Rare Disease Genes

Question 1

What is autozygosity mapping and when is it used

Answer 1

Autozygous = homozygous-by-descent

Used to map autosomal recessive disease genes in families with close parental relatedness (consanguinity)

Runs of homozygosity in affected individuals indicate the likely location of the disease gene in the genome

Naturally occurring homozygosity = homozygous-by-state

Question 2

Describe hereditary spastic paraplegia (HSP)

Answer 2

An example of autozygosity mapping and functional characterisation

Group of inherited neurological disorders characterised by degeneration of motor neurons

Upper motor neurons in the brain and spinal cord deliver signals to the lower motor neurons, which carry messages to the muscles
Lower limb spasticity (stiffness)

Symptoms typically appear age 10-30s - progressive disorder

Question 3

Discuss HSP heterogeneity

Answer 3

Clinical Heterogeneity -variable phenotype
‘Pure’ forms - isolated spasticity
‘Complicated’ form - associated with other neurological abnormalities

Locus heterogeneity
Multiple genes - >75 disease loci described, >60 genes identified

`Autosomal recessive or autosomal dominant inheritance

Question 4

Describe HSP with thin corpus callosum (TCC)

Answer 4

HSP-TCC is a complicated form of spastic paraplegia

The corpus callosum transmits neurological signals between the left and right hemispheres of the brain
Composed of a thick band of ~200 million axons

Autozygosity mapping was undertaken in a consanguineous family
Microsatellite markers on Chr15 were used, and refined a critical linkage interval of 4Mb by merging different family data

‘Gene distiller’ gives a list of the genes within the region and the gene oncology (gene function)
Filtering - protein coding genes, 58 genes
Candidate gene section - filtered according to gene function , 26 gene

Question 5

What are the findings re:HSP-TCC found by autozygosity mapping

Answer 5

Screening candidate - mutations found in KIAA1840, locus SPG11, consisting of 40 exons

Different mutations from the different families were found within this gene

All of which led to were different mutations within the genes leading to frameshift or nonsense mutations leading to loss of the protein/truncation/nonsense mediated decay

One was a splice site mutation - needed validation to ensure it was a splicing defect

Question 6

How can splice mutations be investigated

Answer 6

cDNA sequencing can be examined to detect splicing defects

PCR was completed, and then the protein was examined via gel electrophoresis
Carrier got two bands - a regular protein and a truncated version

Mutant = one band = truncated
Sequencing showed that exon 2 was skipped

Question 7

Describe SPG11

Answer 7

The SPG11 gene encodes the ‘spatacsin’ protein - 2,443 amino acid protein of unknown function
>100 distinct loss-of-function mutations reported

Predicted to have four transmembrane domains and a coil-coil domain

Leucine zipper motif and Myb domain suggest a possible regulatory function during gene expression

Question 8

Describe how spatacsin function was examined in zebrafish

Answer 8

Isolate the zebrafish spg11 orthologue and evaluate the level of homology between species

Assess the effects of transcript knockdown using morpholino oligonucleotides

Comparative analysis of human and zebrafish genes revealed conserved functional domains

GHF1 site Myb domain Leucine zipper Coiled-coil domain
Unique conserved domain identified in the C terminus
92% amino acid similarity between species - suggests a potentially important domain

Question 9

Describe how morpholinos was used to KO SPG11

Answer 9

Splice-blocking morpholino designed to the exon 2-intron 2 boundary

Prevents the binding of small nuclear ribonucleoproteins at the donor site

Morpholino leads to alternative splicing of spg11transcript
One version excludes exon 2
Another uses an alternative splice site within exon 2, leaving some of exon 2 but does still lead to early termination
No splicing out of exon 2, but inclusion of intron (frameshift and early truncation)

Question 10

Describe the effect of SPG11 morphants

Answer 10

SPG11 Morphants have CNS defects including perturbed neuronal development

Curly tail, defective brain ventricle formation, smaller eyes, reduced mid-hind brain boundary, mild hydrocephaly (fluid accumulation), flat somite pattern

Markedly reduced immunohistochemical staining (anti-acetylated alpha-tubulin)

Neuronal projections appear to be impeded (optic axons, retinal ganglion cells)

