Indian Pediatrics 2001; 38: 1236-1243  

Spectrum of Floppy Children in Indian Scenario

T. Dua, M. Das, M. Kabra, M. Bhatia+, C. Sarkar*, S. Arora, M.C. Sharma*
and V. Kalra

From the Departments of Pediatrics, Neurology+ and Pathology*, All India Institute of Medical Sciences, New Delhi 110 029, India.

Correspondence to: Prof. Veena Kalra, Department of Pediatrics, All India Institute of Medical Sciences, New Delhi 110 029, India. Email: [email protected]

Manuscript received: March 14, 2001, Initial review completed: April 23, 2001,
Revision accepted: June 14, 2001.

Objective: To study the clinical profile of paralytic floppy infants undertaking available investigations and detect the frequency of exon7 of survival motor neuron (SMNT) gene deletion among the spinal muscular atrophy (SMA) cases. Design: Descriptive study. Setting: Tertiary care teaching hospital. Subjects: 70 paralytic floppy infants (40 males/30 females) with age less than 13 years were included in the study. Exclusion criteria included central hypotonia of any cause. Detailed clinical evaluation was done followed by serum creatine phosphokinase levels, electrophysiological studies, muscle biopsy including immunohistochemistry and electron microscopy. Exon7 of SMNT gene deletion studies was done by PCR. Results: Final diagnosis of SMA was assigned to 37 patients followed by congenital myopathy (n = 7), cogenital muscular dystrophy (n = 5), mitochondrial myopathy (n = 4), neuropathies (n = 5) and diaphragmatic SMA (n = 1). Only 15.7% of cases remained unclassified. When EMG was correlated with final diagnosis, it was 80.6% and 75% sensitive and 68.8% and 87.5% specific for neurogenic and muscle disease, respectively. Muscle biopsy revealed neurogenic atrophy in 47.8% cases followed by normal in 37.3% and myopathic pattern in 14.9% cases. Exon7 of SMNT gene was deleted in only 50% of SMA cases. Conclusions: Spinal muscular atrophy was the commonest cause of floppy children. The low rate of SMNT gene deletion detected needs confirmation with further studies.

Key words: Congenital muscular dystrophy, Floppy children, Hypotonia, Spinal muscular atrophy, SMN T gene.

FLOPPY infant, an usual broad symptomatic label given to hypotonic children irrespective of etiology, is not an uncommon diagnostic dilemma. The floppy infants may be so due to central hypotonia, hypotonia of neuromuscular origin and others that are uncharacterizable. The advent of investigative techniques, immunohisto-chemical/ultrastructural studies on muscle and nerve, and genetic studies have resulted in many benign essential hypotonia getting classified into specific disorders. No systematic studies to define the various etiologies are yet reported from our country.

Central hypotonia accounts for the large majority of floppy infants, they are generally not accompanied by significant paralysis. Among the floppy infant with motor weakness, spinal muscular atrophy (SMA) is reported to be the most common(1-2). The estimated incidence is 1 in 6-10,000 births second only to cystic fibrosis amongst the recessive disorders(3). A lower incidence of 1 in 25,000 has also been, however, reported(1). A precise diagnosis of this disease and its type is clinically relevant for prognostication and prenatal counselling. The gene for SMA has been mapped to chromosome 5q11.2-q13.3 and two genes are proposed for causation of spinal muscular atrophy, survival motor neuron (SMN), which has a telomeric and centromeric form and neuronal apoptosis inhibitory protein (NAIP) gene(4-9). Exon7 and exon8 of SMNT gene is deleted in 80-100% of SMA cases irrespective of the type (7,9–13). It has a high sensitivity and specificity for diagnosis of SMA(12,14). No reports in this context are published from our country.

We undertook the present study: (i) To evaluate the clinical profile of paralytic floppy infants and correlate the clinical criteria and investigations; and (ii) To determine the frequency of SMNT gene deletion in SMA cases.

A descriptive study in 70 patients recruited from pediatric OPD, neuromuscular clinic and inpatient wards at the All India Institute of Medical Sciences, New Delhi was conducted from January 1998 to December 2000.

The criteria for entry into study were paralytic floppiness with hypotonia and hyporeflexia in children less than 12 years of age. The exclusion criteria included central hypotonia, Down’s syndrome, cerebral palsy and metabolic causes. Acute flaccid paralysis of any etiology were also excluded.

A detailed clinical evaluation was recorded including age, sex, age at onset, present status of functional ability, associated complaints like respiratory muscle weakness and bulbar palsy, detailed antenatal history, reduced fetal movements, natal and perinatal history, feeding difficulties and develop-mental history. Family history of floppy infants/early childhood death and consan-guinity was also recorded.

Examination included recording of pos-ture, spontaneous motor activity, respiratory pattern and effort, facial dysmorphism, joint/chest deformities, higher function, cranial nerves, motor evaluation (atrophy, tone, power, reflexes), sensory system, superficial reflexes and fasciculation.

