Mutation studies in X-linked myotubular myopathy in three Indian families.

Abstract

Congenital myopathies are a group of genetic disorders characterized by generalised muscle hypotonia and weakness of varying severity. They are distinct entities and do not include muscular dystrophies, metabolic myopathies and mitochondrial disorders. Myotubular myopathy is a rare sub type within this group of disorders. Clinical differentiation of the various types is difficult and requires muscle biopsy with histopathological and immunohistochemical studies for specific diagnosis. Gene studies are a prerequisite for genetic counseling adn prenatal diagnosis. Here presented three cases of X-linked myotubular myopathy in three Indian families where the diagnosis was established by mutation analysis in the MTM1 gene in all, and supported his histopathology in two. All three families had history of previous male neonatal deaths with similar complaints. Molecular analysis revealed hemizygous mutations in the MTM1 gene including c.1261-10A>G in case, 1, c.70C>T (R24X) in case 2, and a previously unreported mutation, c.924_926delCTT(p. F308del), in case 3. Genetic counseling was performed regarding the X-linked inheritance, their 50% risk of recurrence in boys in subsequent pregnancies, and a feasibility of prenatal diagnosis. This is the first report of cases of X-linked myotubular myopathy from India.

Full text

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Correspondence and Reprint requests : Dr Sunita Bijarnia, Center
of Medical Genetics, Sir Ganga Ram Hospital, Rajinder Nagar, New
Delhi-110060, India.
[DOI-10.1007/s12098-010-0057-6]
[Received April 15, 2009; Accepted February 1, 2010]
Clinical Brief
Mutation Studies in X-linked Myotubular Myopathy in
Three Indian Families
Sunita Bijarnia, Ratna D. Puri, Monika Jain, Neelam Kler1, Subimal Roy2, J. Andoni Urtizberea3,
Valerie Biancalana4 and I.C. Verma
Center of Medical Genetics, Departments of 1Neonatology, Child Health and 2Histopathology, Sir Ganga Ram
Hospital, New Delhi, India, 3AP-HP, Hôpital Marin, Hendaye, France 4Laboratoire de Diagnostic Génétique and
EA3949, Faculté de Médecine et CHRU, Strasbourg, France
ABSTRACT
Congenital myopathies are a group of genetic disorders characterized by generalised muscle hypotonia and weakness of
varying severity. They are distinct entities and do not include muscular dystrophies, metabolic myopathies and mitochondrial
disorders. Myotubular myopathy is a rare sub type within this group of disorders. Clinical differentiation of the various types
is difficult and requires muscle biopsy with histopathological and immunohistochemical studies for specific diagnosis. Gene
studies are a prerequisite for genetic counseling adn prenatal diagnosis. Here presented three cases of X-linked myotubular
myopathy in three Indian families where the diagnosis was established by mutation analysis in the MTM1 gene in all, and
supported his histopathology in two. All three families had history of previous male neontal deaths with similar complaints.
Molecular analysis revealed hemizygous mutations in the MTM1 gene including c.1261-10A>G in case, 1, c.70C>T (R24X)
in case 2, and a previously unreported mutation, c.924_926delCTT(p. F308del), in case 3. Genetic counseling was performed
regarding the X-linked inheritance, their 50% risk of recurrence in boys in subsequent pregnancies, and a feasibility of prenatal
diagnosis. This is the first report of cases of X-linked Myotubular myopathy from India. [Indian J Pediatr 2010; 77 (4) :
431-433] E-mail: Bijarnia@gmail.com
Key words: X-linked myotubular myopathy; Mutation studies; MTM1; Indian
Myotubular Myopathy (MTM), also known as
Centronuclear Myopathy (CNM), is a genetically
heterogeneous sub-group of congenital myopathies,
characterized clinically by variable degree of muscle
weakness and hypotonia, and histologically, by small
muscle fibres showing a central area devoid of myofibrils
and mitochondrial aggregates around the centrally
located, often large nuclei.1 X-linked myotubular
myopathy is the severest form in the spectrum where
male infants present at birth with marked hypotonia and
require ventilatory support, usually dying in early
infancy. Two more forms of the disease are also
recognized and are autosomal recessive (AR) and
autosomal dominant (AD) in inheritance. The X-linked
form is caused by mutations in the myotubularin (MTM1)
gene on chromosome Xq28 whilst AD and AR forms have
been associated with mutations in the dynamin 2 (DNM2)
gene on chromosome 19p13.2 and the amphiphysin 2
(BIN1) gene on chromosome 2q14, respectively.2 Single
cases with features of CNM have been associated with
mutations in the skeletal muscle ryanodine receptor
(RYR1) and the hJUMPY (MTMR14) genes.2 Gene studies,
are therefore a prerequisite for accurate genetic
counseling and prenatal diagnosis.
Here is presented a series of three cases of X-linked
Myotubular Myopathy in three unrelated Indian families
where the diagnosis was established by histopathology
and/or gene studies in the MTM1 gene.
REPORT OF CASES
Case 1
A two-day-old baby boy, born to non-consanguineous
couple, was admitted in the newborn nursery with severe
floppiness and feeble respiratory effort since birth. The
baby was born at term by spontaneous vaginal delivery
after an uneventful pregnancy. He required resuscitation
for poor respiratory effort. Subsequently, he was put on
ventilatory support. On examination, he had no
dysmorphic features or arthrogryposis. There was
Indian Journal of Pediatrics, Volume 77—April, 2010 431

