Digenic mutational inheritance of the integrin alpha 7 and the myosin heavy chain 7B genes causes congenital myopathy with left ventricular non-compact cardiomyopathy
Orphanet Journal of Rare Diseases
Digenic mutational inheritance of the integrin alpha 7 and the myosin heavy chain 7B genes causes congenital myopathy with left ventricular non-compact cardiomyopathy
Teresa Esposito 0
Simone Sampaolo 2
Giuseppe Limongelli 1
Antonio Varone 3
Daniela Formicola 0 2
Daria Diodato 2
Olimpia Farina 2
Filomena Napolitano 0
Giuseppe Pacileo 1
Fernando Gianfrancesco 0
Giuseppe Di Iorio 2
0 Institute of Genetics and Biophysics Adriano Buzzati-Traverso, National Research Council of Italy , Naples , Italy
1 Department of Cardiological Sciences, Second University of Naples , Naples , Italy
2 Department of Medical Sciences , Surgery, Neurological, Metabolic and Aging , Second University of Naples , Naples , Italy
3 Department of Neuro-sciences, Santobono-Pausilipon Hospital , Naples , Italy
Background: We report an Italian family in which the proband showed a severe phenotype characterized by the association of congenital fiber type disproportion (CFTD) with a left ventricular non-compaction cardiomyopathy (LVNC). This study was focused on the identification of the responsible gene/s. Methods and results: Using the whole-exome sequencing approach, we identified the proband homozygous missense mutations in two genes, the myosin heavy chain 7B (MYH7B) and the integrin alpha 7 (ITGA7). Both genes are expressed in heart and muscle tissues, and both mutations were predicted to be deleterious and were not found in the healthy population. The R890C mutation in the MYH7B gene segregated with the LVNC phenotype in the examined family. It was also found in one unrelated patient affected by LVNC, confirming a causative role in cardiomyopathy. The E882K mutation in the ITGA7 gene, a key component of the basal lamina of muscle fibers, was found only in the proband, suggesting a role in CFTD. Conclusions: This study identifies two novel disease genes. Mutation in MYH7B causes a classical LVNC phenotype, whereas mutation in ITGA7 causes CFTD. Both phenotypes represent alterations of skeletal and cardiac muscle maturation and are usually not severe. The severe phenotype of the proband is most likely due to a synergic effect of these two mutations. This study provides new insights into the genetics underlying Mendelian traits and demonstrates a role for digenic inheritance in complex phenotypes.
Left ventricular noncompact cardiomyopathy; Congenital type fiber disproportion; Integrin alpha 7 (ITGA7); Myosin heavy chain 7B (MYH7B); Whole exome sequencing
Congenital fiber type disproportion (CFTD) is a form of
congenital myopathy in which consistent type 1 fiber
hypotrophy relative to type 2 fibers is the main histological
abnormality . The health impairments commonly
encountered in CFTD are similar to those of many other
congenital myopathies. Muscle weakness is usually
relatively stable or slowly progressive during childhood and
adolescence. In general, scoliosis and joint contractures
(other than mild Achilles tendon contractures) are
relatively uncommon. Mutations associated with CFTD have
been found in the TPM3 (MIM# 191030), ACTA1 (MIM#
102610), SEPN1 (MIM# 606210), RYR1 (MIM# 180901),
TPM2 (MIM# 190990) and MYH7 (MIM# 160760) genes
[2-8]. Despite recent advances, no genetic cause has been
found in at least 50% of CFTD patients.
Left ventricular non-compaction cardiomyopathy (LVNC)
has been reported in patients with different types of
neuromuscular disorders but has never been associated with
CFTD. Within the last 2 decades, more than 200 cases of
LVNC have been described . The hallmark features of
this cardiomyopathy include prominent trabeculations
and deep endocardial recesses. Symptoms associated with
LVNC are variable and can include arrhythmias,
thromboembolic events and heart failure. At least 4 genes that
cause very rare cardiomyopathy diseases have been found
to be linked to non-syndromic LVNC in familial or
sporadic cases. Thus far, the X-linked gene known as G4.5 or
taffazin (TAZ) has been found to be associated with the
largest number of LVNC cases . Other genes found to
be mutated in LVNC include alpha-dystrobrevin (DTNA),
Cypher/ZASP and lamin A/C [11-13]. Cumulatively, these
genes have been shown to be associated with only a small
percentage of sporadic or familial cases. Over the past few
years, mutations in genes encoding sarcomere proteins,
the beta-myosin heavy chain (MYH7), alpha-cardiac actin
(ACTC1), cardiac troponin T (TNNT2), cardiac
myosinbinding protein C (MYBPC3), alpha-tropomyosin (TMP1)
and cardiac troponin I (TNNI3) have been identified in a
significant proportion of patients with LVNC [14-16].
