Inheritance patterns and genetic diseases Flashcards
Define aniticipation:
Phenomenom where the features of a genetic condidiotn becomee more severe and have an earlier onset with each successive generation.
typically associated with TNR disorders as the dynamic mutations are unstable and prone to expand on transmissio
seen in FRAV, DM1, HD, SCA1,3 and 7 and DRPLA
What are the different repeat length categories in TNR disroders?
Exapansion can occur ion the germline (passed to offspring) or in somatic cells resulting in size mosaicism)
Normal (stable) not prone to expansion and will not expand to FM in a single generation
Intermediate- not associated with disease and offpsring will not be affected but repeat length ay show instaability on tranmission
PM- prone to expansion to F. Not pathogenic except ion FXTAS/FXPOI
FM- expansion need to reach a threshold length for the phenotype to be expressed.
What is the sex specific expansion bias for HD and DM1
HD expansions are predominantly male due to CAG instability in spermatogenesis
DM1 large expansions are maternally inherited. only small rpt can be paternally inherited possibly due to selection against sperm with FM
what is the mechanism of TNR expansion?
Replication slippage
strand miss-pairing results in formation of secondary structures (e.g. hairpin loops) which cause rep fork blockage, this result in slippage of the lagging strand and misplacing of okzaki fragments = unequal crossing over and one expanded and one contracted allele
what biases can result in the false appearance of anticipation?
preferential ascertainment of parents with late onset disease as early onset would have reduced sexual fitness
preferential ascertainment of children with severe disease earlier
preferential ascertainment of child-parent pairs with simultaneous onset
define age related mosaicism?
mosaicism (somatic or germline) due to the accumulation of mutations over the course of an individuals lifetime
- e.g. loss of X or Y is a characteristic of ageing to give a 45,X cell line
- knudson hypothesis (2 hits for cancer= sporadic is later onset)
define variable expressivity
pehnotype expressed to different degrees in different individuals with the same genotype.May show variability within the same family
e.g. NF1 range from cafe au lait skin patches to large disfiguring neurofibromas
marfans range from tall with long slender fingers to having life threatening heart conditions.
Define penetrance
Penetrance is the proportion of individuals with the same genotype who express the phenotype.
e.g. BRCA1 shows 80% lifetime risk of breast cancer
in complete penetrance all individuals with the genotype will have the associated phenotype
define reduced penetrance
not everyone with the genoytpe will show the phenoytpe
- affected by genetic modifiers, environmental factors, lifestyle, age, sex hard to predict.
Non penetrant parents can have penetrant children e.g. 22q11.2 making genetic counselling a challenge.
describe the role of ascertainment bias on determining penetrance
- ascertainment bias- the penetrance of an allele can depend on the age of testing, the severity of the disorder a patient present with or a patient dying from a different disorder before the one in question presents
describe attributable risk
attributable risk looks at the amount of risk that can be atributed to an allele- can develop breast cancer without BRCA mut so not all the risk can be attributed to having the allele
define sex limiting
Genes present in both sexes but only expressed in one e.g.lactation in females and beards in males
define epistasis
a variant or allele in one locus that prevents a variant or allele at another locus exerting its affect
- interactions between non-allelic genes in which one has a dominant effect over another
- explains deviation from simple mendelian ratios
- play a major role in susceptibility to complex disease
the masked locua is called hypostaic and the masking locus is epistatic
can occur at the gene level where one gene may encode a protein which prevents the transcription of another gene or at the phenotype level e.g. the gene for albinism would hide the gene controlling a persons hair or skin colour
define pleitropy
Pleitropy is when one gene influences 2 seemingly unrelated traits as the encoded protein is used in different cells of for different signalling functions
e. g. PKU is associated with ID and hypopigmented skin and hair
- affects phenyalanine hydroxylase which converts phenyalanine to tyrosine
- build up of phenyalanine is toxic to the nervous system resulting in ID and DD
- lack of try which is required for melanin production which is required for skin and hair pigmentation
define linkage disequilibrium?
non random association between 2 alleles at 2 didfferent loci- when variants co-occu togehter in one allel more often than would be expected by normal distribution
e.g. CF F508 is in LD with the 9T variant of the polyT tract. Useful as if the 9T and 5T plyTs are detected with R117H and F508, the R117H must be in cis with 5T which confirms a diagnosis of CFTR-RD
what are the features of X-linked dominant disorders?
- Do not always fit the rules of dominant or recessive inheritance
- penetrance and severity are generally high in males with low severity in females. penetrance in females is highly variable
Give an example of a XLD disorder
X-linked Alports
X-linked hypophosphatemia/ vitD resistant rickets
Fragile X- inheritance is debated but widely considered XLD
Given an example of an XLD disorder that is lethal in males?
Rett syndrome
Incontentia Pigmenta
Give an example of an XLD disorder with males unaffected?