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Sunita Bijarnia et al
432Indian Journal of Pediatrics, Volume 77—April, 2010
generalized hypotonia with absent deep tendon reflexes.
No neonatal reflexes could be elicited. Muscle biopsy was
performed and diagnosis of myotubular myopathy was
made, based on characteristic finding of large centrally
placed nuclei in myofibers. His blood was collected for
gene studies for X-linked Myotubular myopathy. There
was presence of the mutation c.1261-10A>G in the MTM1
gene which confirmed the diagnosis of myotubular
myopathy. The mother was also noted to have the same
mutation. The couple subsequently had one normal girl.
Case 2
A no-consanguineous couple had two boys, affected with
congenital hypotonia and weakness. They died in the
neonatal period. The first baby had a normal antenatal
period but was noted to have floppiness and poor
respiration at birth. He required ventilatory support but
could not survive beyond 5 days of age. The second baby
had a similar presentation, the antenatal period being
uneventful. There was history of reduced fetal
movements but no evidence of polyhydramnios. At birth,
the baby required resuscitation and ventilatory
management in nursery. His birth weight was 2.52 kg
(10th -50th centile at 36 wk), length 46 cm (10th-50th centile,
and head circumference was 35 cm (90th centile). On
examination at two days of age, the baby was extremely
floppy – frog like posturing with a long face (Fig. 1). Large
ears were noted. There was generalized areflexia. A
battery of tests were carried out for congenital hypotonia
for disorders including spinal muscular atrophy (SMA),
myotonic dystrophy, Pompe disease, Prader-Willi and
chromosomal disease, all of which were negative. An
open muscle biopsy of vastus lateralis was performed.
Routine haematoxylin and eosin staining and
immunohistochemistry (Modified Gomori staining) were
done, which showed features characteristic of Myotubular
Myopathy (Fig. 2-3). There were large central nuclei in
myofibers resembling fetal cells. The diagnosis was
Fig. 1. Case 2 - Floppy baby in frog like posture and on ventilator
for respiratory insufficiency.
Fig. 2. Muscle biopsy of Case 2 - H & E staining showing large
centrally placed nuclei.
Fig. 3. Muscle biopsy of Case 2 - Modified Gomori stain showing
similar features.
confirmed by molecular studies, revealing a c.70C>T
mutation in exon 3 in the MTM1 gene, leading to a stop
codon R24X. Genetic counseling was performed
regarding the X-linked inheritance of the disorder, a 50%
risk of recurrence in boys in subsequent pregnancies, and
the availability of prenatal diagnosis.
Case 3
A non-consanguineous couple from South India
presented with a history of neonatal deaths in two boys at
6 days and 7 days of life, respectively. Both pregnancies
had been uneventful and there was no history of reduced
fetal movements. Both boys were born by elective
caesarean sections and cried feebly at birth. They required
immediate ventilation on account of severe hypotonia.
The consultand’s (wife’s) mother had history of two male
stillbirths, but no further details were available. The
evaluation of the second baby included a normal heart