We report an Italian family in which the proband showed
a severe disease phenotype characterized by CFTD and
LVNC. The clinical and instrumental analysis of the family
members identified the LVNC phenotype in the mother,
the sister and the first-degree cousin of the proband,
suggesting that the LVNC cardiomyopathy segregated
as a dominant mode of inheritance with high
phenotypic heterogeneity and reduced penetrance. CFTD
phenotype showed a recessive mode of inheritance.
To our knowledge, this is the first example of LVNC
cardiac phenotype associated with CFTD. We provide a
full description of this new phenotype and of the
identification of the disease-causing genes by whole-exome
Patients and methods
Family and unrelated cohort
A single, multigenerational Italian pedigree was involved
in this investigation, as depicted in Figure 1. Phenotypic
data were available for 10 individuals (3 males and 7
females) ranging in age from 4 to 50. All individuals were
of Caucasian ancestry. DNA was available for 6 individuals
(IV-10, IV-13, IV-14, V-1, V-4, V-5). We extended the
clinical and instrumental (EMG, ECG, Echocardiography,
muscle biopsy) evaluation to the whole family. We
identified a mild LVNC phenotype (according to the Jenni
criteria) in the mother (IV-14), sister (V-5) and cousin (V-1)
of the proband, with hypertabeculations of the mid
inferolateral wall and of the ape and without
neuromuscular disorders . During the growth, there was
a reduction of clinical signs in patients V-1 and V-5.
No data have been collected for individual IV-9, who died
at the age of 35 of unknown causes. The father (IV-13) of
the proband was asymptomatic.
An unrelated cohort (UC) of 24 patients (including
LVNC14-BG), all fulfilling the criteria for LVNC, were
also analyzed in this study.
Informed consent was obtained from each patient. In
the case of minors, parental consent was obtained. The
study protocol conforms to the ethical guidelines of the
1975 Declaration of Helsinki, as reflected in the a priori
approval by the institutions human research committee.
Whole-exome sequencing strategy
For all participants over 18 years of age, DNA was extracted
from peripheral blood specimens using a standard
saltingout procedure. Genomic DNA samples from two affected
females in the pedigree (the proband V-4, affected by
CFTD and LVNC, and her cousin V-1, affected by LVNC)
were captured with the NimbleGen SeqCap EZ Exome
capture kits (Roche, Indianapolis, IN, USA)) and
sequenced with one lane per sample on an Illumina GAIIx
(Illumina, San Diego, CA, USA) with 90-bp
Sequences were aligned to the human reference genome
sequence (GRCh37/hg19) with the MAQ7 and NextGENe
software v2.00 with sequence condensation by
consolidation (SoftGenetics, State College, PA, USA). This approach
resulted in more than 40 of target exome coverage.
Single nucleotide variants (SNVs) were called with MAQ
and NextGENe. Small insertions and deletions were
detected with NextGENe. Called SNVs were annotated with
SeattleSeq Annotation and filtered with dbSNP130.
A mutation analysis of already-known genes for CFTD
and LVNC and validation and segregation analyses of
the selected variants obtained from exome sequencing,
were performed by Sanger sequencing.
Figure 1 The complete 5-generational Italian pedigree. The proband affected by CFTD and LVNC is indicated with an arrow and a dark circle.
The mother, sister and cousin of the proband, affected by LVNC, are indicated with gray circles.
Each variant was amplified and sequenced, as described
in our previous studies [19,20]. Variations were detected
by multiple-sequence alignments using the Autoassembler
program (Applied Biosystems).
The SIFT and PolyPhen2 software packages (http://sift.bii.
bwh.harvard.edu/pph2/) were used to assess the
deleterious effects of the mutations.
To assess the minor allele frequencies of the variants,
the dbSNP (http://www.ncbi.nlm.nih.gov/snp) and the 1000
databases were surveyed.
To examine the expression pattern of the MYH7B and
ITGA7 genes, real-time PCR was performed on total RNAs
from human adult tissues purchased from Stratagene and
on total RNAs derived from the blood of family members
using the LightCycler system DNA Engine Opticon 2 (MJ
Research). The detailed protocol has already been
The expression levels were normalized to
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to account
for differences in the starting material and in the cDNA
reaction efficiency. Agarose gel electrophoresis was performed
to further confirm the specific PCR products.
