CMT - Part 2

Question 1

Describe the sequential approach that was previously used in CMT testing. Describe this testing process in the example of a CMT1 patient where male to male transmission had been observed.

Answer 1

• Because of the large number of genes involved in CMT, previous genetic testing utilised a sequential testing approach guided by the patient’s clinical phenotype and observed inheritance pattern.
• For example, in CMT1 patients in examples where male to male transmission has been observed the patient would first be tested for the common 1.5Mb PMP22 duplication which causes the vast majority of CMT1 cases (CMT1A).
• If no duplication was detected using this method then we would follow with testing of MPZ (CMT1B) and point mutations in PMP22 (CMT1E).
• If both are normal would follow up with tests for LITAF (CMT1C) and EGR2 (CMT1D).
• In more than 90% of individuals with a CMT1 phenotype a mutation is found in one of the three genes (PMP22, MPZ, GJB1).
• This gene by gene approach was previously performed by Sanger sequencing.
• Because of advancements in screening tech, current approaches can now take advantage of multiple gene panel screens. For instance the Bristol Genetics Lab has a UKGTN approved mutation screen of 56 genes associated with different types of inherited neuropathy. This would normally follow exclusion of PMP22 duplication dosage analysis in CMT and HNPP patients. To be eligible for the 56 gene screen, patients must fulfil appropriate criteria which cover clinical presentation, NCV result, absence of non-genetic causes and absence of CNS involvement.

Question 2

What form of CMT is caused by mutations in MPZ?

Answer 2

CMT1B.

Question 3

What form of CMT is caused by point mutations in PMP22?

Answer 3

CMT1E.

Question 4

What form of CMT is caused by mutations in LITAF?

Answer 4

CMT1C.

Question 5

What form of CMT is caused by mutations in EGR2?

Answer 5

CMT1D.

Question 6

What CMT testing method has now replaced the sequential method due to the advancement in testing technologies?

Answer 6

Because of advancements in screening tech, current approaches can now take advantage of multiple gene panel screens. For instance the Bristol Genetics Lab has a UKGTN approved mutation screen of 56 genes associated with different types of inherited neuropathy. This would normally follow exclusion of PMP22 duplication dosage analysis in CMT and HNPP patients. To be eligible for the 56 gene screen, patients must fulfil appropriate criteria which cover clinical presentation, NCV result, absence of non-genetic causes and absence of CNS involvement.

Question 7

Would molecular genetic testing be indicated in families that don’t have any family history of CMT? Justify your answer.

Answer 7

• Molecular genetic testing is also often indicated in patients that don’t show any previous family history of CMT.
• This is because de novo duplications of the CMT region causing CMT1A are common.
• De novo mutations in other AD CMT genes can also occur.
• In particular, family pedigrees can be complicated in X-linked forms of CMT as females with GJB1 mutations can be asymptomatic.
• AR forms of CMT, although rare in most european populations, can also occur in the absence of any clear family history.

Question 8

Describe the molecular genetics of CMT1A.

Answer 8

• The 1.5Mb duplication including the PMP22 gene is the most common cause of CMT1.
• In addition, deletion of this 1.5Mb region leads to another clinical phenotype, hereditary neuropathy with liability to pressure palsies (HNPP).
• 84% of HNPP cases are caused by the 1.5Mb deletion. The remaining cases are due to loss-of-function point mutations in PMP22.
• The clinical symptoms seen in HNPP include transient recurrent episodes of focal weakness / sensory loss, usually caused by pressure.
• HNPP patients also show widespread nerve conduction abnormalities (slowing around sites vulnerable to compression).
• In addition to the deletions and duplications in the PMP22 gene observed point mutations in PMP22 can lead to a variety of phenotypes including CMT with deafness, HNPP, or a severe CMT1 phenotype depending on the effect of the mutation on PMP22 function.
• The CMT1A causing duplication in PMP22 occurs at high frequency and sometimes arises de novo. This occurs because of repetitive DNA regions that flank the PMP22 gene termed CMT1A-REPs. Non-allelic homologous recombination (NAHR) between the CMT1A-REPS lead to recurrent deletion / duplication of the region and contributes to the high frequency of these mutations in the population.

Question 9

What condition does deletion of the 1.5Mb region including the PMP22 gene lead to?

Answer 9

• Deletion of this 1.5Mb region leads to another clinical phenotype, hereditary neuropathy with liability to pressure palsies (HNPP).
• 84% of HNPP cases are caused by the 1.5Mb deletion. The remaining cases are due to loss-of-function point mutations in PMP22.

Question 10

What are the clinical features of HNPP?

Answer 10

• The clinical symptoms seen in HNPP include transient recurrent episodes of focal weakness / sensory loss, usually caused by pressure.
• HNPP patients also show widespread nerve conduction abnormalities (slowing around sites vulnerable to compression).

Question 11

Why does the CMT1A causing duplication in PMP22 occur at high frequency?

Answer 11

The CMT1A causing duplication in PMP22 occurs at high frequency and sometimes arises de novo. This occurs because of repetitive DNA regions that flank the PMP22 gene termed CMT1A-REPs. Non-allelic homologous recombination (NAHR) between the CMT1A-REPS lead to recurrent deletion / duplication of the region and contributes to the high frequency of these mutations in the population.

Question 12

What is the most common method used to test for CMT1A? What other methods have been used?