Craniofrontonasal syndrome (CFNS)
Describe the characteristics of XLD inheritance
XLD is a condition that is expressed in hemizygous males and heterozygous females
- males are generally more severely affected and females show variability
- 50% risk of all offspring of an affected female being affected
- 100% of female and 0% or male offspring from an affected male will be affected
can be mistaken from AD inheritance as passed from both parents but is differentiated by the lack of male to male transmission. Because of this there will be more affected females than males.
describe X-linked phosphatemia
Most common cause of phosphatemia
fully penetrant with variable severity and patients may not present until 6-12 months
Symptoms are similar to Vit D deficient Rickets
- bone deformities
- dental abnormalities
- hearing loos
- low phosphate levels
- resistance to treatment with Vit D
Caused by PHEX mutations- result in inhibition of kidney being able to reabsorb phosphate from the bloodstream affecting normal bone growth and development
Describe X linked Alport
Most common form of Alport syndrome
- associated with Kidney disease, hematuria and proteinuria, hearing loos and eye abnormlaities
CLO4A5 variants (Col4A3/4 in AR forms)- encodes type IV collagen which plays a role in kidneys, vision and hearing
What are the characteristics of XLD inheritance with male lethality
- only seen in females, rare in males and if present they are mosaic if XXY
- increased miscarriage rate in families as males pregnancies do not get to term
- sex ratio of offspring is skewed= 1/3 unaffectd females, 1/3 affected females and 1/3 unaffected males
Describe Rett syndrome
Characterised by normal development in the first months, followed by rapid regression of language and motor skill ans then stability of symptoms. characteristic hand flapping also develops
- life expectancy 15-20yrs
caused by variants in MECP2 (classic Rett)- generally de novo and low recurrence risk although some reports of germline mosaicism
Describe male Rett syndrome
~1% of males with severe MR
- Males with MECP” variants are born with neonatal encephalopathy resulting in death before age two years
- adult males with classical Rett are 47,XXY or mosaics and have the same phenotype as females
or have less severe neurological maifestations but carry variants not seen in females with Rett- presumably to mild to cause disease in het state
describe non- classical Rett
Variant Rett is caused by variants in FOXG1 (Chromosome 14, distinguished by congenital microcephaly and corpus callosum abnormalities) and CDKL5 (X chromosome, distinguished by early onset seizures)
Describe Incontinentia pigmenti
affects hair, skin, teeth, nail and CNS- nail dystrophy, and eye and dental abnormalities and born with a rash.
males spontaneously miscarry in the first trimester or have some mosaicism
Due to mutations if IKBKG
high penetrance and variable severity
Describe Craniofrontonasal syndrome (CFNS)
CFNS is a XLD disorder with affects females but not males. females have frontonasal dysplasia, craniofacial asymmetry and craniosynostosis, whereas males typically show only hypertelorism
Caused by mutations in EFNB1- encodes a TM protein involved in bi-directional cell signalling
females are affected as skewed X-inactivation leads to a population of mutant and non-mutant cells. This interface between the normal and mutant protein population is the cause for the phenotype in males. Therefore a mosaic male may also express an phenotype.
Describe the role of X-inactivation in manifestation of XL conditions in females
- can result in the expression of an XLR disorder
- high skewing can affect the severity of XLD and there have been reports of asymptomatic carriers of Rett due to inactivation of the mutant allele
How are conditions associated with genes in the PAR region inherited?
AR or AD as the PAR regions is found on both the X and Y chormosome
What is the incidence and carrier frequency of SMA
incidence 1/10,000
carrier freq ranges from 1/40 to 1/60 (1/50 in the UK)
second most common AR disease in hr UK and the most common cause of infant deaths
What is the pathogenesis of SMA
SMA is caused by a mutation in the survival motor neuron (SMN) gene.
SMA results in progressive muscular weakness due to loss of anterior horn neurons in the brain and spinal cord. The neurons are required to relay signals to proximal muscles from the brain for them to contract. muscle weakness is proximal to distal with inclusion of he intercostal muscles, the diaphragm is usually spared.
what is the SMA phenotype?
Progressive symmetrical muscle weakness of the proximal limbs and trunk
- difficulty breathing due to inclusion of intercostal muscles
- normal or above average IQ
- facial weakness
- fine muscle tremor in fingers
- poor suck/swallowing - difficulty feeding
What is the differential diagnosis for SMA?
Athrogryposis mutliplex congential
What is the structure of the 5q13 SMA region
SMA is due to mutations in SMN
there are 2 copies of SMN located in tandem in a 500kb inverted dup in 5q13
- 5q13 is a has a highly complex structure containing many repetitive regions, pseudogenes, retro- transposable elements, deletions, inverted dups
How many copies of SMN gene do most people carry?
In the normal population the majority of individuals have 1 copy of SMN1 on each chromosome. However approximately 4% of the population have 2 copies of SNM1 on a single chromosome - this can cause issues with carrier testing.
What is the role of the SMN protein?
SMN protein is ubiquitously expressed in motro neurons of the spinal cord. it is though to be involved in RNAP transport in motor neurons. SMA is essental and loss of SMN1 and 2 is embryonic lethal
what is the difference between SMN1 and 2?
SMN1 is the native gene and is telomeric to the SMN2 pseudogene.
Both genes are highly homologous and only differe byt few bases in thr 3’ of the. The key difference is a C>T transition in SMN2 exon 7. This synonymous change disrupts a splice enhancer site at the intron6-exon7 border resulting in inefficient splicing and a shorter transcript lacking exon 7. This results in only ~10% of the full lengthtranscript being translated.
In SMN1 90% of the full length transcript is translated.
