uncle (Aunt) 100% 100% 1/2 1/2 1/25 III:1 III:2 III:3 III:4 III:5 III:6 III:7 Sibling Affected First Cousin 2/3 1/25 2/3 1/25 100% 1/25 1/200 IV:1 IV:2 Nephew (Niece) 1/150 1/25 V:1 1/3750

29. Question 2001 1 64 • In the pedigree shown below, the man indicated by an arrow has been shown to have an autosomal recessive biochemical disorder with complete penetrance. The causative gene has not been identified. His parents are obligate carriers and do not exhibit any biochemical abnormalities. His sister also has normal biochemical studies. The carrier frequency in this population is 10%. • In the absence of consanguinity, what is the risk of the sister having a child with the biochemical abnormality? • A) 1 in 40. • B) 1 in 60. • C) 1 in 80. • D) 1 in 100. • E) 1 in 120.

30. Question 2002 2 35 • Approximately 10% of the Caucasian population has a mutation in the haemochromatosis gene (HFE). Three men in a family (shown below) have been diagnosed with haemochromatosis. • What is the risk that the woman (indicated by the arrow in the pedigree below) has inherited the genetic predisposition to develop this disorder? • A)<1%. • B)5%. • C)10%. • D)25%. • E)50%.

31. Question 2000 2 51 • The following mutations (Cys282Tyr and His63Asp) are associated with hereditary haemochromatosis. Which one of the following genotypes provides the greatest risk for the development of clinical disease? • A) Heterozygous Cys282Tyr. • B) Heterozygous His63Asp. • C) Double-heterozygote for Cys282Tyr and His63Asp. • D) Homozygous Cys282Tyr. • E) Homozygous His63Asp.

32. Question 2004 70 c • In the pedigree shown above, the affected male has a rare autosomal recessive disorder. His niece and nephew have a newborn son (indicated by the arrow). • What is the chance that the baby will have the same disorder? • A. 1 in 18. • B. 1 in 32. • C. 1 in 36. • D. 1 in 64. • E. 1 in 128.

33. X linked disorders • Male to male transmission always/sometimes/never occurs • All daughters of an affected male receive the abnormal gene. • Unaffected males never transmit the disease to their offspring (of either sex). • The risks to sons of women who are definite carriers is ….. • 0/.5/all the daughters of carrier women will be carriers themselves.

34. Risk in a Haemophilia Pedigree I:1 I:2 II:1 II:2 II:3 II:4 II:5 II:6 ? ? ? III:1 III:3 III:2 III:4 III:5 III:7 III:6 IV:1 IV:2 IV:3 IV:4

35. Question 2003 1 2 • A woman (indicated by the arrow in the Figure) seeks your advice about the risk of her unborn child having haemophilia A. Her family history is summarised in the pedigree shown below. The two affected males had presented during the second year of life and had died in their teens. The woman is married to her first cousin. • What is the risk of her unborn child having haemophilia A? • A) <1%. • B) 6.25%. • C) 12.5%. • D) 25%. • E) 33%.

36. Question 2004 1 24 • In the pedigree shown below, the affected man has an X-linked recessive disorder. A polymorphic DNA marker has been identified close to the mutant gene responsible for this disorder. The genotypes at this marker are given below each symbol in the pedigree. The recombination fraction between the gene and the DNA marker is 10%. • What is the best estimate of the risk of the woman indicated by the arrow being a carrier of her grandfather’s disorder? • A. 100%. • B. 90%. • C. 81%. • D. 66%. • E. 10%. a a,b a,b c a,c

37. Trinucleotide Repeat Diseases • Result from instability of repeated DNA sequences of three nucleotides • Instability proportional to length, and in some cases the sex of the transmitting parent. • The repeat number changes from generation to generation - hence ‘dynamic mutation.’ • Anticipation.

