Human Genetics

, Volume 121, Issue 5, pp 559–564

A novel locus for distal motor neuron degeneration maps to chromosome 7q34-q36

Authors

  • Sumana Gopinath
    • Northcott Neuroscience LaboratoryANZAC Research Institute, Concord Hospital
    • Campbelltown Hospital
    • Northcott Neuroscience LaboratoryANZAC Research Institute, Concord Hospital
    • Faculty of MedicineUniversity of Sydney
  • Marina L. Kennerson
    • Northcott Neuroscience LaboratoryANZAC Research Institute, Concord Hospital
    • Faculty of MedicineUniversity of Sydney
    • Molecular Medicine LaboratoryConcord Hospital
  • Jennifer C. Durnall
    • Northcott Neuroscience LaboratoryANZAC Research Institute, Concord Hospital
  • Garth A. Nicholson
    • Northcott Neuroscience LaboratoryANZAC Research Institute, Concord Hospital
    • Faculty of MedicineUniversity of Sydney
    • Molecular Medicine LaboratoryConcord Hospital
Original Investigation

DOI: 10.1007/s00439-007-0348-9

Cite this article as:
Gopinath, S., Blair, I.P., Kennerson, M.L. et al. Hum Genet (2007) 121: 559. doi:10.1007/s00439-007-0348-9

Abstract

The motor neuron diseases (MND) are a group of related neurodegenerative diseases that cause the relative selective progressive death of motor neurons. These diseases range from slowly progressive forms including hereditary motor neuropathy (HMN), to the rapidly progressive disorder amyotrophic lateral sclerosis (ALS). There is clinical and genetic overlap among these MNDs, implicating shared pathogenic mechanisms. We recruited a large family with a MND that was previously described as juvenile ALS and distal HMN. We identified a novel MND/HMN locus on chromosome 7q34-q36 following a genome-wide scan for linkage in this family. The disease causing mutation maps to a 26.2 cM (12.3 Mb) interval flanked by D7S2513 and D7S637 on chromosome 7q34-q36. Recombinant haplotype analysis including unaffected individuals suggests that the refined candidate interval spans 14.3 cM (6.3 Mb) flanked by D7S2511 and D7S798. One gene in the candidate interval, CDK5, was selected for immediate mutation analysis based upon its known association with an ALS-like phenotype in mice however, no mutations were identified. Identification of genes causing familial MND will lead to a greater understanding of the biological basis of both familial and sporadic motor neuron degeneration including ALS.

Introduction

The motor neuron diseases (MND) are a group of related neurodegenerative diseases that cause the relative selective progressive death of motor neurons. These diseases range from slowly progressive forms to the rapidly progressive disorder, amyotrophic lateral sclerosis (ALS). The MNDs are generally classified according to whether the degeneration affects upper motor neurons alone, lower motor neurons alone, or a mixed pathology. Classic ALS, the most common MND, variably affects both upper and lower motor neurons, with relentless progression typically leading to death within 3–5 years of the onset of symptoms. In contrast, the disorders affecting lower motor neurons alone are typically slowly progressive leading to lifelong disability.

The disorders affecting lower motor neurons include progressive muscular atrophy (PMA), spinal muscular atrophy (SMA), and distal hereditary motor neuropathy (dHMN). It is increasingly being recognized that these non-ALS motor neuron diseases show significant clinical and pathological overlap with forms of ALS and some forms share the same genetic basis, i.e., the same gene causes rapid or slowly progressive disease (Hanemann and Ludolph 2002; James and Talbot 2006; Van Den Bosch and Timmerman 2006). For example, there are clinical similarities between ALS type 4 and some distal HMN families. Both disorders were genetically linked to the same locus on chromosome 9q34 (Blair et al. 2000; De Jonghe et al. 2002) and we now know that mutations in the same gene SETX can lead to both phenotypes (Chen et al. 2004). Similarly, mutations in the DCTN1 gene have been identified in both ALS (familial and sporadic) and HMN (Puls et al. 2003; Munch et al. 2004). Neurofilaments have also long been implicated in the pathogenesis of MND (reviewed by Shaw 2005). Mutations in a neurofilament subunit NF-H gene occur in about 1% of sporadic ALS patients, while mutation of the NF-L subunit gene causes a form of motor neuropathy. Mutations of the VAPB gene have also been identified in a range of motor neuron disorders including familial and sporadic ALS, SMA, and a HMN phenotype (Nishimura et al. 2004; Valdmanis et al. 2005). These clear genetic overlaps between ALS and HMN, and obvious clinical and pathological similarities suggest that determining the molecular basis of HMN will provide significant insights into the molecular basis of ALS (Hanemann and Ludolph 2002; James and Talbot 2006).

