18.01.18 LCRs and the origin of genetic disorders

Question 1

What are genomic disorders?

Answer 1

Conditions that result from rearrangements of the genome rather than base pair changes of DNA, and in which genomic instability results from the endogenous genome architecture. Depending on the size of the rearranged genomic segment and the number of genes included, the result can be a Mendelian disorder (e.g. NF1, CMT1A/HNPP, STS, AZFa, SMN), a contiguous gene syndrome (e.g. WAGR, Williams, PWS/AS, SMS, DG/VCFS) or a chromosomal disorder (e.g. idic(15), idic(22), inv dup(8p), translocations, markers).

Question 2

What is a contiguous gene disorder?

Answer 2

Contiguous gene syndrome: Genetic syndrome that is caused by the loss or gain of multiple adjacent genes, some of which are dosage sensitive, leading to specific recognisable clinical phenotype. Contiguous gene syndromes can be caused by genomic disorders.

Question 3

What are LCRs?

Answer 3

Low-copy repeats: Chromosome-specific DNA blocks of around 1-400kb and with high degree of homology (>90%) that occur twice or a few times in a haploid genome.

Question 4

How are LCRs thought to have arisen?

Answer 4

They are thought to have arisen during evolution by duplication of genomic segments resulting in paralogous regions.

Question 5

What is the structure of LCRs?

Answer 5

They can contain genes, gene fragments, pseudogenes, endogenous retroviral sequences or other paralogous fragments and are thought to consist 5-10% of the genome.

They provide the substrate for NAHR depending on repeat size, degree of homology, distance between them, orientation with respect to each other and MEPS (see below).

Question 6

Where are pseudogenes located?

Answer 6

They are often located in pericentromeric and sub-telomeric regions

Question 7

How are LCRs are distinguished from highly repetitive sequences in the genome (e.g. LINE, SINE, Alu) ?

Answer 7

They are not present in large numbers and do not anneal as quickly as the repetitive sequences during reassociation kinetics experiments.

Question 8

What is NAHR? How does it result in genomic rearrangenements?

Answer 8

Non-allelic homologous recombination. Homologous recombination occurs between lengths of homology in different genomic positions. Unequal crossovers during NAHR would lead to genomic rearrangements.

Question 9

How can NAHR lead to genetic disease? Which types of genetic disease are typically caused by NAHR?

Answer 9

1. a change in copy number of dosage-sensitive genes
2. gene disruption
3. creation of fusion genes
4. position effects
5. unmasking of recessive traits / functional polymorphisms or interruption of transvection (communication between alleles).

Recurrent genomic rearrangements (such as deletions, duplications, isodicentric chromosomes and inversions) are usually caused by NAHR between LCRs.

Question 10

What is the difference between NAHR occurring in meiosis and NAHR occurring in mitosis?

Answer 10

NAHR can be different between males and females and can occur either during meiosis or mitosis. When occurring during meiosis, NAHR would lead to constitutional rearrangements, while when occurring in mitosis, NAHR could lead to somatic mosaicism or neoplasia (e.g. i(17p)). It has been suggested that open chromatic conformation due to active transcription could facilitate recombination.

Question 11

What is the role of cohesin proteins?

Answer 11

Proteins called cohesins hold sister chromatids together. A sister chromatid is the preferred partner for recombination events and the opportunity for intra- and inter-chromosomal NAHR is restricted.

Question 12

What are NAHR hotspots?

Answer 12

Positions within LCRs where crossovers preferentially occur. These hotspots are usually in regions of identical nucleotide sequence of at least 200 to ~450bp in length. This is where the rearrangement breakpoints are typically found.

Question 13

What are MEPS?

Answer 13

Minimal efficient processing segment: The minimal stretch of identity required among substrates to enable homologous recombination. T

he length of MEPS can be different between meiosis (estimated around 300-500bp) and mitosis (possibly shorter), but also different between different events.