38. Non-Coding TNRs Fragile X CGG MD CTG Friedreich Ataxia GAA SCA8 CTG SCA12 CAG Fragile E GCC Translated TNRs HD CAG SCA1 CAG SCA2 CAG SCA3 CAG SCA6 CAG SCA7 CAG SBMA CAG DRPLA CAG Trinucleotide Repeat Diseases

39. Fragile X • The commonest monogenetic cause of MR • Unstable CGG repeat in exon1 of FMR • Phenotype in males with full mutation but also… Full mut. Females MR PreMut Females POF ??PreMut Females personality Premutation males FRAXTAS

40. II. Interpreting Fragile X Tests • Full Mutations • >200 CGG repeats • associated with abnormal methylation • Pre-mutation • 60 to 200 CGG repeats (?55 - 60) • unstable in transmission • Intermediate alleles • 40 to 55 CGG repeats • expansions are infrequent and small

41. Interpreting Fragile X Tests The risk of expansion to full mutation is a function of the repeat size: In Maternal transmission Repeat no. Pre : Full 61-70 >3 : 1 71-80 1.25 : 1 81-90 1 : 2 91-100 1 : 4 >100 Always

42. I:1 I:2 ? II:5 II:1 II:6 II:3 II:4 77/29 male ? III:1 III:2 III:3 III:4 III:5 >200 250 52

43. Myotonic Dystrophy • Commonest AD muscular dystrophy. • Wide variation in features, severity and age of presentation within families. • Due to TNR expansion involving a CTG repeat in a non-coding region of the DMPK gene 19q13… but ?other genes. • New mutations rare. • Unstable in maternal transmission. • A cause of MR – 50-60% of CDM

44. …CTGCTGCTGCT… 50 – 2000+ <38 NORMAL DMPK Chromosome 19q13 5’ 3’ polyA DMWD DMAHP

45. Phenotype Sympt Rpts. Onset Death Premutation Nil 38-49 Mild Cataracts Myotonia 50-150 20-70 60-N Classical Muscle, heart Eye, face 100 – 1500 10 – 30 48-55 Congenital Hypotonia, MR, resp. 1000* – 2000+ 0 – 10 (45) Interpretation of MD tests. *may be as low as 750

46. Question 2002 2 52 • A 10-year-old boy (indicated by the arrow in the pedigree below) has been diagnosed with myotonic dystrophy. The diagnosis is confirmed by DNA testing. His mother’s cousin has myotonic dystrophy, but the other surviving relatives have no history suggestive of a myopathy. • What is the most likely explanation for this pedigree? • A) Consanguinity. • B) Imprinting. • C )Non-paternity. • D) Incomplete penetrance. • E) Mitochondrial inheritance.

47. Risk of Congenital DM • Mothers with Mild phenotype have only small risk • Empirical recurrence risk 20 – 40% • Maternal repeats <300 10% >300 59% J Med Genet 1995 32: 105-8

48. Treatment • No specific treatment. • Orthotics and other physical therapies • Cardiac: baseline and annual ECGs • Myotonia: phenytoin, carbamazepine • Cramps: clonazepam, quinine • Surveillance: opthalmalogical, glucose, TFTs • Anesthetic risk

49. Huntington Disease • Movement, cognitive, psychiatric disorder • Clinical: Early -  coordination, involuntary mvts, planning difficulties, depression, irritability. Later - Chorea (90%), Oculomotor disturbance (75%), Hyperrefelxia (90%), Progressive dementia, dysarthria Psychiatric problems: personality change (75%), affective disorders (20-90%), schizophrenic psychosis (4-12%) Suicide (12%), Behavioral disturbance esp. outbursts. End stage- Severe motor disability, mute, dysphagia, incontinent, weight loss, sleep disturbance

50. Question Which one of the following is the most appropriate way to undertake DNA genetic diagnosis of Huntington's disease? A) RFLP (restriction fragment length polymorphism) analysis. B) PCR (polymerase chain reaction) analysis. C) Size estimation of a triplet repeat. D) Southern hybridisation. E) Identification of gene-specific mutations.