MYH7B-cDNA-F 5 GTCTGGGTGCCTGATGAACA 3
MYH7B-cDNA-R 5 CTCGTTCAGGTGCGTCATCA 3
ITGA7-IS1-F 5 GGATGGTGGGGAATGGAAGT 3
ITGA7-IS1-R 5 GGTCAGCAGGGTCCAAAGTT 3
ITGA7-IS2-R 5 GCGGGGGTCCTGCTCTTCT 3
ITGA7-IS3-F 5 CAGAGGCAGGCAGAAGGATT 3
The primer ITGA7-IS2-R was combined with either
ITGA7-IS1-F or ITGA7-IS3-F.
The GAPDH primers were as follows: forward primer,
5 AGCCACATCGCTCAGACAC 3, and reverse primer,
5 GATCTCGCTCCTGGAAGATG 3.
Clinical features of patient V-4
The proband (V-4) is the first child of a family with a
history of consanguinity (mothers parents are first-degree
cousins) and diabetes in the maternal line (grandmother,
one great-uncle, one uncle and a first-degree cousin)
without neuromuscular diseases (Figure 1). Since birth, she
manifested hypotonia, poor sucking and persistent crying.
Persistent arterial duct and patent foramen ovale were
diagnosed at birth but resolved spontaneously, as shown
by echocardiography at the age of 1 month. At 3 months
of age, she was diagnosed with congenital dislocation of
the left hip, and at 17 months of age, she was
hospitalized because of body weight below the third percentile
and hypoglycemia. The failure to thrive was due to
difficulties in chewing and swallowing. ECG showed a long
QTc (478 msec). Echocardiography showed a slight left
ventricular (LV) dilatation, a moderate reduction of the
LV global function (ejection fraction 40%), and a
noncompacted aspect of the overall infero-lateral wall. She
was administered ace-inhibitors, diuretics and digitalis,
with a significant improvement in the clinical state. The
karyotype and FISH were normal. The serum creatine
kinase (CK) was within the normal range. The patient
was followed up for 8 years.
At 8 years of age, she was admitted at our department
for a neuromuscular and cardiological evaluation.
The neurologic examination revealed the following: a
waddling gait, an inability to stand up from a sitting
position, a slight degree of weakness of the facial and
masticator muscles, a mild weakness of the glutei and ileo-psoas
and marked bilateral quadriceps. She developed marked
scoliosis due to congenital hip dysplasia (Figure 2).
Tendon reflexes were absent. Joints contractures had never
been observed. A blood test confirmed hypoglycemia and
Figure 2 The proband at 10 years of age. Note the marked
scoliosis, the leg length discrepancy and the left quadriceps
hypotrophy; the abduction and elevation of the arms are impaired.
normal levels of serum creatine kinase and isoenzymes.
An EEG and a brain MRI showed no abnormality. A
quadriceps muscle biopsy displayed a predominance of
type 1 fibers (76%), the mean diameter of which was
30% smaller than that of the type 2 fibers; the latter
were hypertrophic for the age (mean diameter of 35 m)
(Figure 3). No internal nuclei, nemaline bodies or cores
were observed. Dystrophin, emerin, calpain and lamin A/C
were examined and found to be normal. This allowed us
to exclude other neuromuscular disorders associated with
the myopathy. These findings were compatible with the
diagnosis of CFTD.
The cardiological investigation confirmed a long QTc
and showed signs of left ventricular hypertrophy and
repolarization abnormalities in the ECG. The
echocardiography showed a dilated left ventricle (left ventricular end
diastolic diameter z-score +3) with hypertrabeculation
of the antero-lateral, postero-lateral and inferior walls
(non-compacted/compacted ratio ~3), a severe
impairment of the LV global function (ejection fraction 35%), a
moderate mitral regurgitation, an abnormal mitral inflow
pattern (E/A ratio 0.8), and a mild increase in the
pulmonary artery pressure (40 mmHg) with normal non-invasive
pulmonary wedge pressure (E/Ea ratio 8) (Figures 4A and
4B). A 24-hour Holter ECG monitoring revealed 5 short
runs of supraventricular tachycardias. The patient was
administered with carvedilol and ace-inhibitors, with
the prescription of routine cardiac evaluations (every
At the last cardiological evaluation (10 years of age),
she was asymptomatic for dyspnoea (NYHA class I) and
in therapy with carvedilol 6.25 mg cp 2 times/day,
losartan 50 mg cp/day, spironolattone 25 mg cp/day,
furosemide 25 mg 1 cp/day and cardioaspirin 100 mg
1cp/day. The ECG confirmed a prolonged QTc (485 msec),
and the echocardiography showed a non-compacted LV
with 38% ejection fraction and slightly elevated
pulmonary artery pressure (45 mmHg). NT-proBNP was elevated
(1858 pg/ml). A cardiac MRI analysis confirmed the
diagnosis of LVNC, with a significant reduction of ejection
fraction to 40% (Figure 4C).