Distal HMN is a group of insidiously progressive neurodegenerative disorders. Some HMNs are called the spinal form of Charcot-Marie-Tooth (CMT) syndrome or distal SMA. Clinically, the distal HMNs are characterized by distal muscle atrophy without sensory impairment, neurophysiologically by denervation on EMG, and normal or slightly reduced motor nerve conduction velocities (Neuromuscular Disorder Consortium 1997). Using the traditional classification, distal HMN accounts for 10–20% of hereditary motor and sensory neuropathies (Harding and Thomas 1980). The hereditary motor and sensory neuropathies are the most common group of disorders of the peripheral nervous system affecting 1 in 2,500 people (Keller and Chance 1999). To date, seven genes have been found to cause HMN; however, these only account for a minority of cases (Sambuughin et al. 1998; Antonellis et al. 2003; Puls et al. 2003; Chen et al. 2004; Evgrafov et al. 2004; Irobi et al. 2004).

We report genetic analysis in a family with a MND that was previously described as juvenile ALS and distal HMN (De Jonghe et al. 2002; Chen et al. 2004). A genome-wide scan for linkage and subsequent fine genetic mapping in this family has identified a novel MND/HMN locus. The candidate interval was inspected to identify any obvious candidate genes based upon our knowledge of previously implicated MND genes or disease mechanisms. One gene was selected for immediate mutation analysis based upon its known association with an ALS-like phenotype in mice.

Subjects and methods

Subjects

We ascertained and recruited a large multigenerational distal HMN family, F-54 (Fig. 1). Some members of this family were previously reported (De Jonghe et al. 2002; Chen et al. 2004). Family members provided informed written consent in accordance with protocols approved by the Sydney South West Area Health Service Human Research Ethics Committee, NSW, Australia. Motor and sensory nerve conduction velocity measurements were performed according to standard procedures. Brain and cervical spine MRI was performed at the Royal Prince Alfred Hospital, Camperdown, Sydney. Sural nerve biopsy was performed and analyzed using light and electron microscopy at the Department of Anatomical Pathology, Surgical Pathology, Department of Medicine, University of Sydney.
https://static-content.springer.com/image/art%3A10.1007%2Fs00439-007-0348-9/MediaObjects/439_2007_348_Fig1_HTML.gif
Fig. 1

Pedigree of family F-54. Blackened symbols indicate individuals affected with MND/HMN. Haplotypes for markers on chromosome 7q34-q36 are indicated. A solid black bar represents the haplotype segregating with the disease. Observed recombination events in affected individuals II: 9 and III: 11 define the candidate interval for the disease gene, flanked by D7S2513 and D7S637. Observed recombination events in unaffected individuals II: 6 and III: 3 suggest the minimum probable candidate interval is flanked by D7S2511 and D7S798

Genetic analysis

Genomic DNA was extracted from the peripheral blood of family members and genotyped using standard methods (Blair et al. 1995). A genome-wide scan for linkage was performed using 382 microsatellites spaced at an average of 10 cM (ABI Prism Linkage mapping set Version2, PE Applied Biosystems). Genotyping was carried out at the Australian Genome Research facility (AGRF). Two-point linkage analysis was performed using the MLINK program of LINKAGE package, FASTLINK Version 4.1 (Lathrop and Lalouel 1984). Parameters for linkage analysis included autosomal dominant inheritance, 90% disease penetrance, a disease allele frequency of 0.0001, and equal male and female recombination. Unaffected at-risk individuals below 30 yrs were coded as “unknown”. Marker allele frequencies were obtained from the Genome Database (GDB, http://www.gdb.org). Multipoint linkage analysis was performed using the LINKMAP program of the LINKAGE package with intermarker distances obtained from the Marshfield sex-averaged linkage map.

DNA sequencing

All exons and at least 100 bp of flanking intronic sequence of CDK5 were PCR amplified using gene-specific primers. Primers and amplification conditions are available from the authors on request. Direct sequencing of amplified fragments was performed using Big-Dye terminator sequencing (v3.1, Applied Biosystems). Sequencing primers were the same as used in PCR amplification.