Reduced differentiation of cranial and spinal motor axons

Question 11

Describe spondyloepimetaphyseal dysplasia

Answer 11

An example of autozygosity mapping combined with exome sequencing

Heterogeneous group of skeletal disorders involving abnormal ossification of long bone epiphyses and metaphysis, and malformations of the vertebrae, resulting in limb and spine defects

More than 15 clinical variants described - at least 5 causal genes identified

Unique family with SEMD and abnormal teeth and hands (Rao et al., 1997)

Question 12

Describe the investigation of spondyloepimetaphyseal using autozygosity mapping

Answer 12

Consanguineous family with a unique form of SEMD identified a run of homozygosity on chromosome 11
Large region spanning the centromere

The linkage interval contained >400 genes – too many to screen, filtered for skeletal-related genes

Exome sequencing of one affected individual
Filtering for homozygous, rare, likely deleterious variants identified a c.776_777insT frameshift mutation (p.H261Pfs*2) in the SLC39A13 gene

Question 13

What is adams-oliver syndrome

Answer 13

Developmental disorder defined by terminal transverse limb defects (TTLD) and scalp aplasia cutis congenita (ACC)

Associated features - neurological anomalies, cardiac malformations, vascular defects (e.g. cutis marmorata telangiectatica congenita, dilated veins)

Question 14

Describe how adams-oliver syndrome was investigated using GWAS

Answer 14

Linkage analysis in two autosomal dominant families detected a statistically significant locus on Chr3

Locus Refinement
Refinement of minimal linkage interval using microsatellite and SNP markers across the region
Maximum LOD score of 4.93 at marker rs1464311
Maximum linkage interval (defined by markers D3S3670 and rs1127030) contains ~100 genes
Choose to look at the area with highest peak

Heterozygous mutations of the ARHGAP31 gene detected in two unrelated probands
Both mutations predicted to result in premature protein truncation

Question 15

What is ARHGAP31

Answer 15

ARHGAP31 = GTPase-activating protein (GAP)

Rho GTPases cycle between an active (GTP-bound) and inactive (GDP-bound) form
Inactivates G proteins by promoting the hydrolysis of GTP > GDP + Pi

Specifically regulates Rac1 and Cdc42
qPCR showed mutant transcripts are stable - thus not degraded by NMD, and are active (but truncated)

Question 16

How is ARHGAP31 localisation and activity investigated

Answer 16

ARHGAP31 is expressed in the developing cranium, heart and limb buds in mouse

Mutant cells display increased GAP activity i.e. gain-of-function mechanism of disease
Over-expression of mutant constructs leads to cell rounding, due to Cdc42 inactivation

ARHGAP31-positive patient fibroblasts have defects in proliferation and migration

Patient fibroblasts with a heterozygous p.Q683* mutation proliferate slower than control cells
p.Q683* mutant fibroblasts migrate quicker but cover less distance

AOS cells proliferate slower but migrate faster and have impaired directionality

Question 17

Describe how adams-oliver syndrome is investigated using exome sequencing and gene sharing

Answer 17

Two families, multiple members sequenced - exome sequencing is most powerful for this

Sharing is observed - within and across affected families
Excluded non-conserved variants, common variants, those not shared within families, those not shared between families

This revealed dysregulation of Notch pathway - missense within conserved LAG1 DNA-binding domain
Bioinformatic prediction using polyphen-2 predicted it would cause detrimental effect

Question 18

Describe exome sequencing

Answer 18

Exomes are captured using RNA probes
Reads are mapped back against a reference

It can be combined with autozygosity mapping to help filter candidate genes

Variant detection
Variant profile (VCF file with SNV's and Indels)
Filtering - zygosity, excluding known/common variants, linkage, predicted functionality (nonsense, missense, splice), prior biological knowledge (candidate genes and pathways)

Question 19

Loss-of-function RBPJ mutations in AOS

Answer 19

Electrophoretic mobility shift assay (EMSA) or gel shift
Looks at protein binding to its recognition DNA sequence

RBPJ proteins containing AOS mutations cannot bind to DNA targets i.e. Loss-of-function mechanism of disease