DOI: 10.1093/brain/awg278 Advanced Access publication September 23, 2003 Brain (2003), 126, 2682±2692 Severe infantile neuropathy with diaphragmatic weakness and its relationship to SMARD1

Matthew Pitt,1 Henry Houlden,2 Jean Jacobs,2 Quen Mok,1 Brian Harding,1 Mary Reilly2 and Robert Surtees1

1Great Ormond Street Hospital for Children and Correspondence to: Dr Matthew Pitt, Department of 2The National Hospital for Neurology and Neurosurgery, Clinical Neurophysiology, Great Ormond Street Hospital London, UK for Children NHS Trust, Great Ormond Street, London WC1N 3JH, UK E-mail [email protected] Downloaded from Summary A group of 13 patients with early onset diaphragmatic cord in one patient failed to ®nd any evidence of an palsy in association with a progressive neuropathy is SMA. Four out of the ®ve not tested genetically were presented. All eight of those tested were found to have positive for all diagnostic criteria. None of the cases of

mutations in the same gene encoding the immunoglobu- early onset neuropathies or spinal muscular atrophies http://brain.oxfordjournals.org/ lin mu-binding protein 2 (IGHMBP2) in patients with with early respiratory failure reviewed in the literature spinal muscular atrophy (SMA) with respiratory dis- shares the exact characteristics, but many do have very tress type 1. Six out of these eight patients had either close similarities. Their classi®cation varies, but the dis- homozygous or compound heterozygous mutations, and covery of mutations in IGHMBP2 in cases that are vari- two had only a single heterozygous mutation. Detailed ously classi®ed as SMA plus or severe infantile analysis of the clinical picture and the neurophysiologi- neuropathy with respiratory distress points to a need cal and histopathological ®ndings indicated that these for the search for this genetic defect to be widened to patients shared similar characteristics, which were fur- include both groups. The fact that we identi®ed other, ther developed as a set of diagnostic criteria. Some of similar cases of neuropathy and early respiratory fail- by guest on September 29, 2015 the most striking of these were early onset of respira- ure with and without IGHMBP2 mutations suggests tory compromise, a markedly low birth weight, very genetic as well as clinical heterogeneity in these infants. slow motor nerve conduction velocities and a general It is possible that infants that do not have mutations in decrease in the size of myelinated ®bres on sural nerve the IGHMBP2 gene will be found to have mutations in biopsy. Extensive histological examination of the spinal a similar functioning gene.

Keywords: infantile neuropathy; diaphragmatic weakness; respiratory insuf®ciency; respiratory paralysis; peripheral nervous system diseases

Abbreviations: CMT = Charcot±Marie±Tooth; IGHMBP2 = immunoglobulin mu-binding protein 2; LLN = lower limit of normal; SIANRF = severe infantile axonal neuropathy with respiratory failure; SMA = spinal muscular atrophy; SMARD1 = SMA with respiratory distress type 1; SMN = survival motor neuron

Introduction Respiratory complications in either hereditary peripheral distal hereditary motor neuropathy type VII (Santoro et al., neuropathy or spinal muscular atrophy (SMA) are usually not 2002). It has not been described in the neonatal period, seen in the neonatal period. Involvement of the phrenic although Dyck et al. (1994) did report that severely affected nerves has been described in adults with Charcot±Marie± members of two kindreds could be affected in infancy or Tooth (CMT) type 1 due to the chromosome 17 duplication, childhood. The positive family history will make the PMP22 and MPZ mutations (Hardie et al., 1990; Tyson et al., diagnosis uncomplicated. A case classi®ed as congenital 1997). The form of autosomal dominant CMT type 2 hypomyelinating neuropathy with diaphragmatic and vocal associated with diaphragmatic palsy and vocal cord paralysis cord paralysis has been reported (Hahn et al., 2001). (CMT 2C) is genetically distinct from CMT 2A, 2B and 2D It was believed previously that involvement of the (Nagamatusu et al., 2000), as well as types CMT 2E, 2F and diaphragm made the diagnosis of SMA untenable (see table 1 Brain Vol. 126 No. 12 ã Guarantors of Brain 2003; all rights reserved Infantile neuropathy with diaphragmatic weakness 2683

Table 1 Neurophysiology results Case Age Duration Median nerve Ulnar motor Medial popliteal Phrenic nerve latency EMG tibialis EMG tested of illness action potential velocity amplitude (mV) (ms) anterior diaphragm (weeks) (days) amplitude (mV) (ms) velocity (m/s) velocity (m/s) Left Right

1 13 34 31.2 21* 0.5* 11.0* 6.0 Fibs, large units 22* 14** 2 6 16 11.2 24 0.2* Absent Discrete IP 20.0* 11** 3 10 30 12.5 17** Absent 5.1 4.1 Fibs, discrete IP 25* 4 13 24 7.0* Not done 0.45* Not done Severely reduced 25* Not done IP 5 26 150 2.0* 24* 0.2* Absent Discrete IP 16** Absent 6 6 11 Absent 25* Absent Not done Fibs, discrete IP

