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
1GreatOrmond Street Hospital for Children and Correspondence to: Dr Matthew Pitt, Department of
2TheNational 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 pittm@gosh.nhs.uk

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Summary
A group of 13 patients with early onset diaphragmatic
palsy in association with a progressive neuropathy is
presented. All eight of those tested were found to have
mutations in the same gene encoding the immunoglobu-
lin mu-binding protein 2 (IGHMBP2) in patients with
spinal muscular atrophy (SMA) with respiratory dis-
tress type 1. Six out of these eight patients had either
homozygous or compound heterozygous mutations, and
two had only a single heterozygous mutation. Detailed
analysis of the clinical picture and the neurophysiologi-
cal and histopathological ®ndings indicated that these
patients shared similar characteristics, which were fur-
ther developed as a set of diagnostic criteria. Some of
the most striking of these were early onset of respira-
tory compromise, a markedly low birth weight, very
slow motor nerve conduction velocities and a general
decrease in the size of myelinated ®bres on sural nerve
biopsy. Extensive histological examination of the spinal
cord in one patient failed to ®nd any evidence of an
SMA. Four out of the ®ve not tested genetically were
positive for all diagnostic criteria. None of the cases of
early onset neuropathies or spinal muscular atrophies
with early respiratory failure reviewed in the literature
shares the exact characteristics, but many do have very
close similarities. Their classi®cation varies, but the dis-
covery of mutations in IGHMBP2 in cases that are vari-
ously classi®ed as SMA plus or severe infantile
neuropathy with respiratory distress points to a need
for the search for this genetic defect to be widened to
include both groups. The fact that we identi®ed other,
similar cases of neuropathy and early respiratory fail-
ure with and without IGHMBP2 mutations suggests
genetic as well as clinical heterogeneity in these infants.
It is possible that infants that do not have mutations in
the IGHMBP2 gene will be found to have mutations in
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
neuropathy or spinal muscular atrophy (SMA) are usually not
seen in the neonatal period. Involvement of the phrenic
nerves has been described in adults with Charcot±Marie±
Tooth (CMT) type 1 due to the chromosome 17 duplication,
PMP22 and MPZ mutations (Hardie et al., 1990; Tyson et al.,
1997). The form of autosomal dominant CMT type 2
associated with diaphragmatic palsy and vocal cord paralysis
(CMT 2C) is genetically distinct from CMT 2A, 2B and 2D
(Nagamatusu et al., 2000), as well as types CMT 2E, 2F and
Brain Vol. 126 No. 12 ã Guarantors of Brain 2003; all rights reserved
distal hereditary motor neuropathy type VII (Santoro et al.,
2002). It has not been described in the neonatal period,
although Dyck et al. (1994) did report that severely affected
members of two kindreds could be affected in infancy or
childhood. The positive family history will make the
diagnosis uncomplicated. A case classi®ed as congenital
hypomyelinating neuropathy with diaphragmatic and vocal
cord paralysis has been reported (Hahn et al., 2001).
It was believed previously that involvement of the
diaphragm made the diagnosis of SMA untenable (see table 1
 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

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26** Absent IP
8 16 28 11 22* 0.16* No response Discrete IP
26* 16** (technical?)
9 36 180 Absent Absent Absent Not Severely reduced No
attempted IP discernible
activity
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
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
Neuromuscular Centre workshop on SMA (1996) recognized
that involvement of the diaphragm can occur in SMA,
although the ®rst report was made in 1977 (Kyllerman, 1977).
SMA with diaphragmatic involvement is now classi®ed under
the SMA plus types and has been shown not to be associated
with the deletion of the survival motor neuron (SMN) gene on
chromosome 5 (Zerres and Davies, 1999).
Grohmann et al. (2001) studied 11 patients with a clinical
diagnosis of SMA with respiratory distress type 1 (SMARD1)
and 21 of their relatives from a total of six families. They
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
Grohmann et al. (2001) suggest that this mutation is the cause
of SMARD1 in humans.
Our group has been interested in a series of neonates who
presented with severe respiratory distress in the ®rst few
months of life. They had diaphragmatic palsy and evidence of
an axonal neuropathy. Sural nerve biopsies showed a general
decrease in the size of myelinated ®bres with minimal
evidence of axonal degeneration and no indication of
demyelination. The prognosis was very poor. Our initial
cohort of six patients, presented previously in abstract form
(Pitt et al., 1998), has now increased to 13 cases, mostly
unrelated. We have been unable to ®nd convincing evidence
to classify them as SMA and were interested to determine
whether they share the genetic abnormalities of the patients
studied by Grohmann et al. (2001). The current paper
presents these results, along with detailed analysis of clinical,
electrophysiological and histopathological ®ndings.