Investigations performed included CPK (which was compared with normative data), electrophysiological studies, muscle biopsy and SMNT gene deletion studies.

Electrophysiological studies including motor and sensory nerve conduction studies and needle electromyogram was performed using Medlec or Nicolet Viking II instrument. EMG findings were interpreted as neurogenic (spontaneous fibrillation at rest, long duration polyphasic motor unit potentials, decreased interference pattern), myopathic (low ampli-tude, short duration polyphasic with normal interference pattern) or normal changes according to the standard criterias(15,16).

Muscle biopsy was done from either deltoid/gastrocnemius/vastus lateralis on the basis of clinical evaluation. Muscle tissue was subjected for H & E staining, immuno-histochemical staining including dystrophin, adhalin and merosin and enzyme histo-chemical staining including NADH-TR, SDH, ATPase, and Gomori-trichome. The specimens were also subjected to electron microscopy to look for ultrastructural changes. Muscle specimens were interpreted as neurogenic atrophy, myopathic or normal.

SMNT Gene Deletion studies

Blood samples were collected in EDTA vacutainers and DNA extracted. DNA samples were subjected to PCR amplification by using primers for amplification of exon7 of SMNT gene. Exon7 was amplified with intron 6 primer R1114 and mismatch primer Xy-Dral in 35 cycles of 1 min at 94º C, 1 min at 55º C and 1 min at 72º C using Taq-polymerase (super taq) in a reaction buffer as recom-mended. PCR products were subsequently digested to completeness with Hint 1. Electrophoresis was carried out in 2.5% agarose gels(17). The pattern of bands obtained were studied to detect the deletion of exon7 of SMNT gene. Primers for exon48 of DMD gene were used as control. Both the centromeric and telomeric copy of SMN gene were obtained and the cases with absent telomeric band of SMN gene were diagnosed as deleted SMNT gene cases.

Data was coded and entered on Foxpro 2.6 and analyzed by Epiinfo 6. The deletion positivity in SMA cases was correlated with various demographic and clinical character-istics by Chi square test for proportions and Student ‘t’ test for means. For non-parametric data, Kruskal Wallis test was applied.

A total of 70 patients (40 males and 30 females) were included in the study as per inclusion and exclusion criteria. The ages at presentation to hospital ranged between 2 months and 12.5 years.

Final diagnosis for different categories was assigned on the basis of muscle biopsy report and gene deletion studies. The various etiologies are shown in Table 1.

Diagnosis of SMA was made in patients with muscle histology suggestive of neurogenic atrophy or exon7 of SMNT gene deletion or both. Further typification was done on the basis of age of onset and clinical progression on the basis of classification proposed by Munsat and Davies. Type I SMA or Werding-Hoffmann disease was character-ized in patients by severe, generalized muscle weakness and hypotonia at birth or within the next 3 months and inability to attain sitting. Children with Type II or intermediate SMA had onset of weakness within 18 months, were able to sit, although they could not stand or walk unaided. Type III SMA or Kugelberg-Welander patients had proximal muscle weakness, starting after the age of 2 and were ambulatory at enrolment(18). Congenital muscle dystrophy was diagnosed based on dystrophic muscle histology with further characterization on the basis of merosin staining. Congenital myopathy was diagnosed on the basis of suggestive muscle histology and electron microscopy and negative SMNT gene deletion. Neuropathies were diagnosed on basis of nerve conduction velocity and nerve biopsy studies. In presence of negative gene deletion and normal muscle biopsy, the cases were labeled as unclassified.

The detailed clinical characteristics of the patients are presented in Table II.

Needle electromyogram was performed in 54 patients and was classified as neurogenic pattern in 34 (63%), myopathic in 14 (26%) and normal in 6 (11%). When EMG was correlated with final diagnosis, it was 80.6% and 75% sensitive and 68.8% and 87.5% specific for neurogenic and muscle disease, respectively (Table III).

Muscle biopsy was performed in 67 patients and was characterized as detailed earlier. Neurogenic atrophy was seen in 32 (47.8%) patients, myopathic in 10 (14.9%) and normal in 25 (37.3%) children (Table IV).

SMA emerged as the most common cause with 37 (60.3%) belonging to this group. Diagnosis on basis of both muscle histology and gene deletion was made in 15 (40.5%) while neurogenic atrophy on muscle biopsy alone formed the basis in 17 patients (46.0%). Gene deletion alone formed the diagnostic criteria in 5 patients (13.5%). Commonest type were SMA type II (n = 17) followed by type I (n = 13) and type III (n = 7). One child had SMA variant (diaphragmatic SMA). Their clinical characteristics are depicted in Table V.