7 39 190 9.1* 23** Absent Not done Severely reduced Downloaded from 26** Absent IP 8162811 22* 0.16* No response Discrete IP 26* 16** (technical?) 9 36 180 Absent Absent Absent Not Severely reduced No attempted IP discernible

activity http://brain.oxfordjournals.org/ 10 10 70 Absent 22* 0.04* 5.8 6.4 Discrete IP + Discrete IP + 11** neurogenic units neurogenic units 11 15 42 Absent 19** Absent Absent Absent Discrete IP + Scattered neurogenic units units with high ®ring rate 12 10 70 Absent Not tested 2.1 Not tested Reduced IP + Reduced IP + 15** neurogenic units neurogenic

units by guest on September 29, 2015 13 16 21 Absent 33 1.57* Absent Absent Fibs only 26*

For sensory and motor conduction velocities, * = mildly abnormal < lower limit of normal range (LLN) but > 70% LLN; ** = severely abnormal <70% of LLN. For amplitudes of motor and sensory responses, * = abnormal; ®bs = ®brillation potentials; IP = interference pattern. For phrenic nerve latency, * = above upper limit of normal.

Rudnik-Schoneborn et al., 1996). Later, the 35th European decrease in the size of myelinated ®bres with minimal Neuromuscular Centre workshop on SMA (1996) recognized evidence of axonal degeneration and no indication of that involvement of the diaphragm can occur in SMA, demyelination. The prognosis was very poor. Our initial although the ®rst report was made in 1977 (Kyllerman, 1977). cohort of six patients, presented previously in abstract form SMA with diaphragmatic involvement is now classi®ed under (Pitt et al., 1998), has now increased to 13 cases, mostly the SMA plus types and has been shown not to be associated unrelated. We have been unable to ®nd convincing evidence with the deletion of the survival motor neuron (SMN) gene on to classify them as SMA and were interested to determine chromosome 5 (Zerres and Davies, 1999). whether they share the genetic abnormalities of the patients Grohmann et al. (2001) studied 11 patients with a clinical studied by Grohmann et al. (2001). The current paper diagnosis of SMA with respiratory distress type 1 (SMARD1) presents these results, along with detailed analysis of clinical, and 21 of their relatives from a total of six families. They electrophysiological and histopathological ®ndings. found mutations in the gene encoding immunoglobulin mu- binding protein 2 (IGHMBP2). An animal model with early onset neuromuscular degeneration, the neuromuscular dis- order mouse, also has mutations in the IGHMBP2 gene, and Case report Grohmann et al. (2001) suggest that this mutation is the cause Case 1 of SMARD1 in humans. This boy, the ®rst child of healthy unrelated parents, was born Our group has been interested in a series of neonates who at term by emergency Caesarean section, and weighed 2.06 presented with severe respiratory distress in the ®rst few kg. He presented at 8 days of age, with a history of poor months of life. They had diaphragmatic palsy and evidence of feeding requiring nasogastric tube feeding. Four days later he an axonal neuropathy. Sural nerve biopsies showed a general developed signs of respiratory distress. A chest X-ray showed 2684 M. Pitt et al.

Table 2 Quantitative neuropathology results Case Age Fascicular Myelinated Mean ®bre Mean axon Unmyelinated Mean (months) area (mm2) ®bre diameter diameter axon unmyelinated density/mm2 (mm) (mm) density/mm2 axon diameter (mm)

1 3 0.15 23 730 3.64 2.92 136 300 0.9 2 2 0.18 19 030 4.07 2.78 ± ± 3 2 0.15 24 680 3.29 2.58 130 110 1.0 4 3 0.19 22 760 3.54 2.51 129 800 1.0 5 5 0.29 23 170 3.46 2.56 101 420 0.9 6 3 0.24 21 630 3.85 2.91 ± ± 7 10 0.28 17 704 3.96 3.06 8 3.5 21 211 9 6 0.19 17 285 10 3 0.22 20 862 3.06 2.41 11 3 23 252

12 3 10 640 3.97 Downloaded from 13 5 15 755 4.6 3.6 Controls, 0±2 0.23 6 0.03, 23 480 6 3800, 4.5 6 0.2, 3.4 6 0.2, 149 000, 0.9, mean 6 SD n =4 n =4 n =3 n =3 n =2 n =2

