A gene for an autosomal recessive lower motor neuron disease with childhood onset maps to 1p36

Patients.

The patients belong to a large consanguineous family, native of Mali, composed of two healthy parents and five affected and six healthy children age 25 to 9. The 13 members participated in the study, and an informed consent was obtained from each individual. No evidence of neurologic disease was reported in the grandparents or their relatives. Neurologic examination of the two parents and the six healthy siblings was normal.

Individual II-5, a 19-year-old woman, had normal motor development during early childhood and walked at age 1. At age 3, she presented with abnormal walking, frequent falls, and difficulty in climbing stairs. Symptoms rapidly worsened, and at age 5, a waddling gait and a tiptoes walk were noted at clinical examination. The patient was unable to stand up from a sitting position without aid or to raise her arms up to shoulders. She developed scapular and pelvic girdle muscle atrophy with hip bilateral flexor muscle contractures and symmetric winging of scapula, bilateral varus feet with a moderate bilateral achilleus tendon contracture, elbows recurvatum, and cubital deviation of the hands. Facial muscles were preserved (see muscle testing in table 1). Hyperlordosis was present, with a minor scoliosis. At this time, vital respiratory capacity was normal. All deep tendon reflexes were abolished, and the diagnosis of anterior horn cell disease was suspected on neurophysiologic assays, showing muscle denervation with normal motor and sensory nerve conduction velocities. Muscle biopsy showed a pattern of denervation. However, molecular analysis failed to detect any homozygous SMN1 gene deletion in the proband's DNA. Ability to walk was definitively lost at age 7.5. At age 8, she presented with bilateral equinus varus feet, finger flexor muscle contractures, and a severe hyperlordosis with dorsolumbar scoliosis requiring surgical vertebral arthrodesis at age 12. Cranial nerve function was normal; tongue wasting and fasciculations were absent. Respiratory function altered slowly owing to the weakness of both intercostal and diaphragmatic muscles, leading to respiratory assistance by tracheotomy at age 17. At present, she can write and draw taking a pencil with her mouth, and she operates her electric wheel chair by small movements of the head.

For Individual II-7, a 16-year-old girl, first symptoms appeared at age 3. As for her sister (II-5), proximal muscle weakness was the main symptom at the onset of the disease, causing walking disability and frequent falls. A similar progressive evolution was noted during childhood, with the loss of walking at age 8 and the appearance of severe dorsolumbar scoliosis with hyperlordosis. Distal muscle weakness was present early, leading to bilateral equinus varus pes and grasping fingers. Progressive restrictive respiratory insufficiency appeared. Nocturnal nasal ventilation assistance was necessary at age 13 and tracheotomy at age 16. Severe kyphoscoliosis with lumbar hyperlordosis was noted, requiring vertebral surgery at age 13. Electrophysiology and muscle biopsy exhibited a same pattern of denervation as for her sister (II-5). Microscopic analysis of the right sural nerve was strictly normal. All these findings argued for the diagnosis of SMA. However, no homozygous SMN1 gene deletion was identified in Individual II-7's DNA.

Individual II-8 is the monozygotic twin of Patient II-7. She developed a similar disease with proximal muscle weakness beginning at age 3.5, strongly progressive during childhood and causing the loss of walking at age 8.5. Feet muscle impairment was noted early, since age 5, leading to bilateral equinus varus pes with a slight elevation of the internal plantar arch. Hand muscles were more severely affected than for her twin sister. Flexor and extensor muscles were involved, causing total loss of use of the hands since age 12. All osteotendinous reflexes were abolished early on, and muscle denervation was shown on neurophysiologic assays. She presented a severe hyperlordosis and scoliosis, requiring surgery at age 13. Respiratory insufficiency progressively worsened, and nocturnal respiratory assistance was undertaken at age 15. Tracheotomy is now under discussion. As her sisters, she has strictly normal mental development (figure).

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Figure. Individual II-8 at the age of 16 years. (A) Complete paralysis of distal lower limb muscles. Feet are fixed in an equinus varus position. No pes cavus deformity is noted. (B) Complete paralysis of the wrist and hand muscles with contractures of the digitorum flexor muscles, leading to grasping fingers. (C) Proximal muscles paralysis. Sitting position is possible but precarious. Head control is weak. Scoliosis is corrected by the surgical vertebral fusion. Hips are fixed in flexion and totally paralyzed. Note the bilateral amyotrophy of scapular muscles.

