18.03.13 One gene, many diseases

Question 1

Give three examples of genes in which different mutations result in different disease.

Answer 1

1. RET GoF = MEN2, RET LoF = Hischprung disease
   Somatic mutations in RET can also occur in many cancer pathways, including NSCLCs and thyroid carcinomas.
2. COL2A1 mutations cause an overlapping spectrum of skeletal disorders. Syndrome dependent on the effect of the mutation on the collagen fibres.
3. Germline FGFR3 activating mutations - AD human short limb skeletal dysplasias, including achondroplasia
   Somatic FGFR3 activating mutations associated with bladder cancer.
4. PMP22 Gain = CMT Type 1A, Loss = HNPP
5. AR mutations that impair the amount, structure or function of AR cause AIS. Usually SNVs (>90%). Majority are missense. Limited to DNA and steroid binding domains and splice sites. Increased density in exons 5 and 7

Sprinal and bulbar muscular atrophy (Kennedy’s disease) = Expansion of the CAG trinucleotide repeat in exon 1 of AR

Question 2

Give two examples of locus/genetic heterogeneity.

Answer 2

1. Hereditary spastic paraplegia caused by mutations in SPG4, ATL1 SPG31, SPG6
2. Alport syndrome caused by mutations in COL4A3, COL4A4, COL4A5.
3. HNPCC
4. CAH
5. Noonan
6. CMT

Question 3

What is the role of RET (10q11.2)?

Answer 3

Protooncogene encoding a tyrosine kinase receptor (transduces signals for cell growth and differentiation). Dimersationand activationg of WT RET results in phosphorylation of intracellular tyrosine residues which act as docking points for various adaptor proteins.

Question 4

What disease do autosomal dominant gain of function mutations in RET cause?

Answer 4

Multiple endocrine neoplasia 2 - all high risk of developing medullary thyroid carcinoma (MTC)

Question 5

What are the 3 subtypes of MEN2 and their phenotype?

Answer 5

MEN2A - MTC, phaeochromocytoma, hyperparathyrdoism occurs in early adulthood.

MEN2B - MTC, ganglioneuromas, thickened cornea nerves, marafanoid characteristics. Most aggressive subtype (MTC in early childhood)

Familial medullary thyroid carcinoma (FMTC) occurs in middle age.

Question 6

What is the effect of germline missense varaints in RET?

Answer 6

Can cause constitutive activation of RET kinase.

Question 7

What disease is caused by AD loss of function mutations in RET?

Answer 7

RET mutations account for 40% of Hirschsprung disease.

Can also include loss of 10q11.2 suuporting haploinsufficiency as a cause of disease.

Question 8

What is the phenotype of Hirschprung disease?

Answer 8

Complete absence of neuronal ganglion cells from portion of intestinal tract. Typically results in enlargement of the bowel and constipation in neonates.

Question 9

Why is Hirschprung disease also an example of locus heterogeneity?

Answer 9

Can be caused by mutations in EDNRB (13q22) and EDN3 (20q13)

Question 10

Which cancer pathways have somatic RET mutations been identified in?

Answer 10

RET fusions in 1-2% non–small cell lung carcinomas (NSCLCs); inc incidence in never-smokers with lung adenocarcinomas lacking other known driver oncogenes. Preliminary data that RET inhibitor cabozantinib could offer novel treatment (Drilon et al 2013)

RET acts as dominantly transforming oncogene in thyroid carcinoma and other malignancies. Ponatinib inhibits RET kinase - promising preclinical activity in models of RET-driven medullary thyroid carcinoma (De Falco et al 2013).

Increasing RET targeting drugs for a number of cancers (Mulligan, 2014).

Question 11

What is the role of COL2A1?

Answer 11

COL2A1 encodes the Alpha 1 chain of type II collagen. Type II collagen adds structure and strength to the connective tissues that support the body’s muscles, joints, organs and skin; it is primarily found in cartilage. COL2A1 forms a triple helical conformation that is typical of all the collagen types.

COL2A1 has an important role in endochondral ossification (the replacement of cartilage by bone) during which it is produced by chondrocytes (cartilage cells) – key during fetal development.

Question 12

Give three examples of disorders caused by mutations in COL2A1.

Answer 12

Achondrogenesis

Hypochondrogenesis

Spondyloepiphyseal dysplasia congenita

Kniest dysplasia

Stickler syndrome (auto dom)

Osteoarthritis with mild chondrodysplasia (type II collagen-related premature onset osteoarthritis)

Question 13

What type of COL2A1 variants are associated with hypocondrogenesis?

Answer 13

Milder form of achondrogenesis. Achondrogenesis and hypochondrogenesis are usually due to the substitution of a glycine residue in the triple helical domain and the difference between these two disorders may be due to the location of the glycine in the protein.

Question 14

What type of COL2A1 variants are associated with spondyloepiphyseal dysplasia congenita?

Answer 14

(SEDC; auto dom but mainly de novo muts) characterised by short stature, abnormal epiphyses (the rounded end of a long bone) and flattened vertebral bodies. SEDC mutations are more heterogeneous and have included substitution of single amino acids as well as single amino acid deletions and duplications

Question 15

What type of COL2A1 variants are associated with Kniest dysplasia?