What is the molecular pathogenesis of SMA?
loss of exon 7 causative of SMA- result in a decrease in the amount of SMN produced
Homozygous deletion of at least exon 7 of SNM1 is responsible for ~95% of cases - approximately 2% of the deletions are de novo
4% are compound het for an SMN1 deletion and a pathogenic sequence variant
1% and homozygous for a pathogenic sequence variant
What defines SMA type IA & B?
prenatal/congenital form AKA Werdnig Hoffman disease
- onset at birth and life expectancy to ~6months
- never sit unsupported
- profound hypotnia, feeding difficulties and death from respiratory failure
homozgous SMN1 del
What define SMA type 1B?
severe/ actue SMA AKA Werdnig Hoffman disease
onset at 0-6 months and die < 2 years from respiratory failure
- never sit unsupported
- frog leg posture
- profound hypotnia, feeding difficulties and death from respiratory failure
homozygous del
What define SMA type II?
intermediate AKA Dubowitz disease - onset 6-18 months - death >2 years and 75% reach 25 - can sit but never stand or walk independently - profound hypotonia and muscle weakness postural hand tremor avg or >avg IQ
SMN gene conversion & SMN del
what define SMA type III?
Kugelbury Walander disease
- onset > 18 month and nearly normal life expectancy
- can walk unsupported
- may have hand tremor
- phenotype similar to muscular dystophy
2 SMN2 gene conversions
What defines type IV SMA?
Adult onset
normal lifespan and reach all motor milestones
have more copies of the SMN2 gene which acts a a disease modulator and compensates for the lack of SMN
What are the genotype phenotype correlations in SMA?
SMN2 can modify the disease severity in a dose dependent manner. The more copies of SMN2 the less severe the pheno.
- there has been a report an asymptomatic individual with homozygous SMN1 del but 5 copies of SMN2
BEWARE- correlations are imprecise so this is not used clinically to predict the course if the disease.
what other disease modifiers are reported in SMA?
- there is an SMN2 point mutation that can result in the inclusion of exon 7 by creating a new splicing enhancer and thereby increasing the amount of full length transcript
- plastin 3 shown to be upregulated in a family member with no SMA and an SMN1 del and same number of SMN2 copies as an affected family member.
How is SMA diagnosed?
As majority of cases have homozygous del the first line test is dosage analysis of at least SMN1 exon 7 - 95% sensitivity and 99% specificity
only loos of SMN1 exon 7 is required to develop SMA as it is the clinically relevant transcript
what are the molecular tests for SMA?
dosage analysis will detect 95%, sequwncing will detect a further 4%
only exons 7 & 8 are tested as they have SNPs whish allow distinction of SMN1 & 2
MRC holland MLPA kit available with probes targeted to SNPs in exon 7 and 8- can detect carriers, dels, dups and gene conversion
Quantitative PCR uses flourescently labelled primers specific to the nt differences in ex 7 & 8. Products are separated by capillary electrophoresis and copy number determined by comparison to controls
what are the considerations for carrier testing in SMA
4% of the popultaion carry 2 copies of SMN1 on a single chromosome. As doage does not provide positional information this can mask the presence of a deletion in the other allele and can’t distinguish 2:0 from 1:1.
- linkage analysis can help resolve carrier status in cases with a proband with a hom deletion and only 1 carrier parent identified
- grandparents can be tested- if a parent id 2:0 the liklihood is there parents will be 1:0 and 2:0, but if the deletion in the proband is de novo the grandparents are liekely to be 1: as this is the most common in the population. Small risk of both grandparents being 2:0 but chance of this is vanishingly small.
- de novo deletion occurs in 2% of cases so this should also be considered.
Bayesian calculations can be used to determine the residual risk of being a carrier after a normal dosage result (considers chance of 2:0 and sequence variants)
the finding of 2 copies if SMN1 significantly reduces the chance of being a carrier
How could you investigate a patient with an SMA phenotype but only one SMN1 allele deleted?
May habour a pathognic SNV in the remaining SMN1 copy. Can carry out sequence analysis but this is complicated by the SMN2 psuedogene.
- therefore allele specific long-range PCR and RNA seq technologies are required for specific analysis of the SMN1 gene
- a multiplex PCR for the most common small intragenic mutations has been developed
what are the developments in prenatal testing for SMA?
Prental diagnosis by NIPD has been diagnosed as part of the NIPSIGEN project
- this uses enrichment of highly heterozygous SNPs across the SMN1/2 region followed by massively parallel sequencing and analysis of relative haplotype dosage. . Maternal, paternal and proband DNA samples are also tested for haplotyping purposes.
describe the use of linkage in SMA
linkage analysis can be used to detect the inheritance of the high risk allele.
- this is useful in cases where a second mutation has not been identified but SMA has been diagnosed clinically to enable carrier and prenatal testing.
- amplified MS markers flanking the region and needs DNA from proband to determine phase. Need to consider risk of a double recombination resulting in the high risk allele being associated with the low risk haplotype.
- sensitive to MCC from 5% (lower than the 10% excluded by QF-PCR in most labs)
what are the therapies available for SMA?
Nusinersen (AKA spiranza) is an 18mer anitsensne oligonucleotide which has been approved by NICE for treatment of molecularily confirmed SMA I,II and III.
- treatment works by promoting the inclusion of the SMN2 exon 7 in SMN2 transcripts to increase the level of the full length transcript.
does not need to cross BB as increasing SMN in the CNS is not reuired to rescue the preipheral muscle phenotype and rescue motor neurons andincreaase long term survival (study in mice)
what other therapies are available?
gene conversion of SMN1 to SMN2 has been reported,
stem cell or gene therapy may compensate for the lack of sufficient SMN.
AAV9 can infect numerous cells and cross the BBB. In Mice has been shown to result in mice expressing the engineered WT gene and therefore an increase in SMN levels
What is the current position on SMA and newborn screening?