Case report
Case 1
This boy, the ®rst child of healthy unrelated parents, was born
at term by emergency Caesarean section, and weighed 2.06
kg. He presented at 8 days of age, with a history of poor
feeding requiring nasogastric tube feeding. Four days later he
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

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

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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
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
in a signi®cant number of sites in the muscle. The nerve
conduction velocities were recorded as severely abnormal if
they were below 70% of the lower limit of normal (LLN)
(modi®ed by the Ad Hoc Committee of the American
Association of Neurology recommended criteria for demye-
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.
Histopathological methods
Sural nerve biopsies, as well as sural and mixed nerves
(popliteal or peroneal) taken post-mortem, were ®xed in 3%
glutaraldehyde in 0.05 M cacodylate buffer. After overnight
®xation, 1-mm-thick slices were cut for processing and
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 density from 1 mm sections
These methods are similar to those described in a previous
paper (Jacobs and Love, 1985). Brie¯y, at least 2000
myelinated ®bres from several fascicles were counted at the
microscope at a magni®cation of 3250 using a squared
eyepiece graticule. Fascicular areas were measured using a
microscope with an extension tube positioned over the bitpad
of a Minimop image analyser. A cursor with a light-emitting
diode, visible in the microscope, was used to trace round the
inner edge of the perineurium of each fascicle. The density of
myelinated ®bres was calculated.
Unmyelinated axon density
Unmyelinated axons were counted in electron micrographs
enlarged to about 310 000 (calibrated with a grating replica)
and covering an area of ~0.01 mm2 in each nerve.
Unmyelinated axons were distinguished from Schwann cell
processes by their circular or near-circular outline, their paler
cytoplasm often with numerous microtubules and ®laments
and by the absence of ribosomes.
Myelinated ®bre diameter
Representative ®elds from at least two fascicles were
photographed and enlarged to just under 33000, the exact
magni®cation being obtained by calibration. Using the
Minimop, areas enclosed by the inner and outer perimeters
of the myelin sheath were traced and the data stored in an
interfaced computer. Fibre and axon diameters were derived
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
2 Single heterozygote
3 Not tested
4 Not tested
5 Not tested
6 Single heterozygote
7 Homozygous
8 Compound heterozygous
9 Compound heterozygous
10 Not tested
11 Not tested
12 Compound heterozygous
13 Compound heterozygous
Exon 8. Leu 361 Pro, base 1082 ctg ccg Exon 10. Cys 496 Stop, base 1488 tgc to tga
Exon 5. Del A base 661
Exon 7. Del AA base 983/984
Exon 10. Del T base 1463
Exon 8. Leu 361 Pro, base 1082 ctg ccg Exon 10. Cys496 Stop, base 1488 tgc to tga
Exon 2. Arg 43 Stop, base 127 cga to tga Exon 10. Cys 496 Stop, base 1488 tgc to tga
Exon 7. Arg 320 Stop, base 958 cga to tga Exon 10. Cys 496 Stop, base 1488 tgc to tga
Exon 7. Arg 320 Stop, base 958 cga to tga Exon 8. Leu 361 Pro, base 1082 ctg ccg

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(axon:®bre diameter) were calculated. At least 400 ®bres
were measured in each nerve.
Unmyelinated axon diameter
The perimeter of unmyelinated axons was traced on
micrographs at about 37000, accurately calibrated, using
the Minimop; diameters were calculated from axonal area
assuming axonal circularity. At least 400 axons were
measured in each nerve.
Brain, cord and muscle biopsies
Where autopsy was carried out, brains and spinal cords were
immersion ®xed in formalin for a minimum of 1 week, then
blocked for paraf®n processing and histological examination
utilizing conventional staining with haematoxylin±eosin,
luxol fast blue±cresyl violet and immunohistochemistry for
glial ®brillary acidic protein (GFAP).
Muscle biopsies were oriented and snap frozen for cryostat
sectioning at 8 mm. The sections were stained with
haematoxylin±eosin, the modi®ed Gomori trichrome stain,
for fat, glycogen and mitochondrial enzyme activity and to
demonstrate ®bre types with an ATPase method with and
without acid preincubation.
Genetics methods
Genetic sequencing
DNA was extracted by standard phenol/chloroform extraction
method, a routine automated technique carried out in the
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
IGHMBP2 gene were ampli®ed by the PCR using oligonu-
cleotide primers purchased commercially (Invitrogen).