bilateral diffuse shadowing. By 11 weeks of age he remained Children between 1989 and 2001 with early onset respiratory http://brain.oxfordjournals.org/ persistently tachypnoeic, was failing to thrive and was distress. The majority (11 out of 13) were seen between 1996 referred for further investigation. and 2001. Two were sisters, one seen in 1989 (Case 4) and the On admission, his weight was below the 10th centile, he second in 2001 (Case 12). Case 7 had a 6-year-old sister who had grunting respiration and had only a weak cry. He was was being ventilated in Bahrain with a presumed diagnosis of tachypnoeic, with paradoxical movement of his chest and SMA type 1 with a deletion in the SMN gene. Case 13 had a abdomen, and required oxygen by headbox to maintain brother with a histopathological diagnosis of CMT type 1 but normal arterial oxygen saturation. He had anti-gravity without genetic con®rmation. movement of his limbs, but his deep tendon re¯exes were Ethics approval was obtained from the joint medical and by guest on September 29, 2015 depressed. Twenty-four hours after admission, his condition ethics committee at Great Ormond Street Hospital and The deteriorated and required ventilatory support. Chest radio- National Hospital for Neurology and Neurosurgery to carry graph showed a raised central portion of his diaphragm and, out this clinical and genetic study. on screening, there was paradoxical movement of the left hemidiaphragm with little movement of the right, medial lea¯et. Results of neurophysiological investigations and Neurophysiological methods nerve biopsies are shown in Tables 1 and 2. Nerve conduction studies and EMGs were performed on a Other investigations, including sweat test, white cell Dantec Counterpoint EMG machine or a Medelec MS6 using enzymes, creatine kinase, SMN gene deletion, ECG and CT techniques as described by Payan (1997). Needle EMG was head scan, were either normal or negative. performed using a ®ne concentric needle electrode (needle He required continuous positive airways pressure support diameter 0.3 mm, recording area 0.019 mm2). Phrenic nerve and underwent plication of the diaphragm. At surgery, the stimulation was performed using the technique of Markand diaphragm was noted to be thin and membranous. Despite et al. (1984). The technique of Bolton et al. (1992) was used having had his diaphragm plicated, he remained ventilator for needle EMG of the diaphragm. dependent. Over the next month, he developed progressive Normal values for nerve conduction velocities were taken distal weakness and lost his ankle tendon re¯exes. from Raimbault (1988). The lower limit of the amplitude of At 4 months of age, his distal limb muscles were weak and the median nerve action potential, from wrist to elbow, was wasted, without anti-gravity power; proximal limb muscles 10 mV, and the medial popliteal compound muscle action were affected to a less severe extent. After discussion with his potential from abductor hallucis was 2.0 mV. These were parents, ventilatory support was withdrawn and he died. calculated from the values in `presumed normal' subjects Permission for post-mortem was declined. who had been examined in the department over the last 10 years. The phrenic nerve latencies were obtained in the same way and showed a marked decline with age, the upper limit at Patients and methods 3 months being ~7.8 ms, falling to ~5 ms at 20 months. Patients Chronic denervation with reinnervation was diagnosed All 13 patients (9 males and 4 females) presented to the when a reduced interference pattern was seen with large intensive care units of Great Ormond Street Hospital for amplitude, high-®ring polyphasic units and long duration Infantile neuropathy with diaphragmatic weakness 2685

Table 3 Clinical and radiological ®ndings Case Birth Gestation Presenting symptoms (age) Onset to Neurological signs Diaph Age at weight (week) ventilation at presentation X-ray death (months) (kg)

1 2.06 37 Poor feeding (8 days), failure 28 days Reduced re¯exes R&L 4 to thrive 2 2.9 40 Respiratory arrest (1.25 4 days No wasting, R3 months) hypotonia or fasiculation 3 1.8 40 Respiratory failure (28 days) 1 days Not recorded R & L Died abroad 4 2.33 40 Feeding dif®culty (2.5 months) 7 days Hypotonia only R & L 4 5 1.89 40 Respiratory tract infection (2.5 days None R & L Died months) abroad 6 2.83 42 Tachypnoea and feeding 7 weeks None R 3 dif®culty (1 week)

7 * 40 Hypotonia (3±4 months) Never Hypotonia with Normal Died abroad Downloaded from ventilated proximal wasting 8 1.98 40 Feeding dif®culty (2.75 months) 1 weeks None R 6.5 9 1.16 35 Feeding dif®culty (3 months) 2 months Distal weakness R & L 9.5 with wasting 10 2.3 38 Respiratory distress (1 month) Immediate Hyptonia only R 3

11 * 40 Respiratory failure (2 months) Immediate None R & L 4 http://brain.oxfordjournals.org/ 12 1.5 35 Apnoeas (2 months) 1 week Hypotonia R 3 13 3.2 40 Failure to thrive, stridor, Immediate Talipes and R & L Alive, hypotonia and feeding hypotonia ventilator dif®culty (4 months) dependent

Diaph X-ray = the presence of a raised hemidiaphragm with or without con®rmation of paradoxical movement on ultrasound. *Birth weights not available.