One month before the cardiovascular appointment, the
patient (11 years old) died suddenly early in the morning,
while lying in bed.
Gene identification strategy
Whole-exome sequencing of the proband, affected by
CFTD and LVNC, and her cousin, affected by LVNC,
was used as a strategy to identify the responsible gene/s.
At first, we performed a thorough survey of all
previously identified CFTD and LVNC genes to definitively
exclude their involvement in the disease phenotype. Only
intronic polymorphic variants were detected in TPM3,
ACTA1, TPM2, MYL2, MYL3, G4.5, DTNA, ZASP, ACTC1,
Figure 3 Right quadriceps muscle biopsy (6-m-thick cryostatic sections, 10). (A) H-E: moderate fiber caliber variability and diffuse mild
fibrosis; (B) Trichrome of Gomori: absence of mitochondrial accumulation; (C) NADH-Tr: no oxidative enzymes change; (D) ATPase pH 4.3: a clear
predominance of type I (dark) fibers, which show diameters smaller than those of type 2 fibers (clear).
MYBPC3 and TMP1. Both intronic and coding
polymorphic variants were detected in SEPN1, RYR1, MYH7,
LMNA/C, TNNT2 and TNNI3 (Table 1). After filtering
the data with dbSNP130 and data from six in-house
exomes (1 healthy individual and 5 individuals with
unrelated diseases; Table 2), we performed two models of
analysis to follow both phenotypes: LVNC that
segregated as dominant and CFTD that showed a recessive
mode of inheritance. For the dominant model, we matched
data from both individuals (V-1/V-4) to obtain shared
variants. In total, 93 autosomal non-synonymous coding
variants were shared between the two samples. Only 56 of
these variants were confirmed by direct sequencing in the
two patients. Ten variants showed complete segregation
with the LVNC phenotype in the family but were also found
in the healthy population with a minor allele frequency
(MAF) >1%. Only one of these, R890C in myosin, heavy
chain 7B, cardiac muscle, beta (MYH7B), was found in the
1000 Genomes database with a MAF < 0.05% and was
predicted to be a deleterious change by bioinformatics tools.
For the recessive model, only the proband was
considered to be affected. We then selected all genes carrying
two mutations. In total, 46 homozygous changes were
selected, but only 10 were confirmed by direct
sequencing and analyzed in the family samples. It is important
to note that the parents of individual IV-14 (mother of
the proband) are first-degree cousins, so she is
homozygous for many variants and represents a good sample to
filter data derived from the proband. In fact, 9 of the 10
variants were excluded because they were homozygous
both in the proband and in her mother, who did not
show the CFTD phenotype. Only the E882K variant, in
the integrin, alpha 7 (ITGA7) gene, segregated with the
CFTD phenotype in the family.
The compound heterozygous model produced 50 genes,
of which only 5 continued to carry two mutations after
direct sequencing analysis; none of the 50 genes
segregated with the disease phenotype in the family because
they were also present in the sister of the proband (V5),
who showed no CFTD phenotype.
Figure 4 Cardiological features. (A) B-mode Echocardiography: ipertrabeculation/non-compaction left ventricle cardiomyopathy (white arrows).
(B) Colordoppler-Echocardiography: blood flowing into the intertrabecular spaces (white arrow) communicating with the left ventricle lumen.
(C) Cardiac MRI: a T1 weighted image clearly showing the left ventricle wall trabeculation (white arrow).
Table 1 Summary of the coding polymorphic variants
found in CFTD-LVNC genes
MAF minor allele frequency.
The myosin heavy chain 7B (MYH7B) gene
MYH7B [GenBank: NG_016984.1] is located on
chromosome 20 and is split into 45 exons, 43 of which code for
protein. The nucleotide substitution c.2668 C > T is located
in exon 27 and causes the non-synonymous change of the
amino acid arginine at position 890 into cysteine (R890C).