Individual II-11, a 9-year-old boy, presented with frequent falls and waddling gait at age 2, although his mother detected axial muscle weakness earlier. Hand and foot muscle weakness was present at the first neurologic examination. At age 4, Gowers sign was positive, walk on tiptoes was not possible, and fine motor movements of the hands were hardly performed. All deep tendon reflexes were abolished, and sensation was normal. Electromyography (EMG) showed a pattern of muscle denervation. Motor nerve conduction velocities were slightly decreased, and sensory potentials were weak but present. Now, he is still able to walk a few steps with aid.

Individual II-4, a 20-year-old man, developed a moderate phenotype by comparison with his affected sibs. He had a normal motor development during infancy. He was able to walk unaided at age 8 months. At age 11.5 years, his sport's instructor reported difficulties for running and climbing stairs. The patient complained about episodes of calf pain after physical efforts. Muscle strength testing revealed a generalized weakness, affecting proximal as well as distal musculature. Hyperlordosis and bilateral winging of scapula were noted at physical examination. All deep tendon reflexes were abolished. Sensation was normal. No pyramidal or cerebellar signs were detected. Tongue fasciculations were absent. EMG showed muscle denervation. Distal sensory potentials were present, and motor and sensory nerve conduction velocities were subnormal (37 and 42 m/s). Diagnosis of Kugelberg–Welander disease was suggested, but no homozygous SMN1 gene deletion was detected by DNA analysis. At present, at age 20, proximal muscular weakness is revealed by hardness for climbing stairs and for walking. Grasping toes and incapacity to walk on heels express distal muscular involvement. No pes cavus deformity is noted. Kyphosis adds to hyperlordosis, without scoliosis. Respiratory autonomy is preserved, but vital capacity is reduced.

Procedure.

Patients were all examined at the Childhood Neuromuscular Disorders Department at Raymond Poincaré Hospital, Garches, France. Muscle strength was measured by manual testing using the Medical Research Council numeric scale (grade 0 to 5)¹⁰. Mean scores per limb region were calculated according to Van den Berg-Vos instructions.¹¹ Standardized electrophysiologic studies were performed at the Departments of Neurophysiology at Raymond Poincaré Hospital, Garches, and Armand Trousseau Hospital, Paris. Neuropathologic analyses were achieved at the Saint Vincent de Paul Hospital, Paris.

Genomic DNA was extracted from peripheral blood samples according to standard procedures. Linkage analyses at the SMN1 and SOD1 loci (chromosomes 5q13 and 21q22) were performed, using four and three microsatellite DNA markers (D5S435, D5F149S1/S2, D5S150S1/S2, D5S351 and D21S2049, D21S1888, D21S261). SMN exon 7 deletion was tested as reported previously.¹² Mutations of the SOD1 gene were investigated in all the probands by direct sequencing of each of the five exons using primers described in references.^13,14

A genome-wide scan was undertaken with 382 pairs of fluorescent oligonucleotides of the Genescan Linkage Mapping Set, Version II (Perkin-Elmer Cetus) under conditions recommended by the manufacturer. The polymorphic markers had an average spacing of 10 cM throughout the genome.

For fine mapping around D1S253, 16 contiguous microsatellite DNA markers were used, mapping to the 1p36 region and spanning a 5-megabase interval in the following order: cen/D1S503–D1S1612–D1S508–D1S2666–D1S548–D1S2694–D1S2663–D1S1646–D1S214–D1S2642–D1S2731–D1S253–D1S2870–D1S2633–D1S2795–D1S2845/tel. These markers were obtained from the Genethon map and ordered from the genome integrated map at the UCSC genome database. PCR amplification of the microsatellite markers was carried out with 100 ng of genomic DNA in a 25-μL total reaction volume containing 0.2 U of Taq polymerase (Invitrogen Life Technology), 1 μM of the 5′- fluorescent labeled forward primer, 1 μM of the reverse primer, 200 μM dNTPs, and 1 × reaction buffer (50 mM KCL, 20 mM Tris-HCl, pH 8.4, 2 mM MgCl₂). Amplification was performed using the following conditions: 94 °C for 5 minutes, followed by 35 cycles at 94 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds, with a final 5-minute extension at 72 °C. PCR products were diluted, denatured at 94 °C during 5 minutes, and loaded on an ABI Prism 3100 Genscan analyzer (Applied Biosystems). For data analysis, Genscan Analysis and Genotyper software were used.