units. Acute denervation required the presence of ®brillations eyepiece graticule. Fascicular areas were measured using a by guest on September 29, 2015 in a signi®cant number of sites in the muscle. The nerve microscope with an extension tube positioned over the bitpad conduction velocities were recorded as severely abnormal if of a Minimop image analyser. A cursor with a light-emitting they were below 70% of the lower limit of normal (LLN) diode, visible in the microscope, was used to trace round the (modi®ed by the Ad Hoc Committee of the American inner edge of the perineurium of each fascicle. The density of Association of Neurology recommended criteria for demye- myelinated ®bres was calculated. lination; Ad Hoc Subcommittee, 1991) and mildly abnormal if above this level but still below the LLN. The skin temperature was above 30°C in all cases. Unmyelinated axon density Unmyelinated axons were counted in electron micrographs Histopathological methods enlarged to about 310 000 (calibrated with a grating replica) and covering an area of ~0.01 mm2 in each nerve. Sural nerve biopsies, as well as sural and mixed nerves Unmyelinated axons were distinguished from Schwann cell (popliteal or peroneal) taken post-mortem, were ®xed in 3% processes by their circular or near-circular outline, their paler glutaraldehyde in 0.05 M cacodylate buffer. After overnight cytoplasm often with numerous microtubules and ®laments ®xation, 1-mm-thick slices were cut for processing and and by the absence of ribosomes. embedding in resin for semithin and ultrathin sections; the remaining length was processed into unpolymerized resin for later separation of nerve ®bres (teasing) in complete resin. Quantitative methods on sural nerve biopsies were as follows: Myelinated ®bre diameter Representative ®elds from at least two fascicles were photographed and enlarged to just under 33000, the exact Myelinated ®bre density from 1 mm sections magni®cation being obtained by calibration. Using the These methods are similar to those described in a previous Minimop, areas enclosed by the inner and outer perimeters paper (Jacobs and Love, 1985). Brie¯y, at least 2000 of the myelin sheath were traced and the data stored in an myelinated ®bres from several fascicles were counted at the interfaced computer. Fibre and axon diameters were derived microscope at a magni®cation of 3250 using a squared from area measurements assuming circularity, and g-ratios 2686 M. Pitt et al. Table 4 SMARD1 mutations Case Mutation type Mutation 1 Mutation 2

1 Compound heterozygous Exon 8. Leu 361 Pro, base 1082 ctg ccg Exon 10. Cys 496 Stop, base 1488 tgc to tga 2 Single heterozygote Exon 5. Del A base 661 3 Not tested 4 Not tested 5 Not tested 6 Single heterozygote Exon 7. Del AA base 983/984 7 Homozygous Exon 10. Del T base 1463 8 Compound heterozygous Exon 8. Leu 361 Pro, base 1082 ctg ccg Exon 10. Cys496 Stop, base 1488 tgc to tga 9 Compound heterozygous Exon 2. Arg 43 Stop, base 127 cga to tga Exon 10. Cys 496 Stop, base 1488 tgc to tga 10 Not tested 11 Not tested 12 Compound heterozygous Exon 7. Arg 320 Stop, base 958 cga to tga Exon 10. Cys 496 Stop, base 1488 tgc to tga 13 Compound heterozygous Exon 7. Arg 320 Stop, base 958 cga to tga Exon 8. Leu 361 Pro, base 1082 ctg ccg Downloaded from

(axon:®bre diameter) were calculated. At least 400 ®bres out in a total volume of 50 ml, which contained 20 ng of DNA, were measured in each nerve. 0.2 mM dNTPs, 1 unit of TaqGold polymerase, 1.5 mM

MgCl2, 75 mM Tris±HCl pH 9.0, 20 mM (NH4)2SO4, 0.01% Tween 20 and 50 pmol of each primer. PCR was performed http://brain.oxfordjournals.org/ Unmyelinated axon diameter on a Perkin-Elmer 9700 thermal Cycler (Perkin Elmer, The perimeter of unmyelinated axons was traced on Applied Biosystems, Foster City, CA). The cycling consisted micrographs at about 37000, accurately calibrated, using of denaturation at 94°C for 15 min, followed by 25 cycles of the Minimop; diameters were calculated from axonal area 94°C for 30 s, 60±50°C touching down protocol for 30 s and assuming axonal circularity. At least 400 axons were 72°C for 30 s. After that, 12 cycles of constant annealing measured in each nerve. temperature at 50°C and a ®nal ampli®cation at 72°C for 10 min was carried out. PCR fragments were checked on a 1%

agarose gel. The PCR products were puri®ed by using a by guest on September 29, 2015 Brain, cord and muscle biopsies Qiaquick puri®cation kit (Qiagen, Hilden, Germany) and resuspended in 50 ml of deionized water. For each exon, 100 Where autopsy was carried out, brains and spinal cords were ng of ampli®ed product was sequenced using forward and immersion ®xed in formalin for a minimum of 1 week, then reverse primers and a BigDye Terminator cycle sequencing blocked for paraf®n processing and histological examination kit (Perkin-Elmer). Sequencing was performed on an ABI377 utilizing conventional staining with haematoxylin±eosin, automated sequencer. Sequence alignment and analysis was luxol fast blue±cresyl violet and immunohistochemistry for carried out with Sequence Navigator (Perkin-Elmer). glial ®brillary acidic protein (GFAP). Muscle biopsies were oriented and snap frozen for cryostat sectioning at 8 mm. The sections were stained with Mutation and polymorphism analysis haematoxylin±eosin, the modi®ed Gomori trichrome stain, In addition to the pathogenic mutations identi®ed in the for fat, glycogen and mitochondrial enzyme activity and to IGHMBP2 gene, a number of single nucleotides polymorph- demonstrate ®bre types with an ATPase method with and isms (SNPs) were identi®ed throughout the gene. The without acid preincubation. mutations were characterized in families and controls by re- sequencing affected individuals and sequencing 50 unaf- fected control patients (Table 3). This allowed clear differ- Genetics methods entiation between the presence or absence of the mutation or Genetic sequencing SNPs. A number of nucleotide repeats were identi®ed within DNA was extracted by standard phenol/chloroform extraction the introns of the IGHMBP2 gene. None of the pathogenic method, a routine automated technique carried out in the mutations identi®ed in Table 3 was found in controls. genetics laboratory from blood or muscle biopsy samples. The deletion in the SMN gene was excluded in all families, as type I SMA was a differential in all the cases. The 15 exons and ¯anking intronic regions of the Results IGHMBP2 gene were ampli®ed by the PCR using oligonu- Patient details cleotide primers purchased commercially (Invitrogen). Details of patients are shown in Table 3. No case was Primers have been described previously. PCR was carried symptomatic at birth, except Case 3, who had grunting Infantile neuropathy with diaphragmatic weakness 2687