Most patients diagnosed with SMA are already in the advanced stages with significant motor neuron degeneration having already taken place. no there is treatment available there is an argument for NBS as late treatment is less beneficial.
Already included in NBS in the US and being considered in Belgian and Taiwan
what are the genotype phenotype correlations in Rett syndrome?
truncating mutations are more severe and there is stronger selection against them in the 5’ of the gene. missense mutations are less severe so may only be seen in male Rett
what is the parthenogenesis of Rett?
MECP2 is implicated in the transcription in neuronal cells e.g. regulated BDNF transcription - affects synaps plasticity and neuronal development
What is the molecular testing for Rett
bi directional sequencing detect 85-90% of mutations. dosage analysis by MLPA can also be performed.
X-inactivation can affect the severity of XL disease. How is it tested for?
PCR of polymorphic CAG repeat in the 1st exon of the androgen receptor.
DNA is digested by a methylation snsitive restirction enzyme to determine which CAG repeat length (and therefore which X) is silenced. Can be used to determine the degree of skewing by comparing the level of digestion of each allele.
What is the difference between XLD and XLR?
In XLR females are generally not affected whereas in XLD females are affected.
However the standard definitions of AR or AD do not capture the variable expressivity due to skewed x inactivation.
what are the features of XLR disease?
vertical transmission from mothers- 50% of sons will be affected and 50% of daughters will be carriers.
Male transmission- all daughters will be carriers. there is no male-male transmission so no sons will be affected.
pairing of an affected male with an affected female can give the false impression of male-male transmission.
How may females manifest XLR disease?
skewed X-inactivation
45,X
Inheritance of mutation from both parents
UPD for X chromosome
describe spinal bulbar muscular atrophy
SBMA is a late onset progressive neuromuscular disorder that results in muscular atrophy and distal to proximal muscle weakness and wasting
onset is 30-50 yrs and require a wheelchair within 10yrs
what are the molecular genetics of SBMA?
caused by a CAGn polyglutamine expansion in the 1st exon of the AR gene.
35-37 repeats is variable penetrance
38+ repeats is full penetrance
shows anticipation
GOF , precise mechanism is unknown but it is thought to result from the CAG tract being cleaved into a polyglutmaine fragment which is retained in the nucleus where it forms nuclear inclusions.
female carriers asymptomatic
describe Androgen insensitivity
AIS is caused by pathogenic LOF mutations in the AR gene. Encodes the androgen receptor which allows cells the respond to testosterone and is required for male sexual development
characterised by female external genitalia and abnormal secondary sexual development at puberty
mainly missense mutations and dela and dups are rare. Testosterone levels are normal or elevated
what are the 3 types of AIS
complete AIS- normal female external genitalia, raised as females. Associated with variants throughout the coding region
partial AIS- Nearly normal female genitalia or ambiguous. Variants mainly in the steroid ir DNA binding domains
Mild AIS- typically have male external genitalia and mutations in ex 5 or 7 or amino terminal domain
describe hemophilia A and B
Due to mutations in the coagulation factors factor VIII (F8 in haem A) and factor IX (F9 in haem B). Mutations cause F8 and F9 to be ineffective in coagulation cascade. Clinically indistinguishable.
diagnosed by a deficiency in clotting of the relevant factor
complications arise from bleeding into joints, muscle, brain or other internal organs.
female carriers are biochemically abnormal but clinically unaffected. 10% of females are symptomatic and at risk of bleeding
F8- large gene. Intron 22 is the most common mutation. 1 in 6000 males
F9- small gene, mainly SNVs and 1 in 30,000
describe Hunter syndrome (mucopolysacharidosis II)
Due to mutations in the IDS gene- this encodes the enzyme for the breakdown of mucopolysacharides. without this enzymes MPS build up resulting in tissue damage.
variable severity, age of onset and rate of progression. In the severe disease there is CNS involvement and progressive airway and cardiac disease result in death. In the attenuated form the CNS is spared.
What is the prevalence and penetrance of DMD and BMD?
Most common muscular dystrophy - 1 in 3500 males
penetrance is 100% in males and variable in females
prevalence of BMD is 1 in 8000 and females rarely display phenotype
DMD ans X-linked cardiomyopathy?
Mutations in the dystrophin gene can also result in X-linked cardiomyopathy. This presents as cardiac disease with no skeletal muscle involvement.
due to a mutation in promote and 1st exon resulting in no dystrophin present in cardiac muscle.
- skeletal muscle spared as it can use other promoters
- onset is 20-25yrs in males was fast progress and death in a few years
- in melanges onset is 40-50 yrs and is slower
Describe the phenotype of DMD?