For statistical analyses, two-parametric linkage analyses were performed using the M-LINK and LINKMAP options of the 5.1 version of the LINKAGE program.^15,16 All allele frequencies are available from the CEPH Database.

Mutations of the chromodomain helicase DNA binding protein 5 gene (CHD5) were investigated in proband II-5 by direct sequencing of each of the 41 exons, using the Big Dye Terminator Cycle Sequencing Kit (version 3.1) on a 3100 automated sequencer (ABI Prism; Applied Biosystems, Foster City, CA). Primers were chosen using the Primer 3 software.¹⁷ Data were collected and analyzed with an ABI DNA sequencing analysis software (version 3.4.1; data not shown, available on request)

Clinical study.

Four of the five affected sibs (II-5, II-7, II-8, II-11) developed a severe phenotype, and one (II-4) had a mild phenotype. In the severe phenotype, first symptoms appeared during infancy (2 to 3.5 years), with proximal muscle weakness predominating at the lower limbs and with an early involvement of foot and hand muscles. The disease rapidly worsened, and walking ability was lost at the mean age of 8.5 (7.5 to 9) years (table 2). Paralysis progressively spread to the whole body, sparing the cranial nerves, leading to a generalized areflexive tetraplegia with contractures, severe scoliosis, and hyperlordosis. Respiratory function was impaired by both diaphragmatic and intercostal muscle weakness, and assisted ventilation by tracheotomy was undertaken at the mean age of 16.5 (16 to 17) years. Intelligence was strictly normal, and the disease fulfilled all the clinical, electrophysiologic, and histologic criteria for LMND. Phenotype analysis based on muscle testing is reported in figure E-1 (available on the Neurology Web site at www.neurology.org). The pattern of weakness is a generalized severe progressive SMA with a slight predominance at the pelvic girdle muscles. The “mild phenotype” is characterized by a delayed onset (11.5 years), a moderate generalized weakness, and a slower course.

Genetic results.

Haplotype analyses at the 5q13 and 21q22 chromosomal regions in the genomic DNA of affected siblings showed that the disease was not linked to the SMN1 or the SOD1 loci in this family. In addition, none of the affected individuals carried a homozygous exon 7 deletion in the SMN1 gene, and no mutations were detected in the coding sequence of the SOD1 gene. (data not shown; available on request).

Results of the whole genome scan performed on the genomic DNA of five siblings (II-1, II-3, II-4, II-5, II-7) were analyzed using homozygosity mapping strategy. A bias for homozygous alleles among the affected individuals was observed for 35 of the 382 markers. Subsequently, genomic DNAs from parents and the six remaining siblings were tested for each of these markers and for nearby flanking markers chosen in the Genethon and UCSC Genome Browser maps. Haplotype analyses showed that all affected siblings were homozygous for an unique cosegregating segment of 10 markers in the 1p36 region, consistent with parental consanguinity (figure E-2). Recombination events in Subjects II-2 and II-5 placed the disease locus distal to D1S508 and proximal to D1S2633 on chromosome 1p36, delineating a genetic interval of 3.9 cM. This interval corresponded to a physical distance of 1.5 megabases according to the UCSC Genome database. Pairwise linkage analysis between the disease locus and polymorphic markers in this region gave positive results, and the maximum lod score was obtained at the D1S253 locus (Z_max = 3.79) at the recombination fraction θ = 0.00. These data supported the view that the gene for autosomal recessive childhood-onset LMND reported in this family mapped to the interval defined by loci D1S508 and D1S2633. Sequencing analyses of the CHD5 gene, a strong candidate gene located in the limited interval, failed to detect any mutation.