Exon7 of SMNT gene deletion was performed in 40 patients with 20 (50%) being deletion positive. The demographic and clinical characteristics like sex, age, history of decreased fetal movement, positive family history, presence of fasciculations, respiratory muscle weakness and bulbar palsy were correlated with deletion positivity. None of them except decreased fetal movement had significant statistical correlation with presence of exon7 deletion. The clinical SMA types were correlated with the investigations including EMG, muscle biopsy and deletion studies (Table VI). The EMG was neurogenic in only 50% of SMA type I compared with other types. Only one-third of type III SMA show deletion of exon7 compared to 50% positivity in other types.

Among children with myopathic muscle biopsy, dystrophic pattern was seen in 5 cases with one child merosin negative and 3 of them had normal merosin staining. Merosin staining was not performed in one case. The CPK values were more than 10 times normal in 4 with only one of them having a normal value. One of congenital muscle dystrophy patients had mental delay on clinical history and examination and white matter changes on MRI.

Fifteen biopsies were subjected to electron microscopy. Mitochondrial myopathy on the basis of electron microscopy and positive special stains like NADH-TR, SDH, ATPase and Gomori-Trichome was diagnosed in 4 children. One case was diagnosed as Congenital fibre type disproportion and rest were normal. When correlated with final diagnosis, muscle biopsy was 80% and 37.5% sensitive for spinal muscle atrophy and muscle disease, respectively (Table IV).

Sural nerve biopsy was done in 3 patients with herediatry neuropathies. It was normal on histopathology and electron microscopy in two while one with hereditary sensory and autonomic neuropathy type IV revealed focal segmental demyelination.

Floppy infant syndrome can result from several unrelated diseases. Our data reflects that among the mixed bag of etiologies, spinal muscular atrophy was commonest followed by congenital myopathy, and congenital muscular dystrophy. Hereditary sensorimotor neuropathies were relatively uncommon. Studies from various other countries suggest a similar etiological profile as our data(19). Eight (11%) of the cases remained uncharacterizable, their EMG was neurogenic in 3 and normal in one. Muscle biopsy or gene deletion studies for SMNT gene was not informative. Even after sophisticated staining techniques used in muscle histology, a categorical diagnosis may not be possible is some cases.

EMG is a good initial diagnostic test for floppy children with neurogenic cause (SMA) having 80% sensitivity. Paradoxically, in most severe SMA type I, EMG was only 50% sensitive suggesting that diagnosis of SMA may be missed on EMG alone. Other studies have reported only 82-93% positive correlation in type I SMA(15-16). The likely explanation is sparing of the sampled muscle or non manifestation of neurogenic changes. In myopathic patients, EMG conforms to a myopathic pattern with 75% sensitivity and is thus, a good screening test before perfor-mance of muscle biopsy. Muscle biopsy is needed for precise characterization with immune and enzyme histochemistry and electron microscopy.

Congenital muscle dystrophy (CMD) patients included one merosin negative and three merosin positive patients. One of the merosin positive patients had mental subnormality and white matter changes on MRI suggesting a different subset of CMD seen in our population(20,21).

Exon7 deletion has been considered as highly specific and sensitive for diagnosing spinal muscular atrophy. According to the literature, exon7 is deleted in atleast 90% of cases and mostly both exon7 and exon8 are deleted together (7,9,11-13,22). Deletion of only exon8 has been reported in very few cases(23). In our subjects exon7 of SMNT was deleted in only 50% of clinical SMA (type I-III). Only 38% of type III case had gene deletion compared to type I (54%) and II (56%). Various clinical characteristics were correlated with deletion positivity. Decreased fetal movements emerged as the only feature with significant correlation. The frequency of gene deletion was quite low in all SMA types in comparison to the reported literature (7,9,11-13). The deletion studies were reconfirmed by Institute of Myology, Paris and complete concordance was present. Point mutations, however, were not looked for. A comparatively lower rate of SMA gene deletion (65.5%) has also been, however, reported from South African Black patients(24). The possible explanation for the low frequency of deletion of SMN gene may be ethnic variation, or presence of more point mutations in our population (which was not checked for) or presence of some other genetic locus. This low frequency of gene deletion can not be attributed to not looking for exon8 deletion, as it is alone present in only a few cases.

Based on our clinical experience on floppy children in developing countries, it is suggested that the investigative algorithm should include EMG as a primary test followed by SMN gene deletion studies in neuropathic subjects. Muscle biopsy should be undertaken with detailed immuno-histochemistry and electron microscopic studies, as routine histopathology may be non-contributory. The low gene deletion correlation in our SMA phenotype is an interesting new observation that requires further confirmation.

Contributors: VK designed the study and revised it critically and will act as guarantor for the paper. TD and MD collected and analyzed the data and prepared the manuscript. MK helped in designing the study and collection of data and molecular studies. MB did the electrophysiological studies. CS and MCS did the muscle immunohistochemistry and electron microscopic studies. SA did the SMNT gene deletion studies on DNA samples.

Funding: None.

Competing interests: None stated.

 

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