The distance between the LCRs could play a role in determining the length of the MEPS (the further away the LCRs, the longer the MEPS).

Question 14

What are paralogous genomic segments?

Answer 14

non-allelic genomic segments with highly identical DNA sequence, typically hundreds of kilobases in size and flanking breakpoint junctions.

Question 15

Comment of the frequency of reciprocal deletions and duplications

Answer 15

At least during meiosis, even theoretically (fig. 1a, 1d and 1g), the frequency of deletions should always be higher than duplications. In addition, the prelevance of several reciprocal del/dup syndromes is different (e.g. CMT1A/HNPP, PTLS/SMS, dup(22)(q11.2q11.2)/VCFS).

This could be biased because deletions are expected to have a more severe phenotype than duplications and thus to be identified more often, or because one or the other could be embryonically lethal.

However, single-sperm data support that they have different prelevance.

Question 16

What two mechanisms have been proposed for NAHR?

Answer 16

NAHR is considered to be mainly a recombination event during meiosis or mitosis. At the molecular level, two mechanisms of NAHR have been proposed:

1. Unequal crossing-over: a recombination-based mechanism
2. BIR (Break-induced replication)while BIR is a replication-based mechanism.

Question 17

How does BIR occur?

Answer 17

BIR occurs when a replication fork collapses or breaks and the broken molecule uses ectopic homology to restart the replication fork. BIR will form duplications and deletions in separate events, while unequal crossing-over could form the duplication and the reciprocal deletion in the same event

Question 18

What other mechanisms can lead to genomic rearrangement? How may LCRs have a role in these mechanisms?

Answer 18

1. Non-Homologous End Joining - Mechanism for repair of double stranded DNA breaks
2. FoSTeS (fork stalling and template switching)

Although LCRs do not mediate these mechanisms, LCRs or other repetitive elements are found in the proximity of the breakpoints and therefore may stimulate the rearrangements.

Question 19

Describe NHEJ.

Answer 19

1. DNA break identified
2. Broken DNA ends are bridged, modified and ligated
3. Does not require LCRs in the way NAHR does
4. Often the incorporation of additional bases leaves a ‘molecular scar’

Question 20

Describe FoSTeS.

Answer 20

Fork stalling and template switching (FoSTeS) – plays an important role in the origin of genomic-disorder-associated non-recurrent rearrangements that have a complex structure

1. DNA replication fork stalls and lagging strand disengages from the original template and anneals to another replication fork nearby
2. Micro-homology-mediated break-induced replication (MMBIR) – depending on location of the new fork, a deletion of duplication will occur.

Question 21

Give clinical relevant examples of disease associated with FoSTeS/MMBIR.

Answer 21

FoSTeS/MMBIR shown to have a role in MECP2 duplication (Xq28), deletions and duplications of 17p13.3, deletions and duplications of 17p11.2p12, deletions and duplications in 9q34 and many others.

FoSTeS/MMBIR likely to be a major mechanism for generating structural variation, particularly non-recurrent copy-number variations (CNVs) and other genomic rearrangements.

Question 22

Give three examples of genomic disorders arising from LCR/NAHR.

Answer 22

1. WBS
2. PWAS
3. CMT1A/HNPP
4. SMS/PTLS
5. NFI
6. 22q11 del/dup
7. STS
8. 17q21.31 microdel
9. 15q13.3 microdel
10. 15q24 microdel

Question 23

Describe the origin of WBS.

Answer 23

Williams-Beuren Syndrome at 7q11.23. ~1.6MB deletion including the elastin (ELN) gene. Three ~300kb complex repeat gene structures (cen, mid and tel) are composed of several differentially orientated subunits. The common deletion results from NAHR between directly orientated blocks with the cen and mid LCRs.

Question 24

Describe the origin of PWAS.