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Mutation Studies in X-linked Myotubular Myopathy in Three Indian Families
Indian Journal of Pediatrics, Volume 77—April, 2010433
size on chest radiograph and absence of structural
malformation on ultrasound of brain and abdomen. Based
on history of floppiness in male offsprings, and presence
of male stillbirths of the mother of the proband, X-linked
myotubular myopathy was suspected as one of the
differentials and the wife was subjected to gene studies
for mutation in the MTM1 gene. A previously unreported
heterozygous mutation, p. F308del (c.924_926delCTT),
was detected in exon 10 on sequencing of all 14 exons of
the MTM1 gene in both forward and reverse directions. In
addition, a polymorphism (IVS11+3G>A) was also
detected. The couple was counseled regarding the X-
linked inheritance of the myotubular myopathy.
DISCUSSION
Congenital myopathies comprise one-tenth of all cases of
neuromuscular disorders, occurring with a combined
incidence of 0.06/1000 live births.2 Myotubular myopathy
occurs less frequently than the more common central core,
multi-minicore disease and nemaline rod myopathies. The
incidence in molecularly confirmed myotubular
myopathy in France is estimated at 2/100,000 male births
per year.2 The incidence of this disorder is not known in
India. To our knowledge, this is the first case report of this
disorder from India. The paper also documents a
previously unreported mutation in the MTM1 gene
(c.924_926delCTT) detected in case 3.
The X-linked form of myotubular myopathy is usually
associated with a severe disease course leading to death in
wks to months. In a genotype-phenotype correlation
study performed in 116 cases, only 17 individuals were
classified as mild and seven with intermediate
phenotype.3 Patients with recessive mutations in the
amphiphysin 2 (BIN1)4 gene and dominant mutations in
the dynamin 2 (DNM2) gene5 have better prognosis and
may even improve over time. There is no known
treatment for the disorder. The management is essentially
supportive and/or rehabilitative, requiring a
multidisciplinary approach.2, 6 The decision regarding
duration of ventilatory support requires serious
consideration and knowledge of the gene involved, in
view of the difference in prognosis in all three types of
disease.
Congenital hypotonia is not an uncommon
presentation in the neonatal period, and needs
comprehensive evaluation for the cause, because many of
them have a genetic basis and thus, associated with a
recurrence risk. Neuromuscular causes of hypotonia are
an important group to exclude, even though majority of
the cases of hypotonia have a central origin.7 As most of
the babies presenting with congenital hypotonia are sick
and may not survive for long, hence there is a need for
quick evaluation of these newborns. As has happened in
our case 3, the family seeks advice only after the death of
one or more neonates, in which case the disorder often
remains undiagnosed. Thus, it is very important to save
the DNA of the affected child (blood in EDTA vial to be
sent to nearest genetic laboratory) in any undiagnosed
floppy infant to enable gene studies for accurate
diagnosis.
CONCLUSION
X-linked inheritance is the most common and the most
severe fom of myotubular (centronuclear) myopathy. The
diagnosis is established on a muscle biopsy, which greatly
helps in counseling for a couple’s future pregnancy
outcomes. Prenatal diagnosis is possible by mutation
analysis of gene implicated in the disorder, performed on
chorionic villus sampling at 10-12 wk of pregnancy.
Acknowledgements
The authors acknowledge the contributions of Dr J Mandel,
Laboratoire de Diagnostic Génétique, Faculté de Médecine et
CHRU, Strasbourg, France and Dr Soma Das, Department of
Human Genetics, University of Chicago, USA for carrying out
mutation studies in cases 1 & 3, respectively.
Contributions: SB; Clinical management of case 2 and preparation
of manuscript, RDP; Clinical management of cases 2 & 3 & assisted
in manuscript preparation, MJ; Histopathology studies in case 2,
NK; Clinical management of case 2 SR; Histopathology studies in
case 2, JAU; Facilitated mutation studies in case 2, VB; Performed
mutation studies in case 1 and 2, ICV; Clinical management of cases
1, 2 & 3, finalized the manuscript.
Conflict of Interest : None.
Role of Funding Source : None.
REFERENCES
1. Wallgren-Pettersson C, Jungbluth H. Congenital myopathies.
In Rimoin DL, Connor JM, Pyeritz RE, Korf BR, eds. Emery and
Rimoin’s Principles and Practice of Medical Genetics, 5th ed. Vol.
3., Churchill Livingstone 2007; 2971-2973.
2. Jungbluth H, Wallgren-Pettersson C, Laporte J. Centronuclear
(myotubular) myopathy. Orphanet J Rare Diseases 2008; 3: 26.
3. McEntagart M, Parsons G, Buj-Bello A et al. Genotype-
phenotype correlations in X-linked myotubular myopathy.
Neuromuscul Disord 2002; 12: 939-946.
4. Nicot AS, Toussaint A, Tosch V et al. Mutations in
amphiphysin 2 (BIN1) disrupt interaction with dynamin 2 and
cause autosomal recessive centronuclear myopathy. Nat Genet
2007; 9: 1134-1139.
5. Bitoun M, Maugenre S, Jeannet PY et al. Mutations in dynamin
2 cause dominant centronuclear myopathy. Nat Genet 2005;
37: 1207-1209.
6. Fujimura-Kiyono C, Racz GZ, Nishino I. Myotubular/
centronuclear myopathy and central core disease. Neurol India
2008; 56: 325-332.
7. Harris SR. Congenital hypotonia: clinical and developmental
assessment. Dev Med Child Neurol 2008; 50: 889-892.