Fig. 1 Resin sections (1 mm) of sural nerve. (A) Case 5 aged 5 months. Scale bar = 25 mm. (B) Control nerve from 9-week-old infant, showing the difference in myelinated ®bre size. Downloaded from respirations. Decisions to withdraw treatment or transfer prevented detailed follow-up in most cases. Where serial neurological observations were made (Cases 1, 2, 6 and 8), Fig. 2 Motor nerve fascicle from popliteal nerve (post-mortem) weakness and subsequent wasting developed over a period of http://brain.oxfordjournals.org/ from Case 2, showing a reduced density of myelinated ®bres. weeks and showed a distal to proximal progression. Scale bar = 25 mm.

Neurophysiology evidence of occasional myelinated ®bre degeneration. In Results of neurophysiological investigations are shown in Case 12, there was frequent evidence of ®bre degeneration. Table 1. The most severe abnormalities were markedly There was no evidence of loss of unmyelinated axons. The reduced motor conduction velocities, particularly in the legs, ultrastructure of axons of myelinated and unmyelinated axons and a very marked reduction or loss of the compound muscle appeared normal: in particular, neuro®laments seemed to be by guest on September 29, 2015 action potential. Studies of the sensory ®bres showed similar present at normal densities. In mixed nerves taken post- abnormalities of conduction velocity and compound action mortem (Cases 2, 4 and 6), the motor components were potentials, but of a much milder type. The EMG in each case identi®ed from the larger size of the motor ®bres. In resin revealed a denervation pattern only in distal muscles initially. sections, there was some evidence of ®bre degeneration in An EMG of the diaphragm was made in four cases (9±12) and these motor ®bres; Fig. 2 shows a reduced density of con®rmed the presence of denervation in three. This was myelinated motor ®bres. Electron microscopy con®rmed the con®rmed by post-mortem biopsy in Case 10. presence of numerous Bands of Bungner in such regions, indicating earlier myelinated ®bre degeneration.

Histopathology Nerve biopsy results Quantitative ®ndings in sural nerve biopsies Qualitative ®ndings Control data were obtained from sural nerves of four infants Sural nerve biopsies from Cases 1±11 showed a similar aged 0 and 3 days, and 3 and 9 weeks, removed within 24 h of appearance, compared with age-matched control sural nerves. death from cases without a history of peripheral nerve disease The myelinated ®bres were small (Fig. 1) .There was no (Jacobs and Love, 1985). Inclusion of controls rather younger evidence in 1 mm resin sections of ®bre degeneration, than our patients emphasizes the marked difference between regeneration or demyelination. In a few cases, a very the two groups. Table 2 summarizes the quantitative ®ndings. occasional degenerating ®bres was seen. There was no Fascicular areas: fascicular areas were generally smaller evidence of demyelination or remyelination in any of the than those of the controls, and this is probably explained by teased nerves. Cases 12 and 13 showed different features: in the smaller size of the ®bres. Case 12, there was signi®cant evidence of degeneration Myelinated ®bre density: there were no obvious differ- re¯ected in the reduced myelinated ®bre density (Table 2); ences between myelinated ®bre densities in control sural and in Case 13, the myelinated ®bre size spectrum was nerves and nerve biopsies from Cases 1±11. The reduced normal, with only very occasional evidence of ®bre degen- density in Case 12 is associated with more marked evidence eration. of degeneration of myelinated ®bres. Electron microscopy of Cases 1±11 and 13 revealed a few Myelinated ®bre size: it is justi®able to use the mean ®bre denervated Schwann cell processes (Bands of Bungner), diameter measurement for purposes of comparison when 2688 M. Pitt et al. Downloaded from