The inheritance of the R890C mutation was examined
by Sanger sequencing of the DNA of the family individuals
(Figure 5A). It was found to be homozygous in samples
IV-14 and V-4 and to be heterozygous in IV-13, V-1 and
V-5; it was absent in IV-10. This finding suggests the
segregation of the mutation with the LVNC disease
phenotype in our family; however, its reduced penetrance and
variable expressivity need to be considered because the
mutation is also carried by the asymptomatic subject
IV-13. To further validate our finding, we analyzed the
R890C mutation in a panel of 24 unrelated LVNC patients
from the same geographical area and found it in patient
Table 2 Summary of the whole exome sequencing of CFTD-LVNC samples
NS after filter dbSNPs
NS after filter 6 exomes*
Dominant Model + validation**
Dominant Model (deleterious)
Recessive Model homozygous
Recessive Model + validation
Recessive Model (deleterious)
Recessive Model heterozygous
Recessive Model + validation
Recessive Model (deleterious)
SNV single nucleotide variation; *: non synonymous variants obtained after to filter with six in house genome; Dominant Model: number of gene in which at least
one variant is shared between the two samples; Dominant Model + validation**: autosomal variants shared between the two samples and remaining after filtering
with 1000 genome database and validated with direct sequencing; Dominant Model (deleterious): variants predicted as deleterious effect of the change with SIFT;
Recessive Model: genes in which two alleles are mutated.
LVNC-14 (BG), who showed a classical LVNC phenotype
with severe systolic dysfunction (25%) (Figure 5A).
We then analyzed 600 chromosomes from an unrelated
healthy population from the same geographical origin.
This mutation was absent in this panel but was present in
the 1000 Genomes database with a MAF < 0.006%.
Most variants underlying rare Mendelian diseases either
affect highly conserved sequences and/or are predicted to
be deleterious. For this reason, we analyzed the R890C
mutation with the SIFT and PolyPhen2 software to
confirm a deleterious effect. At this position (R890), only
arginine (R), lysine (K), serine (S), asparagine (N) or
glutamine (Q) are tolerated (data not shown). The
PolyPhen2 analysis supported the notion that this variant is
damaging, with the highest probability score of 1.
The MYH7B gene belongs to the MYH gene family,
which, in humans, also includes the MYH6 and MYH7
genes, both clustered on chromosome 14. Evolutionary
conservation analysis shows high similarity for these
proteins. Interestingly, the amino acid arginine (R) at position
890 is highly conserved in all species, but in MYH6 and
MYH7 and their homologs, the arginine (R) is replaced
with lysine (K) (Figure 5B). However, lysine is one of the
amino acids that SIFT predicted to be tolerated at this
protein position. Using a bioinformatics simulation model
(http://minnou.cchmc.org/), we demonstrated that the
R890C mutation induces a conformational change in
the MYH7B molecule [GenPept: AAI51243.1], leading
to the alteration of the beta sheet and alpha helix
structures (Figure 6).
A significant expression of the MYH7B gene was
observed in heart and muscle tissues, and very low expression
was observed in the brain, testes, ovary, liver and blood.
No expression was observed in the kidney, lung and
pancreas. No difference in expression was observed in blood
RNA derived from both affected and healthy subjects from
our family (Figure 5C).
The integrin, alpha 7 (ITGA7) gene
The ITGA7 gene [GeneBank: NG_012343.1] is located
on chromosome 12 and is split into 28 exons, 26 of
which code for protein. This gene has 23 transcripts,
but only 10 are protein coding. The homozygous
nucleotide substitution c.2644 G > A is located in exon 20
and causes the non-synonymous change E882K in
transcript 1 [GeneBank: NM_001144996.1]. The position of this
mutation is E886K in isoform 2 [GeneBank: NM_002206.2]
and E789K in isoform 3 [GeneBank: NM_001144997.1].
The inheritance of this mutation was examined by
Sanger sequencing of the DNA of the family individuals.
Our analysis confirmed that this mutation was present
in a homozygous form only in the proband with the
CFTD phenotype; her parents were heterozygous for this
change (Figure 7A).
