Next generation sequencing to identify the causative gene for distal hereditary motor

neuropathy

Luigi CitrignoCNR – Institute of Neurological Sciences – Mangone - CS

Fred Sanger

“Hereditary peripheral neuropathies are the most common inherited disorder of the peripheral nervous system”

•Muscle weakness •Atrophy •Additional features

(Left) Neurons in culture transfected with DNA expressing a gree-fluorescent protein (GFP). (Right) GFP-expressing neurons after a treatment that induced neurodegeneration, as depicetd by the blebbing of axons and cell soma.

Clinically genetically heterogeneous

Neurodegeneration in the peripheral nervous system

Three distinct groups are recognized:

- hereditary motor and sensory neuropathy type 1 or Charcot- Marie Tooth type 1 (HMSN type 1 or CMT1);

- hereditary motor and sensory neuropathy type 2 or Charcot- Marie- Tooth type 2 (HMSN type 2 or CMT2);

- hereditary distal motor neuro(no)pathy (dHMN).

HMSN(Hereditary Motor Sensory Neuropathy)

HSN(Hereditary Sensory Neuropathy)

CMT(Charcot-Marie-Tooth)

dHMN(Hereditary distal motor

neuropathy)

“The dHMN are a heterogeneous group of disorders characterized by an exclusive involvement of the motor part of the peripheral nervous system”

Degeneration of spinal motor neurons Involvement of peripheral sensory nerves

Peroneal muscular atrophy

VCN sensory normal

dHMNPes cavus, scoliosis, lordosis, tremors

paralysis of the diaphragm and vocal cord

VCN motor slightly reduced

• Progressive weakness and wasting of the extensor muscles of toes and feet.

• Foot deformity

Overall clinical picture:

• additional features are present in ‘complicated' distal HMN, like vocal cord paralysis, diaphragm paralysis and pyramidal tract signs.

On clinical examination, distal HMN can hardly be distinguished from CMT because unequivocal sensory signs are often lacking in these disorders. Therefore, electrophysiological examinations are essential to support the diagnoses of CMT or distal HMN BUT not resolutive

Genetic classification of dHMN:

6 Autosomal dominant forms 4 autosomal recessive forms

•dHMN - I

•dHMN - II

•dHMN - V

•dHMN - VII

•dHMN with pyramidal signs

•Distal SMA

• dHMN - III

• dHMN - IV

• dHMN - VI

• dHMN - J

IV:9

II:1 II:2

III:1 III:2 III:3 III:4 III:5 III:6

IV:1 IV:2 IV:3 IV:5IV:4

V:1 V:2

IV:6 IV:7 IV:8

I:1I:2

II:3 II:4

III:7III:8

II:5II:6

III:9 III:10

IV:10

II:7II:8

III:11 III:12

IV:11

III:13

IV:12 IV:13

III:14

IV:14

40 subjects30 wild type10 affected7 died before the study28 available for molecular analysis

Previous studies (*) excluded by linkage analysis the known locion chromosomes 7p14, 9q34, 11q12, 12q24, 2q14 and 2p13.

(*) Passamonti L, Muglia M, Magariello A et al. “Further evidence of genetic heterogeneity in autosomal dominant distal motor neuronopathy.”Neuromuscul Disord. 2004 Nov;14(11):705-10.

Genome wide search

Genotyping of 206 initial microsatellites (VNTR)

(Resolution of 20 cM, LD-20cM, Applied Biosystem)

STATISTICAL ANALYSIS OF DATA (Two point parametric linkage analysis)

Lod score values

Identification linkage regions

LOD scoreProbability of linkage between two loci

LOD score = log10

probability that the two loci are associated

probability that the two loci are not associated

Threshold values

LOD <-2 to exclude linkage

LOD> +3 to accept the linkage

0<<0.5

= 0.5

III:10? ?2 2? ?1 11 13 54 51 11 2? ?5 53 5? ?1 4

IV:74 15 22 41 44 15 64 52 3? ?4 12 3? ?1 22 4

II:22 42 61 51 12 45 56 32 11 14 55 32 4? ?1 3

D4S2992D4S2991D4S3028D4S415D4S1607D4S1584D4S3041d4s2943snx25

D4S2920D4S1535D4S2924D4S3051D4S426

IV:54 35 22 31 24 15 64 42 22 14 12 12 11 42 1

II:7

IV:24 45 32 31 24 15 64 42 22 14 12 12 11 42 1

IV:4? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?1 2

IV:82 52 71 51 34 35 24 52 1? ?4 42 32 31 12 2

II:1

III:1? ?? ?? ?1 ?? ?? ?? ?? ?? ?? ?2 ?? ?? ?2 ?

III:23 42 33 53 21 14 62 41 21 11 45 11 31 43 1

III:34 45 62 51 14 45 54 32 12 14 52 32 41 22 3

III:42 32 45 73 43 31 21 42 21 11 26 32 41 21 2

III:54 25 22 11 14 25 54 62 22 14 42 52 41 22 3

III:61 52 74 54 31 32 65 53 11 11 43 33 31 24 2

IV:64 35 22 51 34 35 24 42 22 14 12 62 41 12 2

I:1I:2

II:3 II:4

III:7? ?2 3? ?1 11 32 54 41 11 2? ?1 32 2? ?? ?