Answer 24

Prader-Willi Syndrome/Angelman syndrome. The common ~4MB deletion and duplication results from NAHR between complex 15q11–q13 LCRs with a size of >500kb, that are mainly duplications of the gene/pseudogene HERC2 forming blocks called END-repeats.

Four large clusters of complex repeats have been termed BP1 to BP4.The distal breakpoints in 95% of cases map to BP3. In some unusual larger deletions the distal breakpoint maps to BP4. The proximal breakpoints cluster within BP2 (60%) or BP1 (40%).

Question 25

Describe the origin of CMT1A/HNPP.

Answer 25

Charcot-Marie-Tooth (dup) / Hereditary neuropathy with liability to pressure palsies (del). At 17p12 encompassing the PMP22 gene. Recurrent breakpoints map to two direct LCRs: CMT1A-REPs

Question 26

Describe the origin of SMS/PTLS

Answer 26

Smith-Magenis (del) / Potocki-Lupski (dup). Contiguous gene syndromes caused by microdeletion / microduplication within 17p11.2, including the RAI1 gene with overlapping clinical features. Three ~250kb LCRs present: distal (D), middle (M) and proximal (P) SMS-REPs. The SMS-REPM is inverted in orientation. The common ~4Mb del/dup occurs due to NAHR between the SMS-REPD and P. Smaller deletions occurring by NAHR between the SMS-REPD and M.

Question 27

Describe the origin of NF1

Answer 27

NF1: Neurofibromatosis type 1. ~1.5Mb deletion of the NF1 gene on 17q11.2 mediated by direct ~85kb LCRs called NF1REPs that span 15-100kb and contain several genes and pseudogenes

Question 28

Describe the origin of DGS/ VCFS / dup(22)(q11.2q11.2)

Answer 28

DiGeorge syndrome/velocardiofacial syndrome and reciprocal duplication. Both the common ~3Mb deletion and duplication and the smaller ~1.5MB deletions within 22q11.2 result from NAHR between LCRs.

These LCRs are ~250-440kb long and each one has different complex internal organization. They are termed LCR22-A (more proximal), -B, -C and -D (more distal). The common del(90%)/dup arises due to NAHR between LCR22-B and –D, while the smaller deletions (7%) arise due to NAHR between LCR22-A and –B.

Question 29

Describe the origin of STS.

Answer 29

Deletion of ~1.9Mb on Xp22.3 including the Steroid Sulphatase (STS) gene. Direct LCRs called S232 at either side of the gene could mediate NAHR and lead to the deletion.

Question 30

Describe the origin of 17q21.31 microdeletion syndrome.

Answer 30

500-650 kb in size, flanked by LCRs, deletion arises only on a specific inversion haplotype (H2 lineage) (900 kb inversion is a necessary factor for the deletion to occur). CRHR1 and MAPT proposed as candidate genes for pathogenicity.

Question 31

Describe the origin of 15q13.3 microdeletion syndrome.

Answer 31

1.5 Mb critical region maps between BP4 and BP5 LCRs (distal to the Prader-Willi and Angelman syndrome region), results in loss of six known genes including CHRNA7.

Haplo-insufficiency of CHRNA7 might be responsible for most of the neurodevelopmental disorders associated with the deletion. The BP4–BP5 region undergoes frequent inversion, suggesting a possible link between this inversion polymorphism and recurrent deletion.

Question 32

Describe the origin of 15q24 microdeletion syndrome.

Answer 32

Ranges in size from 1.7-6.1 Mb. NAHR between paralogous low-copy repeats (LCRs).

The majority of 15q24 deletions have breakpoints that localize to one of five LCR clusters labelled LCR15q24A, -B, -C, -D, and -E.

The smallest region of overlap (SRO) spans a 1.2 Mb region between LCR15q24B to LCR15q24C. Candidate genes within the SRO include CYP11A1, SEMA7A, CPLX3, ARID3B, STRA6, SIN3A and CSK, that may predispose to many of the clinical features seen in individuals with 15q24 deletion syndrome.