- onset < 5yrs and delayed motor development
- wheelchair bound by 12 years
- progressive muscle weakness and muscle replaced by fat and fibrotic tissue
- gower sign
- legs and pelvis affected first
- scoliosis may develop
- 95% of males develop cardiomyopathy
- dev delay present in 30-50% and coincides with later onset of symptoms (may be referred for array for LD and DMD discovered then)
- mean age at death is 25yrs due to respiratory and cardiac insufficiency
- creatine kinase levels are 10-100x normal
what is the phenotype of BMD?
milder than DMD
- 20% have dilated cardiomyopathy
- may be lat learning to walk
- muslce weakness onset from ~ 11yrs
- lose ability to walk at 49-50 yrs
- no LD
- survive to middle
- creatine kinase 5x normal
describe the DMD gene
largest human gene with 79 exons- 2.4MB in size but only 3% is coding
7 tissue specific promoters including brain, cardiac an skeletal muscle
Describe the dystrophin protein.
dystrophin is a rod shaped cytoskeletal protein which provide structural support dystroglycan complex (DGC)
the DGC forms a critical link between the cytoskeleton and the ECM and stabilizes the sarcolemma during contraction and relaxation.
loss of dsytrophin disrupts the link between the ECM and cytoskeleton which increase the fragility of the cell membrane and muscle becomes mechanically damaged during contraction. This also increases the permeability to Ca2+ which activate proteases which digest contractile proteins
increased creatine kinase levels indicate muscle damage
How much dystophin is expressed in DMD and BMD?
In DMD dystrophin is virtually absent
In BMD there is between 20% to normal levels (although abnormal protein is expressed)
What is the mutation spectrum in DMD?
No correlation between del/dup sizer and the severity of the phenotype.
Majority of pathogenic variants are exon deletion (>1 exon) clustering in exons 2-20 (20%) and 45-55 (80%). duplications are clustered in these regions with opposite frequencies
nonsense mutations are next most common mut in DMD followed by dups and indels.
single exons deletions- do not routinely test asymptomatic patients therefore do not know the frequency of these in the normal population. If detected in a patient with phenotype it is considered to confirm the diagnosis but could be masking the true diagnosis
what is the reading frame rule in DMD?
mutations that disrupt the reading frame result in a no functionla dystrophin due to NMD and asre associated with DMD
in-frame dels and dups result in the expression of a partially functional protein and are associated with BMD
the reading frame hypothesis holds true in 90% of cases therefore a diagnosis should be made on clinical assessment not the reported prediction.
What are the limitations to the reading frame hypothesis?
- hypothesis is based on DNA not RNA. Due the RNA processing this should be tested to give a definitive diagnosis
- in-frame deletions may result in DMD if they affect the protein binding domain
- assumes that duplications are tandem- may be inverted or inserted elsewhere in the gene or genome
- central-rod domain may be associated with no/mild symptoms
- exon skipping could result in rescue of the reading frame or vice versa
- exceptions to the reading frame rule are more common in BMD
What are the genotype-phenotype correlations in DMD?
- Mutations affecting the barin specific isoform as associated with later onset of muscle phnotype and LD (Dp140 and Dp71)
- In BMD dels including the a.a. terminal are associated with early onset cardiomyopathy
- central rod domain is associated with mil manifestions
What is the inheritance of DMD?
XLR
2/3 of cases are inherited from a carrier mother and 1/3 are do novo
- new mutation can occur in the oocyte, embryo post conception (mosaic) or be in the mothers germline (7-10% recurrence risk)
when the at risk haplotype is known the recurrence risk is 8.6%. However testing my MLPA does not revel this (need linkage) therefore the recurrence risk for a mother of an affected who has not been found to be a carrier is 4.3%
sibling of an affected is also at risk of being a carrier even if mother is not. If the sibling then tests negative for the family mutation there is no recurrence risk and her risk of an affected is reduced to that of the general population.
DMD has a high recombination rate- 4x greater than would be predicted by its size alone
Manifesting females in DMD
2-8% range from mild muscle weakness to an inability to walk.
If cardiomopathy is included the incidence is 38%- therefore monitoring for cardiac involvement should be included in female carrier reports
How can DMD be diagnosed? CK/IHC
- Normal CK levels rule out a diagnosis of DMD
- elevated CK support s diagnosis but does not confirm it as can be raised for other reasons e.g. after muscle injury or heavy exercise
- IHC - lack of staining for dystrophin confirms DMD and reduced BMD. requires invasive muscle biopsy so not commonly tested.
what is the genetic testing for DMD?
Dosage analysis (MLPA - 2 kits, can only use one if it will detect known mut in familial cases) will detect 72%
- sequencing analysis for CNV negatuve cases
- linkage analysis using microsatellites can be used for carrier testing and PND in cases where the pathogenic mutaion has not been detected but there is a confirmed clinical diagnosis
in MLPA is a single exon del or dup is detected it should be confirmed by another method e.g. dosage pCR or sequencing across the breakpoint
Carrier testing in DMD?
if the mutation is not known and the poband is not available them most closley related at risk family member is tested as they have the highest risk of carrying the mutation (mother then sibling). If a mutation is not found it does not rule out that they are carriers just reduces the risk
Even if the mutation is known there is still a recurrence risk in mothers if affected due to the possibility of germline mosaicism so PND is still offered
PND in DMD?
for all known carriers of mothers of affected.
Only male pregnancies tested, NIPD for sex is becoming more common as can avoid the need for an invasive test in a female pregnancy
Linkage analysis in DMD
Preferably need a sample from the proband but if not available can use an unaffected male sibling for exclusion testing
- 10% rate of recombination across the DMD gene
- to reduce the risk of recombination markers spanning the whole gene are used not just flanking it. Still need to calculate the risk of a double recombination which could result in the mutation being present on the low risk allele
- recombination risk is calculated from the average risk of a double recombination between markers and multiplying by the percentage of the coding region that is covered by the markers
Microarray IFs
- may be detected in a male referred for LD- explains pheno
- may be detected in females referred for LD- does not explain pheno byt result is reported for risk to family members
What are the differential diagnosis for DMD?