Results

Clinical features

The disease in family F-54 shows autosomal dominant inheritance. Nine affected family members were examined and their clinical features are summarized in Table 1. The disease was slowly progressive with an early but variable age of onset (range 3–40 years, median 10 years). The presenting features were, difficulties with walking and running due to lower limb weakness. The main findings on neurological examination were prominent foot deformities: pes cavus in all and hammertoes in most patients (7/9) with callus formations. Muscle tone was increased in six patients. Power was reduced in the ankle extensors and intrinsic feet muscles in all but one patient. Deep tendon reflexes were preserved, except in individual II:9 whose ankle reflexes were absent following ankle fusion surgery. Plantar responses were extensor in 5/9 patients. Two patients (II:8 and II:9) reported a mild reduction in handgrip, with no other demonstrable upper limb weakness. There were no sensory signs apart from reduced perception of vibration in the feet of four patients. Electrophysiological studies showed normal median motor conduction (range 49–64 m/s), slowed tibial conduction in the older patients (range 37–50 m/s) with normal compound muscle action potentials. Sensory conduction velocities were normal in the median and sural nerves with reduced median sensory nerve action potential (SNAP) in the older patients (range 6–31 mV). Sural SNAP was reduced in one patient (data not shown). MRI was normal in two patients. Sural nerve biopsy in individual II:9 at 43 years was consistent with chronic axonal neuropathy. Using the Harding classification (Harding and Thomas 1980), the disease seen in this family is dHMN type I.
Table 1

Clinical features of available affected individuals from family F-54

Pedigree reference

Onset of symptoms (years)

Pes cavus/hammer toes

Tone

Power knee/ankle/feet

Tendon reflexes knee/ankle

Plantar response

Perception vibration

II.2

20

+/+

Inc

5/3/2

2+/+

E

+

II.4

40

+/+

N

5/4/4

3+/2+

F

+

II.8

10

+/+

Inc

5/4/2

3+/2+

E

II.9

4

+/+

Inc

4/0/2

2+/−

E

III.6

11

+/+

Inc

5/5/5

3+/2+

E

+

III.9

20

+/−

Inc

5/4/3

2+/2+

III.10

10

+/+

N

5/2/4

2+/1+

F

+

III.11

10

+/−

Inc

5/4/4

1+/1+

E

+

III.12

3

+/+

N

5/2/1

2+/2+

F

Foot deformity: + present, − absent. Tone: N normal, Inc increased. Muscle power graded on Medical Research Council scale: 5 normal power, 4 active movement against gravity and resistance, 3 active movement with gravity, 2 active movement without gravity, 1 flicker of movement, 0 complete paralysis. Tendon reflexes: 0 absent, 1 reduced, 2 normal, 3 hyperreflexia, 4 clonus. Plantar response: F flexor, E extensor, − absent. Vibration perception: + normal, − reduced

Genetic analysis

We examined known distal HMN loci on chromosomes 2q14, 7p15, 7q11-q21, 9q34, 11q13 and 12q24 in family F-54 using two-point linkage analysis. This excluded these known loci from playing a pathogenic role in family F-54. Similarly, haplotype analysis among affected individuals excluded the DCTN1 locus on 2p15 (data not shown).

A genome-wide scan for linkage was performed in family F-54. Significant evidence for linkage was obtained at D7S636 on chromosome 7q34-q36 with a LOD score of 3.22 (Table 2). An additional 16 flanking genetic markers were analyzed in F-54 and four of these provided significant evidence of linkage (Table 2), strongly supporting the presence of a novel MND/HMN gene at this locus. Multipoint linkage analysis using linked markers provided a maximum multipoint LOD score of 3.74 at D7S661 (data not shown). Recombinant haplotype analysis among affected individuals established that the novel MND/HMN locus spans 26.2 cM flanked by D7S2513 and D7S637 (Fig. 1). Recombinant haplotype analysis including unaffected individuals >25 years of age suggests that the refined candidate interval spans 14.3 cM flanked by D7S2511 and D7S798 (Fig. 1).
Table 2

Two-point LOD scores between chromosome 7q34-q36 markers and the MND/HMN disease locus