Answer 12

• MLPA is the most common method.
• Other techniques that have been used for diagnosis of CMT1A include quantitative flluorescent dosage PCR, breakpoint PCR, FISH and array CGH.
• Breakpoint based assays are often less sensitive than dosage assays because the breakpoints of the copy number changes may not always be identical.
• Genomic techniques such as array CGH are also capable of detecting PMP22 deletions and duplications. There is therefore a risk that PMP deletions and duplications could be detected as incidental findings during array CGH, particularly when testing children that are younger than the normal age of onset for these diseases.

Question 13

Describe MLPA testing for CMT1A.

Answer 13

• One of the most common methods used to test for CMT1A duplications of PMP22 (17p11.2) is MLPA.
• This is also used to detect PMP22 deletions in HNPP.
• The P033 MLPA kit (MRC-Holland) detects 70% of CMT1 (98% if CMT1A) and 84% of HNPP mutations.
• Sequence variants close to the probe ligation site can cause apparent deletions of single probes. These can be confirmed by subsequent sequence analysis of the affected exon. These sequence variants could be pathogenic mutations or neutral variants.

Question 14

What is the location of the PMP22 gene?

Answer 14

17p11.2

Question 15

What CMT phenotype do mutations in the MPZ gene lead to?

Answer 15

• Mutations in the MPZ (myelin protein zero) gene lead to a wide phenotypic spectrum including:
  1) . AD CMT1 (CMT1B),
  2) . AR CMT1
  3) . AD CMT2
  4) . Intermediate CMT.
• Most of these mutations are missense mutations. However, recent studies have demonstrated that duplication of the MPZ gene can also lead to an early-onset CMT1 phenotype.

Question 16

What CMT phenotype do mutations in the GJB1 gene lead to?

Answer 16

• Mutations in the GJB1 gene cause X-linked CMT (CMTX1).
• These causative mutations are usually loss-of-function mutations and can include missense mutations affecting functionally important amino acids as well as truncating mutations and frameshift mutations.
• The GJB1 gene encodes the connexin-32 protein. This protein forms multimeric gap junctions that are involved in cell to cell communication.

Question 17

What protein does the GJB1 protein encode?

Answer 17

The GJB1 gene encodes the connexin-32 protein. This protein forms multimeric gap junctions that are involved in cell to cell communication.

Question 18

What CMT phenotype do mutations in the MFN2 gene lead to?

Answer 18

• Mutations in the Mitofusin-2 (MFN2) gene are the major cause of CMT2.
• Mutations in this gene can often occur de novo and often include novel mutations, causing diagnostic difficulties.
• MFN2 protein functions in mitochondrial fusion and transport.

Question 19

What is the function of MFN2?

Answer 19

MFN2 protein functions in mitochondrial fusion and transport.

Question 20

How is gene sequencing in CMT now performed?

Answer 20

• Previously, most mutation screening in CMT has been done using a sequential gene by gene approach and by Sanger sequencing.
• MLPA kits for some of the genes involved are available but most are not routinely used as the majority of causative mutations are detectable by sequencing, for instance missense and truncating point mutations.
• Recently because of the advent of NGS some of the labs are now able to offer panel based screens that analyse multiple genes known to cause CMT and other associated phenotypes.
• Bristol offer a screen of 58 genes that are associated with inherited peripheral neuropathies. This screening procedure uses a custom Agilent SureSelect capture array for target enrichment and the resulting library is run on an Illumina MiSeq NextGen sequencer. Mutations are then confirmed separately by Sanger sequencing.

Question 21

What techniques are currently being examined that may be used to test for CMTs in the future?

Answer 21

• Several groups have also looked at the potential for techniques such as whole exome sequencing (WES) or whole genome sequencing (WGS) for mutation detection in CMT and related disorders.
• These methods are particularly applicable for mutation detection in rare, genetically heterogeneous disorders such as CMT. Need a very well defined data filtering and analysis strategy for the identification of putative causative variants due to the large amounts of data generated.

Question 22

Why can molecular genetic diagnosis in CMT be complicated compared to some other disorders such as hereditary breast cancer?

Answer 22

• Molecular genetic diagnosis in CMT can be complicated compared to some other disorders such as hereditary breast cancer because many mutations causing CMT are missense mutations.
• It can be difficult to establish definitive pathogenicity, especially with novel mutations.
• The pathogenicity of a missense mutation depends on the location and nature of the amino acid change.
• Methods for investigating the pathogenicity of missense variants include:
  1) . in silico analysis (e.g. amino acid conservation, splice site prediction - Alamut)
  2) . presence on mutation databases (e.g. Diagnostic Mutation Database DMuDB)
  3) . Absence in normal controls
  4) Published literature in large families or including functional analysis can help, however, there are many novel and previously unpublished mutations that occur in CMT
  5) . Segregation analysis (can be difficult in CMTX because of variable penetrance in females, often few affected/unaffected males available)

Question 23

What methods can be used to investigate the pathogenicity of missense mutations?

Answer 23

Methods for investigating the pathogenicity of missense variants include:

• In silico analysis (e.g. amino acid conservation, splice site prediction - Alamut)
• Presence on mutation databases (e.g. Diagnostic Mutation Database DMuDB)
• Absence in normal controls
• Published literature in large families or including functional analysis can help, however, there are many novel and previously unpublished mutations that occur in CMT
• Segregation analysis (can be difficult in CMTX because of variable penetrance in females, often few affected/unaffected males available)