Primers have been described previously. PCR was carried
out in a total volume of 50 ml, which contained 20 ng of DNA,
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
on a Perkin-Elmer 9700 thermal Cycler (Perkin Elmer,
Applied Biosystems, Foster City, CA). The cycling consisted
of denaturation at 94°C for 15 min, followed by 25 cycles of
94°C for 30 s, 60±50°C touching down protocol for 30 s and
72°C for 30 s. After that, 12 cycles of constant annealing
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
Qiaquick puri®cation kit (Qiagen, Hilden, Germany) and
resuspended in 50 ml of deionized water. For each exon, 100
ng of ampli®ed product was sequenced using forward and
reverse primers and a BigDye Terminator cycle sequencing
kit (Perkin-Elmer). Sequencing was performed on an ABI377
automated sequencer. Sequence alignment and analysis was
carried out with Sequence Navigator (Perkin-Elmer).
Mutation and polymorphism analysis
In addition to the pathogenic mutations identi®ed in the
IGHMBP2 gene, a number of single nucleotides polymorph-
isms (SNPs) were identi®ed throughout the gene. The
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-
entiation between the presence or absence of the mutation or
SNPs. A number of nucleotide repeats were identi®ed within
the introns of the IGHMBP2 gene. None of the pathogenic
mutations identi®ed in Table 3 was found in controls.
Results
Patient details
Details of patients are shown in Table 3. No case was
symptomatic at birth, except Case 3, who had grunting

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.
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),
weakness and subsequent wasting developed over a period of
weeks and showed a distal to proximal progression.
Neurophysiology
Results of neurophysiological investigations are shown in
Table 1. The most severe abnormalities were markedly
reduced motor conduction velocities, particularly in the legs,
and a very marked reduction or loss of the compound muscle
action potential. Studies of the sensory ®bres showed similar
abnormalities of conduction velocity and compound action
potentials, but of a much milder type. The EMG in each case
revealed a denervation pattern only in distal muscles initially.
An EMG of the diaphragm was made in four cases (9±12) and
con®rmed the presence of denervation in three. This was
con®rmed by post-mortem biopsy in Case 10.
Histopathology
Nerve biopsy results
Qualitative ®ndings
Sural nerve biopsies from Cases 1±11 showed a similar
appearance, compared with age-matched control sural nerves.
The myelinated ®bres were small (Fig. 1) .There was no
evidence in 1 mm resin sections of ®bre degeneration,
regeneration or demyelination. In a few cases, a very
occasional degenerating ®bres was seen. There was no
evidence of demyelination or remyelination in any of the
teased nerves. Cases 12 and 13 showed different features: in
Case 12, there was signi®cant evidence of degeneration
re¯ected in the reduced myelinated ®bre density (Table 2);
and in Case 13, the myelinated ®bre size spectrum was
normal, with only very occasional evidence of ®bre degen-
eration.
Electron microscopy of Cases 1±11 and 13 revealed a few
denervated Schwann cell processes (Bands of Bungner),
Infantile neuropathy with diaphragmatic weakness 2687
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Fig. 2 Motor nerve fascicle from popliteal nerve (post-mortem)
from Case 2, showing a reduced density of myelinated ®bres.
Scale bar = 25 mm.
evidence of occasional myelinated ®bre degeneration. In
Case 12, there was frequent evidence of ®bre degeneration.
There was no evidence of loss of unmyelinated axons. The
ultrastructure of axons of myelinated and unmyelinated axons
appeared normal: in particular, neuro®laments seemed to be
present at normal densities. In mixed nerves taken post-
mortem (Cases 2, 4 and 6), the motor components were
identi®ed from the larger size of the motor ®bres. In resin
sections, there was some evidence of ®bre degeneration in
these motor ®bres; Fig. 2 shows a reduced density of
myelinated motor ®bres. Electron microscopy con®rmed the
presence of numerous Bands of Bungner in such regions,
indicating earlier myelinated ®bre degeneration.
Quantitative ®ndings in sural nerve biopsies
Control data were obtained from sural nerves of four infants
aged 0 and 3 days, and 3 and 9 weeks, removed within 24 h of
death from cases without a history of peripheral nerve disease
(Jacobs and Love, 1985). Inclusion of controls rather younger
than our patients emphasizes the marked difference between
the two groups. Table 2 summarizes the quantitative ®ndings.