Answer 15

short stature, enlargement and stiffness of joints, contractures of fingers, bell-shaped chest and myopia. Kniest dysplasia is usually caused by mutations (often inframe deletions or exon skipping splicing) located between exons 12 and 24.

Question 16

What type of COL2A1 variants are associated with Stickler syndrome?

Answer 16

auto dom) - myopia, cataracts, glaucoma, vitreous alterations and high risk of retinal detachment. Usually caused by nonsense mutations that lead to haploinsufficiency due to nonsense mediated decay. Splice site variants often also often resulted in at least one isoform with a frameshift and premature termination, although some glycine or arginine missense mutations also reported.

Question 17

What type of COL2A1 variants are associated with Osteoarthritis with mild chondrodysplasia?

Answer 17

Early onset osteoarthritis, mild epiphyseal dysplasia & spinal involvement. Patients usually of normal height. Missense muts have been identified in affected individuals, the most characterised is p.Arg519Cys.

Question 18

What types of diseases are associated with germline activating mutations in FGFR3?

Answer 18

AD human short limb skeletal dysplasias

1. Achondroplasia
2. Hypochondroplasia
3. Thanatophoric dysplasis types I and II
4. Severe achondroplasic with dev. delay and acanthosis ngricans
5. Muenke syndrome
6. Crouzon syndome with acathosis.

Question 19

What are somatic activating mutations in FGFR3 associated with?

Answer 19

Bladder cancer. The same mutationsin the germline cause skeletal dysplasia thanatophoric dysplasia.

Question 20

What is the function of PMP22 protein?

Answer 20

Encodes peripheral myelin protein 22 that is a glycoprotein expressed by myelinating Schwann cells in the PNS. PMP22 is thought to have a role in the initiation of myelin spirals, regulation of growth & differentiation of Schwann cells & control of thickness & stability of myelin sheaths (= insulation of nerves).

Role of PMP22 still not fully elucidated; may be part of a multi-subunit complex, so altered gene dosage could result in altered subunit ratio or affect complex formation – consistent with fact that PMP22 expression is closely related to expression of other myelin proteins, and PMP22 is associated with MPZ in myelin. Studies in injured nerve suggested role during Schwann cell growth and differentiation

Regulatory function of PMP22 may be very tightly controlled, in which case altered gene dosage could be critical.

Question 21

Summarise the cause and phenotype of CMT Type 1a/HMSN1a.

Answer 21

peripheral demyelinating neuropathy – loss of the myelin sheath of the peripheral nervous system & subsequent degeneration of nerve fibres

results in progressive muscle weakness.

Caused by 1.5Mb duplication or gain of function missense mutations

Question 22

Summarise the cause and phenotype of HNPP.

Answer 22

peripheral demyelinating neuropathy- segmental demyelination with tomaculous “sausage-like” myelin thickening

results in numbness, muscle weakness and atrophy.

Caused by 1.5Mb deletion; truncating mutations (frameshift, nonsense and splice-site) have been found in a small proportion of HNPP.

Question 23

Describe the gene dosage model in PMP22: overexpression

Answer 23

CMT1A: Increased levels of PMP22 expression leads to overproduction of PMP22 protein, which prevents the protein from being processed correctly (usually processed in ER and Golgi). Reduced amount of functional PMP22 protein impairs formation of myelin and has been shown to alter Schwann cell growth and differentiation and increase myelin thickness (remember: PMP22 regulates myelin thickness). Missense PMP22 muts results in altered protein with slower processing of this protein and impaired function. Model is supported by the finding of increased PMP22 protein and mRNA in nerve biopsies of CMT1A patients.

Question 24

Describe the gene dosage model in PMP22: haploinsufficiency

Answer 24

HNPP: Deletion of one copy of PMP22 decreases amount of PMP22 protein available for myelin production; other muts result in unstable protein and loss of myelin. Results in increased susceptibility of nerves to mechanical forces (pressures), probably due to reduced adhesion of myelin lamellae.

Question 25

Which two syndromes are associated with mutations in the androgen receptor gene (Xq12)?

Answer 25

Androgen insensitivity syndrome | SBMA/Kennedy's disease

Question 26

What is the role of the AR protein?

Answer 26

Essential for androgen activation requried for normal primary and secondary male development.

Question 27

What are the three phenotypes of AIS?

Answer 27

1. Complete AIS - born apparently female as no virilisation of external genitalia; not suspected until pubery, usually lack a uterus, oviducts and cervix. 2. Partial AIS - various degrees, one form nearly normal female. Family history important 3. MAIS - Morphologically normal male genitalia but small. two forms: Impaired of sufficient fertility.

Question 28

What is the mutation spectrum of AIS?

Answer 28

90% are SNVs. Predominantly missense. Increased frequency in exons 5 and 7. Few mutatinosin amino-terminal. PAIS more associated with missense variants in the steroid and DNA binding domains. Identical mutations can result in phenotype between or within families.