Fig. 3 Histogram of myelinated ®bre diameters from case 3 (diagonal hatching) and controls (chequerboard hatching). http://brain.oxfordjournals.org/

there is no evidence of ®bre loss as in Cases 1±11. Table 2 reported as consistent with chronic denervation, and vastus shows signi®cantly reduced mean ®bre diameters in all lateralis in three, all of which showed non-speci®c changes. except Case 13. There is a similar signi®cant reduction in size The exact site was not recorded in four examinations and the of the axons of these myelinated ®bres, again excepting Case conclusions made were as follows: possibly neurogenic, 13. In histograms, both the myelinated ®bre diameter size denervation of unusual type, chronic denervation and acute

spectrum (Fig. 3) and their axon diameters show a marked denervation. by guest on September 29, 2015 shift to the left compared with the control nerves. Myelin sheath thickness: the slope of the regression line relating myelin sheath thickness and ®bre diameter was Genetic analysis compared between controls and Cases 1±6. Mean values of Genetic analysis was carried out on eight cases, and the 0.108 6 0.022, (n = 5, controls) and 0.103 6 0.022 (n =6, results are shown in Table 4. As can be seen, ®ve out of the patients) were not different statistically (Levenes test for eight cases have compound heterozygous mutations, Case 7 equality of variances, F = 0.009, P = 0.926), con®rming that has a homozygous deletion and Cases 2 and 6 has a single the myelin sheaths were of appropriate thickness for the size heterozygous deletion. Cases 2 and 6 may have an uniden- of axon. ti®ed deletion involving one or more exons or a cryptic splice Unmyelinated axon density: these are within normal limits site change, or they may have a mutation in another gene in Cases 1, 3, 4 and 5. Mean unmyelinated axon diameters acting in parallel to the IGHMBP2 gene. The pathogenicity of from controls and these cases were similar. single heterozygous changes is not proven in this context, although the change was not present in 50 British controls. Case 7 is from Bahrain; ethnically matched controls were not Brain, cord and muscle biopsy results available, but this change was not present in 50 British, ®ve Examination of serial sections of the spinal cords of Cases 4 Pakistani, four Indian and one Iranian control. The mutations and 6 showed no abnormality, in particular no loss or changes in exons 2, 7, 8 and 10 were not identi®ed in 50 British in anterior horn cells or accumulation of microglia (Fig. 4). controls; detailed ethnicity was not available in these cases, Dorsal root ganglia from cervical, thoracic and lumbar but they were clinically assessed in the UK. The position of regions showed no abnormalities. Diaphragmatic muscle the mutations on the IGHMBP2 gene are shown in Fig. 5, showed denervation in Case 10 but no abnormality in Case 6, which also shows the mutations identi®ed by Grohmann et al. although the intercostal muscles were denervated. (2001). Ten of the patients had muscle biopsies. Case 6 had two, the ®rst in vastus lateralis, which showed no diagnostic features, the second in medial gastrocnemius, which showed Proposed diagnostic criteria chronic denervation. The site of the biopsy was medial The results of our investigations suggest that the cases shared gastrocnemius in four cases, all of which showed changes common clinical, neurophysiological and histopathological Infantile neuropathy with diaphragmatic weakness 2689 Downloaded from http://brain.oxfordjournals.org/ Fig. 4 Anterior horn cell at C5 level from Case 6, showing a normal complement of anterior horn cells. (Luxol fast blue Nissl X170). by guest on September 29, 2015

Fig. 5 Intron/exon structure and size in the IGHMBP2 gene. Mutations identi®ed by Grohmann et al. (2001) indicated by arrows and number in each exon. Mutations described in the current study are indicated by arrowheads.

characteristics, which might perhaps allow for their more sheath thickness is found to be appropriate for axon size, the accurate diagnosis and also distinguish them from other ®bre size change would have originated from the axon); (ii) reported cases. We judged the cases against clinical, minimal evidence of ongoing myelinated ®bre degeneration histological and neurophysiological criteria. The criteria in biopsies taken up to 3±4 months; and (iii) no evidence of chosen are shown below. regeneration or of demyelination which might account for the change in ®bre size. Clinical criteria The clinical criteria were (i) low birth weight below the 3rd centile; (ii) onset of symptoms within the ®rst 3 months; (iii) EMG criteria diaphragmatic weakness either unilaterally or bilaterally; (iv) EMG criteria were ful®lled if, in any of the investigations ventilator dependence within less than one month of onset performed, there was (i) evidence of distal denervation acute with an inability to wean; and (v) absence of other or chronic (ii) evidence of severe slowing (<70% of LLN) in dysmorphology or other conditions. one or more nerves (motor or sensory). The Venn diagram (Fig. 6) shows the criteria that the cases Histopathological criteria met, with the reasons for the cases not ful®lling a particular The histopathological criteria were (i) a shift to the left of criterion and also whether the cases were tested for the myelinated ®bre size in sural nerve biopsies (since myelin mutations. 2690 M. Pitt et al. Downloaded from

Fig. 6 Venn diagram of the cases identi®ed by their case numbers, categorized by their assessment http://brain.oxfordjournals.org/ against the three sets of criteria. The cases within shaded boxes are those tested genetically (Cases 1, 2, 6±9, 12 and 13).

they were reclassi®ed as a severe infantile polyneuropathy. Discussion Grohmann et al. (2001) regard autosomal recessive SMARD1 All of the eight cases tested showed mutations in the gene as synonymous with SIANRF (Wilmshurst et al., 2001). The encoding IGHMBP2 located on chromosome 11q13.2-q13.4. criteria presented here may help clinicians to identify similar