Fascicular areas: fascicular areas were generally smaller
than those of the controls, and this is probably explained by
the smaller size of the ®bres.
Myelinated ®bre density: there were no obvious differ-
ences between myelinated ®bre densities in control sural
nerves and nerve biopsies from Cases 1±11. The reduced
density in Case 12 is associated with more marked evidence
of degeneration of myelinated ®bres.
Myelinated ®bre size: it is justi®able to use the mean ®bre
diameter measurement for purposes of comparison when
2688 M. Pitt et al.
Fig. 3 Histogram of myelinated ®bre diameters from case 3 (diagonal hatching) and controls (chequerboard
hatching).
there is no evidence of ®bre loss as in Cases 1±11. Table 2
shows signi®cantly reduced mean ®bre diameters in all
except Case 13. There is a similar signi®cant reduction in size
of the axons of these myelinated ®bres, again excepting Case
13. In histograms, both the myelinated ®bre diameter size
spectrum (Fig. 3) and their axon diameters show a marked
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
compared between controls and Cases 1±6. Mean values of
0.108 6 0.022, (n = 5, controls) and 0.103 6 0.022 (n = 6,
patients) were not different statistically (Levenes test for
equality of variances, F = 0.009, P = 0.926), con®rming that
the myelin sheaths were of appropriate thickness for the size
of axon.
Unmyelinated axon density: these are within normal limits
in Cases 1, 3, 4 and 5. Mean unmyelinated axon diameters
from controls and these cases were similar.
Brain, cord and muscle biopsy results
Examination of serial sections of the spinal cords of Cases 4
and 6 showed no abnormality, in particular no loss or changes
in anterior horn cells or accumulation of microglia (Fig. 4).
Dorsal root ganglia from cervical, thoracic and lumbar
regions showed no abnormalities. Diaphragmatic muscle
showed denervation in Case 10 but no abnormality in Case 6,
although the intercostal muscles were denervated.
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
chronic denervation. The site of the biopsy was medial
gastrocnemius in four cases, all of which showed changes
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reported as consistent with chronic denervation, and vastus
lateralis in three, all of which showed non-speci®c changes.
The exact site was not recorded in four examinations and the
conclusions made were as follows: possibly neurogenic,
denervation of unusual type, chronic denervation and acute
denervation.
Genetic analysis
Genetic analysis was carried out on eight cases, and the
results are shown in Table 4. As can be seen, ®ve out of the
eight cases have compound heterozygous mutations, Case 7
has a homozygous deletion and Cases 2 and 6 has a single
heterozygous deletion. Cases 2 and 6 may have an uniden-
ti®ed deletion involving one or more exons or a cryptic splice
site change, or they may have a mutation in another gene
acting in parallel to the IGHMBP2 gene. The pathogenicity of
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
available, but this change was not present in 50 British, ®ve
Pakistani, four Indian and one Iranian control. The mutations
in exons 2, 7, 8 and 10 were not identi®ed in 50 British
controls; detailed ethnicity was not available in these cases,
but they were clinically assessed in the UK. The position of
the mutations on the IGHMBP2 gene are shown in Fig. 5,
which also shows the mutations identi®ed by Grohmann et al.
(2001).
Proposed diagnostic criteria
The results of our investigations suggest that the cases shared
common clinical, neurophysiological and histopathological
 Infantile neuropathy with diaphragmatic weakness 2689

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Fig. 4 Anterior horn cell at C5 level from Case 6, showing a normal complement of anterior horn cells.
(Luxol fast blue Nissl X170).

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
accurate diagnosis and also distinguish them from other
reported cases. We judged the cases against clinical,
histological and neurophysiological criteria. The criteria
chosen are shown below.
Clinical criteria
The clinical criteria were (i) low birth weight below the 3rd
centile; (ii) onset of symptoms within the ®rst 3 months; (iii)
diaphragmatic weakness either unilaterally or bilaterally; (iv)
ventilator dependence within less than one month of onset
with an inability to wean; and (v) absence of other
dysmorphology or other conditions.
Histopathological criteria
The histopathological criteria were (i) a shift to the left of
myelinated ®bre size in sural nerve biopsies (since myelin
sheath thickness is found to be appropriate for axon size, the
®bre size change would have originated from the axon); (ii)
minimal evidence of ongoing myelinated ®bre degeneration
in biopsies taken up to 3±4 months; and (iii) no evidence of
regeneration or of demyelination which might account for the
change in ®bre size.