III:8? ?2 3? ?1 11 32 53 41 11 1? ?1 32 5? ?? ?

II:5II:6

III:9? ?5 2? ?3 61 32 34 52 11 1? ?3 7? ?? ?2 3

D4S2992D4S2991D4S3028D4S415D4S1607D4S1584D4S3041d4s2943snx25

D4S2920D4S1535D4S2924D4S3051D4S426

IV:9? ?5 2? ?3 11 12 54 42 11 2? ?3 54 5? ?2 4

D4S2992D4S2991D4S3028D4S415D4S1607D4S1584D4S3041d4s2943snx25

D4S2920D4S1535D4S2924D4S3051D4S426

IV:10? ?? ?? ?6 13 13 35 51 11 2? ?7 54 3? ?2 4

II:8

III:114 75 12 51 44 35 64 52 12 14 12 32 21 32 1

III:127 6? ?5 53 31 52 45 51 21 14 42 52 21 3? ?

IV:117 61 25 54 33 16 25 51 21 11 43 52 21 32 3

III:13

IV:12? ?? ?? ?? ?3 3? ?3 41 1? ?? ?? ?? ?? ?? ?

IV:13? ?? ?? ?? ?3 32 53 41 1? ?? ?? ?4 5? ?? ?

III:14

IV:14? ?? ?? ?? ?1 34 53 31 1? ?? ?? ?3 2? ?? ?

Pedigree of the family and haplotypes of marker loci spanning the linked region on chromosome 4q34.3-q35.2.

We mapped a novel dHMN locus on chromosome 4q34.3-q35.2 . The recombination events that occurred in the family localized the gene disease in the 26-cM region, flanked by the markers D4S1552 and D4S2930.

The identified critical region is rich of genes, three highly interesting candidate were tested:

SNX25CASP3TUBB4Q

...and with Sanger, a lot of money and time only for this three!!!

Next generation sequencing approach

Reasons for NextGen’s rapid growth in human genetics

- Sanger sequencing is fully optimized (after the Human Genome Project)

- Whole genome assemblies (e.g., H. sapiens) are available as references

- Constant growing of annotated database

The $1,000 genome

Targeted capture/ re- sequencing

• Used 4 ug of input DNA to capture the all exome

• Capturing/enrichment process SureSelect target Enrichment system (Illumina/Solexa) technology

• Genomic DNA extracted from peripheral blood lymphocyte of a proband

• Capture efficiency evaluated by quantitative PCR reaction

• Quality assesed with Agilent 2100 Bioanalyzer

Re- sequencingCaptured DNA submitted to high throughput sequencingusing the Genom Analyzer II - GAII (Illumina) platform with Sequencing by synthesis technology

We were able to obtain 0.9 Gb of genomics sequences from two GAII runs.The average read length obtained was 340 bp, fragment size with a range 50bp - 835bp

[1] Read mapping and variant analysis.

• Reads were mapped to the reference human genome (UCSC hg18), initially with ELAND software (Illumina) for quality recalibration.

• Also the raw data was trimmed and aligned with the human genome reference using CLC genomics workbench v3.7

65508 high-confidence SNPs and

87 deletion/insertion polymorphisms (DIPs).

…after the alignement we obtain a wide variety of variations including:

Sequence calls were also performed with Maq and filtered with ≥8× coverage and Phred consensus quality ≥20

[2] Read mapping and variant analysis.

Maq file submitted to SeattleSeq annotations: •dbSNP rs ID•gene names and accession numbers•SNP functions (e.g. missense)•protein positions and amino-acid changes•conservation scores•HapMap frequencies•PolyPhen predictions•Clinical association

PolyPhen Grid Gateway(*) predictions

[3] Read mapping and variant analysis.

“Prediction of deleterious human alleles”Shamil Sunyaev, Vasily Ramensky, Ina Koch, Warren Lathe III, Alexey S. Kondrashov, and Peer Bork Hum Mol Genet (2001) 10: 591-597

All nonsynonymous variants not assigned 'benign’ by PolyPhen prediction were considered to be damaging

as were all splice acceptor and donor site mutations and all coding indels.

All nonsynonymous SNPs

preliminary results…

and based on linkage evidence(region flanked D41552 and D4S2930)

After the reads stringencies

1447

50 SNPs not present in dbSNP v.131

2 DIPs

The variations that are not false positive will be assessed in all members of the family and in a normal control population.

next steps…still running!!!

52 variations are a reasonable number and cheap to be subjected to Sanger sequencing

Exome sequencing in a proband from a families with linkage evidence is a powerful, efficient strategy for identifying the genes underlying rare mendelian disorders.

CNR - Institute of Neurological Sciences Mangone - Cosenza

Maria MugliaAngela Magariello

Alessandra PatitucciRosalucia Mazzei

Francesca Luisa ConfortiCarmine UngaroAnnalia Gabriele

William SprovieroAntonio Gambardella

Aldo Quattrone

Hussfan Institute for Human Genomics Miami - Florida

Stephan ZüchnerJia Huang

Gladys MontenegroEric Powell

Thanks and questions