Marker

LOD score at recombination fraction

0.00

0.01

0.05

0.10

0.20

0.30

0.40

D7S1518

−5.09

0.86

1.36

1.41

1.20

0.70

0.25

D7S2513

−0.05

0.04

0.22

0.30

0.30

0.20

0.06

D7S2473

2.99

2.94

2.75

2.51

2.00

1.30

0.62

D7S661

3.73

3.68

3.43

3.11

2.40

1.70

0.78

D7S498

0.58

0.59

0.60

0.57

0.44

0.25

0.08

D7S2511

2.59

2.57

2.47

2.30

1.84

1.26

0.58

D7S688

1.67

1.61

1.40

1.12

0.53

−0.05

−0.33

D7S2426

2.69

2.64

2.46

2.21

1.69

1.11

0.49

D7S505

3.29

3.23

3.00

2.70

2.03

1.29

0.48

D7S636

3.22

3.16

2.95

2.68

2.10

1.40

0.67

D7S2439

1.72

1.70

1.59

1.46

1.16

0.83

0.45

D7S3070

3.59

3.53

3.30

2.99

2.32

1.57

0.74

D7S483

3.29

3.24

3.03

2.76

2.15

1.46

0.69

D7S798

1.76

1.74

1.63

1.49

1.20

0.90

0.46

D7S2462

2.17

2.14

2.00

1.82

1.45

1.02

0.55

D7S2546

2.48

2.49

2.45

2.32

1.90

1.30

0.65

D7S637

−2.23

0.71

1.31

1.45

1.30

1.00

0.47

Significant LOD scores (>3.0) are in bold

Transcript mapping and mutation analysis

We generated a transcript map of the chromosome 7q34-q36 candidate region by using the human genome annotation projects of the Sanger Centre (Ensembl), NCBI (Map Viewer) and UCSC (Genome Browser). The 26.2 cM candidate interval spans 12.3 Mb and the probable refined interval of 14.3 cM spans 6.3 Mb. The 26.2 cM candidate interval contains 250 entries for known or putative protein-coding genes (after excluding alternatively spliced forms of the same gene and mRNAs that show extensive overlap with known genes). The interval encompasses large clusters of genes including those that encode T-cell receptors (86), olfactory receptors (27), and taste receptors (9) as well as trypsin molecules (10). The remaining 113 transcripts in the interval are listed in Supplementary Table 1. We established a preliminary in silico expression profile for the remaining transcripts by determining the source tissues for cDNA clones from which ESTs were derived (through UniGene and dbEST). Most genes are expressed in neural tissues and are therefore candidates on the basis of expression alone. We inspected the interval to identify any obvious candidate genes based upon our knowledge of previously implicated MND genes or disease mechanisms. One gene, CDK5 was selected for immediate mutation analysis based upon its known association with an ALS-like phenotype in mice (Nguyen et al. 2001; Bian et al. 2002). We screened CDK5 for mutations in family F-54 by directly sequencing all exons and flanking intronic regions. No mutations were identified.

Discussion

We used genetic linkage mapping in a family with a phenotype that has been variably described as juvenile ALS and distal HMN, to identify a new MND/HMN locus on chromosome 7q34-q36. Disorders affecting lower motor neurons alone are individually rare; however, collectively they cause significant morbidity in the population (James and Talbot 2006). The increasing evidence for genetic overlap among these disorders and with ALS indicates that shared molecular mechanisms are likely to be at play. Identification of the gene causing the progressive motor neuron degeneration seen in family F-54 may provide insights into other motor neuron disorders including classic ALS.

The disease haplotype seen in all affected individuals in family F-54 defines a 26.2 cM candidate interval. Observed disease chromosome recombination events are also seen in unaffected individuals, implicating a probable candidate interval of 14.3 cM. However, it must be cautioned that these unaffected individuals may be nonpenetrant for the disease, despite their ages being 26 and 55 years of age. We aim to initially focus on the 14.3 cM probable candidate interval using modern positional cloning techniques in order to identify the disease gene. Genes in this interval will be prioritized for mutation analysis based upon their known function and/or expression in affected tissues.

Once identified, the novel MND/HMN gene on chromosome 7q34-q36 can be tested amongst a spectrum of neurodegenerative disorders to determine whether this gene, like other MND genes, is involved in the slowly progressive disorders of motor neurons (e.g., HMN) and rapidly progressive disease (ALS). The gene in question can then also be tested among sporadic ALS cases to determine whether it plays a role as a susceptibility gene. About 90% of ALS cases are sporadic. Some gene variations have been associated with small subsets of sporadic cases; however, it is difficult to prove if these are chance associations or truly causative. A common view is that sporadic ALS may be a multifactorial syndrome caused by combined effects and interaction of susceptibility genes and environmental factors. The best chance of unraveling the causes of MND lies in the identification of genes involved in the familial forms. Already some genes identified from HMN and ALS families (including DCTN1 and VAPB) have also been found to be associated with the disease in sporadic ALS cases (Puls et al. 2003; Munch et al. 2004; Nishimura et al. 2004; Valdmanis et al. 2005). Identification of the various genes involved in these diseases will lead to an unraveling of the biological pathways that cause the selective progressive death of motor neurons.

Increasing evidence points to mutations in different unrelated genes converging on common pathways of neuronal degeneration (Shaw 2005; James and Talbot 2006; Van Den Bosch and Timmerman 2006). The focus of our research should be to identify these common pathways, which once discovered would provide targets for effective drug treatments.

Acknowledgments

We gratefully acknowledge the participation and contribution of patients and family members. We also thank Dr. D. Zhu for technical support and Dr. S. Reddel for assistance with neurological assessment of patients. This study was supported by a research grant (S.G.) and Bill Gole fellowship (I.P.B.) from the Motor Neurone Disease Research Institute of Australia Inc. (MNDRIA).

Supplementary material

Copyright information

© Springer-Verlag 2007