- Limb girdle dystrophy- clinically simillar but AR and AD inheritance
- emery dreiffus muscular dystrophy- characterized by joint contractures and progressive muscle weakness
_ SMA- poor muscle tone and neonatal hyopotonia
- dilated cardiomyopathy- AR, AD and XLR
What are the treatments for DMD?
No cures, treatments aims to help the symptoms e.g. physiotherapy and steroids. Transplant in severe cases of DCM
What re the molecular therapies for DMD?
Stop codon read through using amnioglycosides to result in some functional dystrophin being produced and can convert DMD to BMD pheno. 15% of DMD have a PTC.
exon skipping by mopholino ASO interferes with splicing and can skip the alterd exon to restore the reading frame and give a BMD phenotype
Define ahploinsufficiency
Genes are described as HI when inactivation/loss of a single allele (leaving the second allele unaffected) produces a clinical phenotype.
A gene is likely to be HI is all mutation types (missense, nonsense, deletion etc) result in the same phenotype.
Show AD inheritance
Note that genes on sex chromosomes cannot show haploinsufficiency.
What is the theory as to why some genes are HI and others are not?
Current theories focus on the specific function of the gene and the context of the gene’s function.
- Genes encoding enzymes are rarely haploinsufficient
- genes linked to srtuctural and regulatory function or are members of complexes or celullar signalling networks are more sensitive to changes in dosage.
What are the types of genes that show HI
imprinted genes
highly expressed genes
dosage sensitive genes
Why are imprinted gene HI?
in imprinting only one of the 2 alleles of a gene is expressed in a parent of origin depending manner, therefore mutations in the expressed gene are dominant. e.g. UBE3A mutation causes AS
Why are highly expressed genes HI?
Highly expressed show HI as they need a very high amount of gene product and a single functional copy is insufficient in producing enough. e.g, Elastin- Loss of one copy has no effect in skin and lung (low levels of product required), but can cause supraventricular aortic stenosis because the aorta requires high expression levels.
Why are dosage sensitive genes HI?
Dosage sensitve genes include:
- gene whose products are part of a quantitative signalling system depending on partial occupancy or competition for binding to DNA or receptor- gene products that compete to determine a metabolic swith
- gene products that cooperate in interactions with stoichiometry. Includes many structural proteins
In these situations the gene product is titrated against something in the cell, therefore relative not absolute levels are important.
Genes whose products are essentially alone e.g. soluble involved in metabolism, rarely show dosage sensitivity
How do dome genes have an AR and AD form?
Some genes with AD inheritance also have a recessive form. e.g. Marfans syndrome. A phenotype cause by complete loss of one allele could also be caused by a 50% reduction in the activity of 2 alleles.
Variation in penetrance & expression of both mono-allelic and bi-allelic mutant alleles highlights the fact that external factors are involved in such systems. These may include expression of other genes, environment, age, developmental status, ability of the individual to up-regulate expression of other genes or (in heterozygous individuals) the unaffected allele to compensate.
Where are HI genes found in the genome?
less likely to be found in segmentally duplicated regions which are prone to mutation (CNV) but NAHR. This suggests a selective pressure to maintain HI genes at their correct CN
Give 3 examples of HI single gene disorders?
hypertrophic cardiomyopathy (HCM)
HNPP
Aniridia
Give 2 examples of HI contiguous gene deletion syndromes?
22q11.2 Di George syndrome
del5p Cri-du-chat syndrome
Give 3 examples of HI in cancer?
HI affects TSGs
TP53
BRCA1
PTEN
Describe Hypertrophic cardiomyopathy (HCM)
Clinical features:
- Left-ventricular hypertrophy (LVH) in the absence of predisposing cardiac/cardiovascular conditions.
- Characteristic abnormal ECG.
- highly variable and can include palpitation, progressive heart failure and heart congestions
- Sudden cardiac death (often in early adulthood and associated with exertion, e.g. sport) is a major cause of mortality
Onset varies from childhood to middle age,
LVH occurs in ~1 in 500. Between half and three-quarters of these patients have an HCM mutation
What are the molecular genetics of HCM?
70-80% of mutations are found in MYH7 and MYBPC3 About 10% of mutations are in Troponin-coding TNNT2 and TNNI3,
Clinical sensitivity of the four-gene panel is ~50%
Haploinsufficiency can be demonstrated in MYBPC3 by the lack of incorporation of truncated proteins into HCM cardiac tissue (evidence against dominant-negative effect), along with a lowered expression level of full-length protein, which suggests haploinsufficiency
Interaction of multiple genes in a large system (presumably requiring cooperation & fixed stoichiometry between gene products) is characteristic of a haploinsufficiency syndrome, and probably explains some of the variability in presentation & phenotype.
Describe Aniridia
PAX6 mutaion result in lack of irises.
other symptoms include: nystagmus, cataracts, defects in vision.
PAX6 is a control gene involved in eye development with very high conservation in all organisms with eyes
Homozygous loss of PAX6 is known to cause wide-ranging defects including complete lack of eye formation and is fatal in mice.
WAGR syndrome is caused by a contigous deletion on 11p13 and includes WT1 (wilm tumour gene) and PAX6 resulting in Wilms tumor, aniridia, genitourinary anomalies and mental retardation syndrome.
Describe HNPP Hereditary neuropathy with liability to pressure palsies
HNPP is charactersied by focal pressure neuropathies- carpal tunnel syndrome, peroneal palsy and foot drop
Due to LOF of PMP22- 80% are due to a recurrent 1.5Mb deletion mediated by NAHR between CMT1A-REPS. 20% are due to LOF sequence mutations.