Question 29

What is the genetic cause of Spinal and Bulbar muscular atrophy/Kennedy's disease?

Answer 29

CAG expansion in exon 1 of AR

Question 30

What is the phenotype of SBMA?

Answer 30

Adult onset (30-50 yrs), not always reduce life expectancy, proximal muscle weakness, muscle cramps, atrophy and fasciculations (twitching), progressive course complicated by involvement of the bulbar muscles. Affected patients also often have signs of androgen insensitivity, such as reduced fertility, testicular atrophy and gynecomastia, dysphagia (difficulty in swallowing). Heterozygous females are usually asymptomatic, but may have minor signs of lower motor neurone involvement.

Question 31

What are the repeat sizes associated with the different classes of SBMA alleles?

Answer 31

Normal range: 10 - 36. Affected range: 38 - 72. No size overlap between normal and affected ranges. Repeat size shows tissue specific somatic mosaicism.

Question 32

What is the underlying molecular pathology of SBMA?

Answer 32

Nuclear accumulation of polyglutamine-expanded AR in motor neurones leading to neuronal dysfunction and cell death. Also formation of toxic oligomers sets off cascade of events that lead to neurodegeneration. Ligand-dependent toxicity of polyQ-AR, binding of testosterone affects trafficking of AR Altered post-translational modification of AR Disruption of axonal transport Transcriptional dysregulation

Question 33

What diseases have contractions of the polyQ repeat in AR exon 1 been associated with?

Answer 33

Contraction of same polyQ repeat shown to increase the risk of prostate cancer, hirsutism, male infertility, and cryptorchidism.

Question 34

Define locus/genetic heterogeneity.

Answer 34

A single disorder, trait, or pattern of traits caused by mutations in genes at different chromosomal loci Clinical syndromes often result from failure or malfunction of a developmental or physiological pathway, and many cell functions depend on multi-component protein aggregates. So, if the correct functioning of several genes is required, muts in any of the genes may cause the same, or v similar, phenotype.

Question 35

What is the phenotype of HSP?

Answer 35

Disorder of the body's motor system in which certain muscles are continuously contracted, causes stiffness or tightness of the muscles and may interfere with gait, movement, and speech Age of onset is 1-76 years; a marked variation in age of onset has been seen even within families.

Question 36

What is the mutations spectrum of HSP?

Answer 36

Can be autosomal dominant, autosomal recessive and X-linked, with now >52 genes implicated. 1. SPG4 (2p22.3) - 40% 2. ATL1 (14q22.1) - ~20% (most frequent in early-onset ~4yrs) Other causes of AD-HSP include mutations in REEP1* (SPG31 2p11.2) ~6.5% of cases, and mutations in NIPA1 (SPG6 15q11.2, Non-imprinted in Prader-Willi/Angelman syndrome 1 gene). Other genes causing HSP = SPG6, SPG8, SPG9, SPG10, (AD HSP) and SPG7, SPG11 (AR HSP) and SPG1, SPG2 (X-linked HSP) *Note that a REEP1 mutation had now also been associated with dHMN so can be an example of clinical heterogeneity also. The majority of HSP gene products have been implicated in intracellular membrane and protein trafficking. Other possible pathogenic mechanisms of the HSP-related gene muts include mitochondrial dysfunction, and abnormalities in axonal pathfinding or myelination.

Question 37

How does Alport syndrome show both locus heterogeneity and clinical heterogeneity?

Answer 37

Alports is a good example of both locus heterogeneity, and also clinical heterogeneity due to the AS and thin basement membrane nephropathy phenotypes presenting depending on the inheritance pattern of the mutation(s). It is also an example of X-linked and autosomal inheritance X-linked dominant inheritance with variable expressivity, autosomal dominant and recessive inheritance with variable expressivity, dominant negative mutations due to polypeptide interaction.

Question 38

What is the phenotype of Alport syndrome?

Answer 38

AS is a hereditary nephropathy associated with haematuria, progressive renal failure, sensorineural deafness and characteristic eye signs. Caused by defects in type IV collagens which are mostly made up of collagenous domains of Gly-X-Y repeats.

Question 39

What is the genetic spectrum of Alport syndrome?

Answer 39

COL4A5 (Xq22.3) -85% COL4A3 (2q36-q37) and COL4A4 (2q35-q37) - 15% which can be inherited in a recessive (10%) or possibly even dominant (<5%) pattern. Autosomal recessive Alport syndrome is caused by two mutations in trans in the COL4A3 and/or COL4A4 genes. One heterozygous mutation in COL4A3 or COL4A4 may be involved in autosomal dominant AS but can also manifest as thin basement membrane nephropathy (TBMN). Individuals with TBMN are sometimes considered to be carriers for autosomal-recessive AS.

Question 40

How is COL4A6 associated with Alport syndrome?

Answer 40

COL4A6 gene lies upstream of COL4A5 in a head to head arrangement. A contiguous deletion that includes the 5’ end of the COL4A5 gene and usually exons 1 and 2 of the COL4A6 gene causes Alport syndrome and leiomyomatosis.