In two cases, only a single heterozygous mutation was found; cases. by guest on September 29, 2015 and even though these mutations were not found in controls, There are 39 cases in the literature [Mellins et al. (1974), the signi®cance of these single changes are uncertain, as the two cases; McWilliam et al. (1985), four cases; Schapira and pathogenicity of these mutations is not proven in the absence Swash (1985), two cases; Bertini et al. (1989), ®ve cases; of a mutation on the other allele. Four out of the remaining Appleton et al. (1994), one case; Novelli et al. (1995), two ®ve cases who were not tested ful®lled all the clinical, cases; Grohmann et al. (1999), nine cases; Geller et al. physiological and pathological criteria for classi®cation, and (2000), three cases; Mercuri et al. (2000), one case; the other, Case 4, was the elder sibling of Case 12, who was Grohmann et al. (2001), three new cases in addition to positive. Although not possible to prove, it would be those reported elsewhere; Mohan et al. (2001), two cases; and anticipated that they might also have tested positive. The Wilmshurst et al. (2001), ®ve cases]. Sex was given in only location of the mutation positions was rarely the same in our 26 cases (14 males and 12 females). Thirteen out of the 29 group compared with those of Grohmann et al. (2001). All the with family history were positive. Only six out of the 20 with identi®ed mutations were likely to be pathogenic, except complete clinical details ful®lled all criteria, but the birth- perhaps for the two cases with single heterozygous mutations weight was low in 12. Seven out of the 17 with as discussed above: they either changed a conserved amino neurophysiological details ful®lled our criteria. Often the acid, caused a mutation that produces a stop codon or caused slowing in nerve conduction velocity was not as severe as we a deletion that resulted in a truncated protein. None of the had seen. The cases that were closest to ours were Mellins mutations was identi®ed in controls. et al. (1974), case 1; Bertini et al. (1989), case 3; Appleton Infants and neonates who present with severe respiratory et al. (1994), single case; Geller et al. (2000), case 3; and distress from diaphragmatic palsy have been described. They Wilmshurst et al. (2001), cases 1 and 5. All had low birth are de®ned as either diaphragmatic SMA (Zerres and Davies, weight and six out of the seven remaining criteria. 1999) or severe infantile axonal neuropathy with respiratory Insuf®cient data prevented the same analysis of the failure (SIANRF) (Wilmshurst et al., 2001). Some have slow histological ®ndings. The sural nerve biopsy from conduction velocities and many do very badly, either dying Appleton's case (Appleton et al., 1994) was reviewed in early or remaining ventilator dependent. We initially felt that our laboratory and found to have the same changes as the our patients had a distal SMA, but the spinal cord was entirely majority of our group. Most importantly, the sural nerve normal histologically in Case 6 and consistent sural nerve biopsy ®ndings were not speci®c. Identical changes were changes were seen in all cases. It seemed more appropriate found in ®ve other patients, four of whom had tested negative Infantile neuropathy with diaphragmatic weakness 2691 for mutations in the IGHMBP2 gene (one was not tested). Although it is well recognized that some slowing may These four patients presented at birth or soon afterwards and occur in the classical SMA patients, slowing to <70% of the needed immediate ventilatory support. Only one had a low LLN remains an exclusion criterion. The combination of the birth weight, but also had complicated heart disease and was slow motor nerve conduction velocity and severe distal dysmorphic, and was diagnosed as having had an axonal denervation is unusual and was a signature of the condition. It sensorimotor neuropathy. Of the others, one case had grade II was not possible to make the same correlation between the intraventricular haemorrhage, de®ciency of the anterior ribs motor nerve conduction studies and the histopathology of but no diaphragmatic abnormality and the EMG was motor nerves. suggestive of a neuromuscular junction abnormality. The impression was not one of loss of nerve ®bres with the Another baby was clearly dysmorphic and had micrognathia accompanying changes of axonal degeneration, but rather and was thought to have a severe SMA on nerve conduction that the nerve ®bres had not developed. Myelinated axons did studies. The ®nal infant had no other somatic abnormalities not attain their expected size for age. Axon calibre is but had neurophysiological changes of a sensorimotor associated with neuro®lament density, which in the sural neuropathy. The diaphragm was not involved in any case nerves appeared normal; the total number of neuro®laments and none showed the characteristic severe slowing of motor produced is therefore less than normal. IGHMBP2 colocalizes nerve conduction. Therefore, the condition on histopatholo- with RNA-processing machinery in cytoplasm and nucleus, Downloaded from gical criteria is heterogeneous but no information about its speci®c effects is available. The recent case described by Hahn et al. (2001), the ®rst to However, the fact that there were similar sural nerve ®ndings report phrenic nerve involvement in a congenital hypomye- in other cases that were clinically and genetically distinct may linating neuropathy, may be another case, as it has many of suggest that the IGHMBP2 gene is not involved in the axon the characteristics of our