PMP22 encodes an essential component of the peripheral myelin sheath
reciprocal dup results in the more severe CMT1A
shows that gene is HI and TS
Describe DiGeorge syndrome
DiGeorge syndrome, 22q11.2 deletion on one allele causing haploinsufficiency of the TBX1 T-box transcription factor gene (important in developmental regulation).
Describe Cri-du-chat
Cri-du-chat syndrome caused by deletions of 5p15,
- a critical region between 5p15.2 is responsible for the observed dysmorphism and intellectual disability
- the proximal region of 5p15.3 is associated with the cat-like cry and speech delay.
Describe TP53
Mutated in over 50% of all tumours.
- Germline mutations responsible for Li-Fraumeni syndrome (LFS) (early-onset breast and multiple other poor prognosis soft tissue & bone cancers).
Describe PTEN
TSG mutated in a wide array of tumours
- Germline mutations cause Cowden syndrome & PHTS.
- PTEN is a “gatekeeper” that negatively regulates cellular proliferation.
- “obligate haploinsufficiency”; heterozygous loss is more tumourigenic than homozygous loss. Reason for this is a “failsafe” p53-dependent cellular senescence mechanism that works when PTEN is completely lost; this “masks” the cellular proliferation signal that would be the result of losing both copies of PTEN, so heterozygous PTEN loss is more tumourigenic. Loss of p53 (e.g. in tumours) prevents the “failsafe” senescence mechanism, so cases with complete loss of both PTEN AND p53 show faster rates of tumour development than heterozygous PTEN losses.
Describe BRCA1
TSG associated with hereditary breast cancer: women carrying a heterozygous inactivating mutation have an 85% lifetime risk of developing breast cancer, and increased risk of other cancer types.
- Conforms to the two-hit model of tumorigenesis (
- BRCA1 -/- mice show embryonic lethality:
What is the result of an AD GOF mutation?
result in increase in gene expression/product or gene developing a new aberrant function
what are the characteristics of a GOF mutation?
- usually dominant as the presence of the normal allele does not prevent the mutant allele from functioning aberrantly
- usually involve genes that control cell signalling- e.g. constitutively active
- product can obtain a novel function e.g. fusion gene due to translocation
- usually require a much more specific mutation than LOF e.g. mutation in the channel pore domain of an ion protein
give an example of a gene that shows GOF and LOF by different mutation mechanisms
PMP22- GOF CMT1A (dup) and LOF HNPP (del)
AR- triplet repeat expansion >35 = SBMA (GOF), LOF SNV = AIS
what are 4 different mechanisms of GOF mutations?
Unstable triplet repeats- HD, DM!, SCA
Overexpression - CMT1A
novel function - BCR-ABL1 t(9;22)(q34;q11)
Highly mutable codon- Achondroplasia
Describe the genetic change in HD?
HD is caused by a CAG repeat expansion (>36 variable penetrance- 40rpts = disease) in the 1st exon of the HTT gene at 4p16.3
result in a polyglutamine tract which is though to develop novel deleterious function when expanded
What evidence supports that the repeat expansion in HD acts by a GOF mechanism?
- deletions and translocation affecting HTT do not result in HD
- the phenotype of a homozygote is the same as a heterozygote
- dominant phenotype
- Levels of HD protein transcribed are the same in normal and mutant patients.
what is the characteristic finding in HD?
he presence of intranuclear inclusions is the characteristic sign of HD and are more abundant in the brains of patients with juvenile onset HD
it is thought that the polyQ expansion results in the formation of intranuclear inclusions containing HTT, chaperone proteins and ubiquitin.
HOWEVER, the inclusions do not appear to cause the disease themselves. In mouse models the location of inclusions does not correlate with cell toxicity.
What is the molecular pathogenesis of HD?
the seq of pathogenesis in HD is unclear
Mutant HTT forms abnormal protein structures - Bsheets and is truncated by caspase-6 into toxic N-terminal fragments. this is though to lead to altered processing of abnormal proteins in HD
Mutant Htt interferes with gene transcriptions (PGLC1a) and may have a direct or indirect affect on the mitochondria = affecting metabolism and leading to oxidative stress
there is also abnormal vesicle transport and release of BDNF and increased exocitotoxicity
What is the genetic change in DM1?
DM1 is caused by a CUG repeat expansion in DMPK
What is the molecular pathogenesis of DM1?
Though to act via a toxic RNA GOF mechanism
Repeat structures in RNA form stable hairpin structures that sequester RNA binding proteins CUG-BPI and MBNL in riobonuclear inclusion. This result in upregulation of CUG-BP1 and down regulation of MBNL. the altered expression of these affects alternative splicing and embryonic splicing patterns are seen instead of adult patterns in patients with DM1
What is Achondroplasia?
Achondroplasia is the most common inherited disproportionat short stature
incidence is 1 in 26-28,000
caused by mutations in the Tm domain of FGFR3
What is the molecular pathogenesis of Achondroplasia?