EMG criteria
EMG criteria were ful®lled if, in any of the investigations
performed, there was (i) evidence of distal denervation acute
or chronic (ii) evidence of severe slowing (<70% of LLN) in
one or more nerves (motor or sensory).
The Venn diagram (Fig. 6) shows the criteria that the cases
met, with the reasons for the cases not ful®lling a particular
criterion and also whether the cases were tested for the
mutations.
2690 M. Pitt et al.
Fig. 6 Venn diagram of the cases identi®ed by their case numbers, categorized by their assessment
against the three sets of criteria. The cases within shaded boxes are those tested genetically (Cases 1, 2,
6±9, 12 and 13).
Discussion
All of the eight cases tested showed mutations in the gene
encoding IGHMBP2 located on chromosome 11q13.2-q13.4.
In two cases, only a single heterozygous mutation was found;
and even though these mutations were not found in controls,
the signi®cance of these single changes are uncertain, as the
pathogenicity of these mutations is not proven in the absence
of a mutation on the other allele. Four out of the remaining
®ve cases who were not tested ful®lled all the clinical,
physiological and pathological criteria for classi®cation, and
the other, Case 4, was the elder sibling of Case 12, who was
positive. Although not possible to prove, it would be
anticipated that they might also have tested positive. The
location of the mutation positions was rarely the same in our
group compared with those of Grohmann et al. (2001). All the
identi®ed mutations were likely to be pathogenic, except
perhaps for the two cases with single heterozygous mutations
as discussed above: they either changed a conserved amino
acid, caused a mutation that produces a stop codon or caused
a deletion that resulted in a truncated protein. None of the
mutations was identi®ed in controls.
Infants and neonates who present with severe respiratory
distress from diaphragmatic palsy have been described. They
are de®ned as either diaphragmatic SMA (Zerres and Davies,
1999) or severe infantile axonal neuropathy with respiratory
failure (SIANRF) (Wilmshurst et al., 2001). Some have slow
conduction velocities and many do very badly, either dying
early or remaining ventilator dependent. We initially felt that
our patients had a distal SMA, but the spinal cord was entirely
normal histologically in Case 6 and consistent sural nerve
changes were seen in all cases. It seemed more appropriate
Downloaded from http://brain.oxfordjournals.org/ by guest on September 29, 2015
they were reclassi®ed as a severe infantile polyneuropathy.
Grohmann et al. (2001) regard autosomal recessive SMARD1
as synonymous with SIANRF (Wilmshurst et al., 2001). The
criteria presented here may help clinicians to identify similar
cases.
There are 39 cases in the literature [Mellins et al. (1974),
two cases; McWilliam et al. (1985), four cases; Schapira and
Swash (1985), two cases; Bertini et al. (1989), ®ve cases;
Appleton et al. (1994), one case; Novelli et al. (1995), two
cases; Grohmann et al. (1999), nine cases; Geller et al.
(2000), three cases; Mercuri et al. (2000), one case;
Grohmann et al. (2001), three new cases in addition to
those reported elsewhere; Mohan et al. (2001), two cases; and
Wilmshurst et al. (2001), ®ve cases]. Sex was given in only
26 cases (14 males and 12 females). Thirteen out of the 29
with family history were positive. Only six out of the 20 with
complete clinical details ful®lled all criteria, but the birth-
weight was low in 12. Seven out of the 17 with
neurophysiological details ful®lled our criteria. Often the
slowing in nerve conduction velocity was not as severe as we
had seen. The cases that were closest to ours were Mellins
et al. (1974), case 1; Bertini et al. (1989), case 3; Appleton
et al. (1994), single case; Geller et al. (2000), case 3; and
Wilmshurst et al. (2001), cases 1 and 5. All had low birth
weight and six out of the seven remaining criteria.
Insuf®cient data prevented the same analysis of the
histological ®ndings. The sural nerve biopsy from
Appleton's case (Appleton et al., 1994) was reviewed in
our laboratory and found to have the same changes as the
majority of our group. Most importantly, the sural nerve
biopsy ®ndings were not speci®c. Identical changes were
found in ®ve other patients, four of whom had tested negative

for mutations in the IGHMBP2 gene (one was not tested).
These four patients presented at birth or soon afterwards and
needed immediate ventilatory support. Only one had a low
birth weight, but also had complicated heart disease and was
dysmorphic, and was diagnosed as having had an axonal
sensorimotor neuropathy. Of the others, one case had grade II
intraventricular haemorrhage, de®ciency of the anterior ribs
but no diaphragmatic abnormality and the EMG was
suggestive of a neuromuscular junction abnormality.