cases, including low birth weight, changes. It is possible that the sural nerve changes simply http://brain.oxfordjournals.org/ absence of large diameter myelinated ®bres and distal re¯ect an early onset neuromuscular disorder and are not denervation. Interpretation of a neuropathy as hypomyelinat- speci®c for any particular cause. The changes seen in the ing may be dif®cult in a nerve biopsy taken at the age of 1 child with abnormalities suggesting a neuromuscular junction month, when myelination is still incomplete. Quantitative abnormality are otherwise impossible to reconcile. comparison with age-matched controls is required to con®rm With the genetic studies presented, it is unlikely our cases the degree of myelination. are examples of the infantile neuropathy initially described by It seems likely that SMARD1/SIANRF are descriptions of Ouvrier et al. (1981) and to which more cases have been a condition with variable clinical expression, of which a added by Gabreels-Festen et al. (1991). Our cases are also by guest on September 29, 2015 proportion will have mutations in the IGHMBP2 gene. The different in their clinical, genetic and morphological aspects recent linkage study of Viollet et al. (2002) in a family with a from autosomal dominant CMT type 2, which also show a SMARD phenotype that was negative for the IGHMBP2 gene distinct loss of the largest diameter myelinated ®bres with a identi®ed a locus close to SMARD1 on 11q. Its is likely that shift in the histogram to the left. Regenerative features are families that are negative for IGHMBP2 will have a defect at almost absent. With the identi®cation of IGHMBP2 muta- this locus that is likely to affect a gene functionally similar to tions, it will be interesting to screen all cases presenting with IGHMBP2. similar clinical and neurophysiological ®ndings, irrespective The differing pathological involvement reported in the of whether they are considered a neuropathy or an SMA. This peripheral nerves and spinal cords may have a parallel in will help further de®ne the phenotype and also allow severe congenital SMA (Zerres and Davies, 1999). On examination to see whether there is a phenotype/genotype molecular analysis, a large deletion including the SMN effect explaining some of the features. Given the clinical and telemeric and NAIP genes is seen in most families with this genetic heterogeneity in this patient group, identi®cation of condition linking them unequivocally to SMA 5q. Yet, on further genes will also help us to de®ne a genetic classi®ca- neurophysiological testing, there is a lack of sensory and tion. motor nerve excitability. Moreover, the sural nerve biopsy shows typical signs of axonal degeneration. The histological examination of the spinal cord may not show a marked reduction in the number of motor neurones, despite most References severe clinical manifestations. Ad Hoc Subcommittee of the American Academy of Neurology AIDS Task Force. Research criteria for diagnosis of chronic The absence of the largest diameter axons in the sural nerve in¯ammatory demyelinating polyneuropathy (CIDP). Neurology biopsy explained the low velocities in sensory nerves. The 1991; 41: 617±18. velocity decrease on nerve conduction studies was so slow that the initial impression was of demyelination as the Appleton R, Riordan A, Tedman B, MacKenzie J, Helliwell T. underlying pathology. When demyelination is encountered in Congenital peripheral neuropathy presenting as apnoea and the neonatal period, it is most unusual to obtain sensory respiratory insuf®ciency. Dev Med Child Neurol 1994; 36: 545±53. responses, which in these cases could often be found. High Bertini E, Gadisseux JL, Palmieri G, Ricci E, Di Capua M, Ferriere stimulation intensities are also needed (Bolton, 1996). G, et al. Distal infantile spinal muscular atrophy associated with 2692 M. Pitt et al. paralysis of the diaphragm: a variant of infantile spinal muscular Mohan U, Misra VP, Britto J, Muntoni F, King RHM, Thomas PK. atrophy. Am J Med Genet 1989; 33: 328±35. Inherited early onset severe axonal polyneuropathy with respiratory failure and autonomic involvement. Neuromuscul Disord 2001; 11: Bolton CF. Polyneuropathies. In: Jones HR, Bolton CF, Harper CM, 395±9. editors. Paediatric clinical electromyography. Philadelphia: Lippincott-Raven; 1996. p. 259. Nagamatusu M, Jenkins RB, Schaid DJ, Klein DM, Dyck PJ. Bolton CF, Grand'Maison F, Parkes A, Shkrum M. Needle Hereditary motor and sensory neuropathy type 2 is genetically electromyography of the diaphragm. Muscle Nerve 1992; 15: distinct from types 2B and 2D. Arch Neurol 2000; 57: 669±72. 678±81. Novelli G, Capon F, Tamisari L, Grandi E, Angelini C, Guerrini Dyck PJ, Litchy WJ, Minnerath S, Bird TD, Chance PF, Schaid DJ, P, et al. Neonatal spinal muscular atrophy with diaphragmatic et al. Hereditary motor and sensory neuropathy with diaphragm and paralysis is unlinked to 5q11.2-q13. J Med Genet 1995; 32: vocal cord paresis. Ann Neurol 1994; 35: 608±15. 216±19. Gabreels-Festen AA, Joosten EM, Gabreels FJ, Jennekens FG, Ouvrier RA, McLeod JG, Morgan GJ, Wise GA, Conchin TE. Gooskens RH, Stegeman DF. Hereditary motor and sensory Hereditary motor and sensory neuropathy of neuronal type with neuropathy of neuronal type with onset in early childhood. Brain onset in early childhood. 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Mercuri E, Goodwin F, Sewry C, Dubowitz V, Muntoni F. Received March 27, 2003. Revised June 25, 2003. Diaphragmatic spinal muscular atrophy with bulbar weakness. Eur Accepted June 26, 2003 J Paediatr Neurol 2000; 4: 69±72.