Due to a highly mutable codon - GOF
FGFR3 is a TM tyr kinase receptor responsible for -vely regulating bone growth. Mutations that constitutively activate the receptor result in reduced bone growth
99% of disease is due to 2 mutations
- achondroplasia is relatively common considering it requires very specific gene mutations- it has been suggested that the high de novo rate is because there is a proliferative advantage in spermatogonal cells.
other mutations in FGFr3 result in different disorders e.g. thanapophoric dysplasia and hypochondroplasia
What is the molecular pathogenesis of BRC-ABL1?
found in nearly all CML (major breakpoint) and some ALL (minor breakpoint)
fusion gene formed by the t(9;22)(q34;q11) rearrangement resulting in the formation if a constitutively active tyr kinase - aberrant signalling promotes genome instability
treated by TKI imatinib
Describe the molecular parthenogenesis of CMT?
CMT1A is caused by gene overexpression 70-80% is due to a recurrent 1.5Mb duplication at 17p12 whihc includes PMP22. remaining cases are due to GOF SNVs.
PMP22 encodes peripheral myelin protein and is present in the myelin membrane of peripheral neurons where it plays a role in arresting schwann cell division
recurrent dup is due to NAHR between CMT1A reps which flank the duplicated region. Reciprocal deletion result in HNPP
what are the characteristics of dominant negative mutations?
Mutations generally impair the function of a protein that are involved in protein complexes or reduce the activity of the WT allele
only seen inhets
more severe than null alleles of the same goen
MND probably developed to protect against dominant negative truncated proteins
proteins that are part of multimeric structures are particularly vulnerable to dominant negative effects as they are dependent on oligomerisation for activity- in a multimer a variant subunit with intact binding but altered catalytic activity can affect the function of the entire multimer
describe the pathogenesis of GJB2
dominant negative- result in deafness
GJB2 and 6 encode connexin 26 and 30- these are major gap junction proteins expressed in the cochlear.
connexins are TM proteins and 6 oligomerise to form to form a hemi channal (connexon)- connexons of neighboring cells align symmetrically to from continuous gap junctions- they can be homopolymeric or heteropolymeric with different functional properties
Cx26 and 30 are involved in K+ recycling in the ear- K+ is required for NT release from hair cells of cochlear.
dominant missense mutation in Cx26 result in the formation of full length aberrant proteins- these form gap junctions with WT Cx26 and 30 forming connexons with impaired permeability = hearing loss in hets
what is the molecular parthenogenesis of osteogenesis imperfecta?
dominant negative mutations in CLO1A1 and COL1A2
encode fibrillar collagens which are the major structural proteins of connective tissues
pre-pro collagen contains N and C terminal globular pro-domains flanking a central repeat seq with a gly every 3rd residue
3 pre-procollagen chain associate to forma triple helix under the control of the globular domain- C and N terminal domain cleaved to form mature collage and this process is disrupted in OI
what are the genetic mutations associated with OI?
Missense mutations result in the expression of abnormal protein and severe OI.
- dominant -ve (type II, II, IV)
- 80% of muts replace gly in triple helix domain
- tupe 1 procollagen comprises 2 chains encoded by COL1A1 and COL1A2- helix assembly starts at c-terminal domain therefore mutations close to the c-terminus are more deleterious than those near the N-terminus
OI type 1
- due to LOF mutations
- reduced production of type 1 procollagen as abnormal protein degraded by NMD
- less severe
Describe TP53 mutations as dominant negative
Most commonly mutated gene in cancer and mutations can be LOF, GOF and dominant negative
Normally TP53 acts as a TSG- transcription factor whcih activates transcription of genes involved in cell cycle control, damage repair, and apoptosis.
missense mutations in the DNA binding domain affect the ability of the protein to recognise DNA or inactivates TP53 by altering the conformation
- dominant neagtive as TP53 binds DNA as a tetramer
- WT and mutant p53 proteins form tetramers with impaired ability to bind DNA and trnscriptional activity
- c-terminal domain is requird for dimerisation therefore truncating mutations in this domain are not dominant -ve as they cannot bind WT
describe the pathogenesis of FBN1
Mutations result in the connective tissue disorder Marfans
fibrillin has a structural role in the walls of large arteries
dominant negative variants:
- missense and exon skipping variants result in a stable but altered protein
- impared interactions between variant and WT fibrillin and other proteins results in impaired ECM
LOF mutations
- nonsense and frameshift result in NMD
- decreases the amount of fibrillin in vasculature and the aortic wall strength is weakened
- increased risk of cardiovascular disease
what is the inheritance pattern in CF?
AR
- shows horizontal inheritance with affected siblings born to generally unaffected parents
- increased risk in consanguineous families
- unaffected siblings of affected have a 2/3 risk of being a carrier (1/3 unaffected non-carrier)
Describe the basics of CF
- most common AR disease in UK with carrier freq of 1/25 (high carrier freq suggests het advantage like in sickle, advantage is unknown- may be associated with cholera)
- affects 1 in 2500
- carrier freq depends on population and so specific frequencies should be used for risk calculations where possible
- there is a good genptype/phenotype correlation between the CF mutation and pancreatic sufficiency but not pulmonary disease.
- phedel508 is most common UK caucasian deletion ~75%
what is the function of the CFTR gene
CFTR gene encodes a cAMP activated chlorine channel located in the plasma membrane of secretory cells e.g. in the respiratory tract, reproductive tract, kidney, pancreas, vas deferens, sweat ducts
CFTR channels are responsible for moving chlorine out of the cell, this creates a trans-membrane gradient. and Na+ and water flow out of the cell down the concentration gradient. Mutations that result in a loss of function or reduce ability to function result in reduced Cl- transport and hence water resulting in increased viscosity of mucus