Another baby was clearly dysmorphic and had micrognathia
and was thought to have a severe SMA on nerve conduction
studies. The ®nal infant had no other somatic abnormalities
but had neurophysiological changes of a sensorimotor
neuropathy. The diaphragm was not involved in any case
and none showed the characteristic severe slowing of motor
nerve conduction. Therefore, the condition on histopatholo-
gical criteria is heterogeneous
The recent case described by Hahn et al. (2001), the ®rst to
report phrenic nerve involvement in a congenital hypomye-
linating neuropathy, may be another case, as it has many of
the characteristics of our cases, including low birth weight,
absence of large diameter myelinated ®bres and distal
denervation. Interpretation of a neuropathy as hypomyelinat-
ing may be dif®cult in a nerve biopsy taken at the age of 1
month, when myelination is still incomplete. Quantitative
comparison with age-matched controls is required to con®rm
the degree of myelination.
It seems likely that SMARD1/SIANRF are descriptions of
a condition with variable clinical expression, of which a
proportion will have mutations in the IGHMBP2 gene. The
recent linkage study of Viollet et al. (2002) in a family with a
SMARD phenotype that was negative for the IGHMBP2 gene
identi®ed a locus close to SMARD1 on 11q. Its is likely that
families that are negative for IGHMBP2 will have a defect at
this locus that is likely to affect a gene functionally similar to
IGHMBP2.
The differing pathological involvement reported in the
peripheral nerves and spinal cords may have a parallel in
severe congenital SMA (Zerres and Davies, 1999). On
molecular analysis, a large deletion including the SMN
telemeric and NAIP genes is seen in most families with this
condition linking them unequivocally to SMA 5q. Yet, on
neurophysiological testing, there is a lack of sensory and
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
severe clinical manifestations.
The absence of the largest diameter axons in the sural nerve
biopsy explained the low velocities in sensory nerves. The
velocity decrease on nerve conduction studies was so slow
that the initial impression was of demyelination as the
underlying pathology. When demyelination is encountered in
the neonatal period, it is most unusual to obtain sensory
responses, which in these cases could often be found. High
stimulation intensities are also needed (Bolton, 1996).
Infantile neuropathy with diaphragmatic weakness 2691
Although it is well recognized that some slowing may
occur in the classical SMA patients, slowing to <70% of the
LLN remains an exclusion criterion. The combination of the
slow motor nerve conduction velocity and severe distal
denervation is unusual and was a signature of the condition. It
was not possible to make the same correlation between the
motor nerve conduction studies and the histopathology of
motor nerves.
The impression was not one of loss of nerve ®bres with the
accompanying changes of axonal degeneration, but rather
that the nerve ®bres had not developed. Myelinated axons did
not attain their expected size for age. Axon calibre is
associated with neuro®lament density, which in the sural
nerves appeared normal; the total number of neuro®laments
produced is therefore less than normal. IGHMBP2 colocalizes
with RNA-processing machinery in cytoplasm and nucleus,
Downloaded from http://brain.oxfordjournals.org/ by guest on September 29, 2015
but no information about its speci®c effects is available.
However, the fact that there were similar sural nerve ®ndings
in other cases that were clinically and genetically distinct may
suggest that the IGHMBP2 gene is not involved in the axon
changes. It is possible that the sural nerve changes simply
re¯ect an early onset neuromuscular disorder and are not
speci®c for any particular cause. The changes seen in the
child with abnormalities suggesting a neuromuscular junction
abnormality are otherwise impossible to reconcile.
With the genetic studies presented, it is unlikely our cases
are examples of the infantile neuropathy initially described by
Ouvrier et al. (1981) and to which more cases have been
added by Gabreels-Festen et al. (1991). Our cases are also
different in their clinical, genetic and morphological aspects
from autosomal dominant CMT type 2, which also show a
distinct loss of the largest diameter myelinated ®bres with a
shift in the histogram to the left. Regenerative features are
almost absent. With the identi®cation of IGHMBP2 muta-
tions, it will be interesting to screen all cases presenting with
similar clinical and neurophysiological ®ndings, irrespective
of whether they are considered a neuropathy or an SMA. This
will help further de®ne the phenotype and also allow
examination to see whether there is a phenotype/genotype
effect explaining some of the features. Given the clinical and
genetic heterogeneity in this patient group, identi®cation of
further genes will also help us to de®ne a genetic classi®ca-
tion.
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Received March 27, 2003. Revised June 25, 2003.
Accepted June 26, 2003