In the 1000 Genomes database, this change is reported
as number rs144983062; the heterozygous genotype C/T
Figure 5 Molecular analysis of MYH7B gene mutation. (A) Mutation analysis of the MYH7B gene. Arrowheads mark the sites of base
alterations. The DNA sequences of the family subjects are shown; the pedigree number is indicated on the right. (B) Evolutionary conservation
analysis. Arginine 890 is underlined. The MYH7 amino acids mutated in cardiomyopathy are shown in red. (C) MYH7B mRNA expression in human
tissues and in RNA derived from family members. M indicates the 1 kb Plus marker (Fermentas).
Figure 6 Secondary structures prediction of MYH7B. (A) Secondary structures predictions for the MYH7B-890R (left) and MYH7B-890C (right)
proteins. The program also performed an accurate prediction of the real-valued relative solvent accessibility. The 890 R to C amino acid
substitution changes the beta strands (green arrows) and alpha helix (red lines) structures of the MYH7B protein. All changes are indicated with
black arrows. (B) Enlarged area with the region containing the mutation highlighted.
was found in 12 of the 4540 samples analyzed (freq of
the CT genotype = 0.003), and the homozygous genotype
TT was not found. We analyzed 600 chromosomes from
an unrelated healthy population from the same
geographical area of the proband but did not find the
mutation. Therefore, we analyzed the E882K mutation with
the SIFT and PolyPhen2 software, which confirmed a
deleterious effect of this variant. Evolutionary
conservation analysis showed that the missense change E882K
affects a highly conserved residue of the integrin alpha 7
protein (Figure 7B). Moreover, bioinformatics simulation
modeling demonstrated that the E882K mutation induces
a conformational change in the ITGA7 molecule, resulting
in the alteration of the beta sheet structures (Figure 8).
Expression profiling was performed for three transcripts
of the gene. Isoform 1 was significantly expressed only in
heart, muscle, liver and blood tissues. No difference in
expression was detected between blood RNAs derived
from affected and healthy subjects of our family.
Isoform 2 was significantly expressed in heart, muscle, testes
and ovary tissues; low levels of expression were detected
in the brain, kidney, lung, pancreas and liver; no
expression was detected in the blood. Isoform 3 was detected at
We describe an unusual association between CFTD and
LVNC and link this unusual severe phenotype to digenic
inheritance, a novel type of transmission that is emerging
with the application of novel powerful genomic
technologies, such as whole-exome sequencing . The CFTD
myopathy is a genetically heterogeneous disorder
characterized by relative hypotrophy of type 1 muscle fibers
compared to type 2 fibers in skeletal muscle biopsies .
The diagnosis of CFTD is made for exclusion because
these findings are not specific and can be found in several
neuromuscular diseases. The term fiber size
disproportion has been suggested for describing this specific
histological picture, reserving the term CFTD for those cases
in which no secondary cause can be found .
Cardiovascular involvement has been rarely reported in patients
with CFTD myopathy. Banwell et al. described 2
unrelated children with cardiac involvement . One child,
with a dilated type cardiomyopathy, developed an
intractable congestive heart failure, necessitating cardiac
Figure 7 Molecular analysis of ITGA7 gene mutation. (A) Mutation analysis of the ITGA7 gene. Arrowheads mark the sites of base alterations.
The DNA sequences of the family subjects are shown; the pedigree number is indicated on the right. (B) Evolutionary conservation analysis. The
glutamic acid at position 882 is underlined. (C) ITGA7 mRNA expression in human tissues and in RNA derived from family members. M indicates
the 1 kb Plus marker (Fermentas).
transplantation at the age of 13 years. The second, a
1-year-old child without cardiomyopathy or congenital
heart diseases, developed a high-rate atrial fibrillation,
requiring treatment with digoxin. The association of DCM
with CFTD has also been reported in two Japanese
patients [25,26]. However, a defined geno-phenotype
correlation has not been established in these cases. LVNC
has been reported in patients with different types of NMDs,
including dystrophinopathy, laminopathy, zaspopathy,
myotonic dystrophy, Barth syndrome, Friedreich ataxia,
CharcotMarie-Tooth disease, and metabolic and mitochondrial
Nevertheless, considering the rarity of these pathological
conditions, which are caused by a premature arrest of
skeletal and cardiac muscle development, the hypothesis
of a common pathogenesis is intriguing.
Our data suggest that rather than the action of a single
gene, a synergic effect of homozygous mutations in two
genes, i.e., myosin heavy chain 7B (MYH7B) and integrin
alpha 7 (ITGA7), underlies the phenotype observed in
the proband (V-4). Both genes are crucial for the
physiological development of skeletal and cardiac muscles.
However, a single-gene mutation responsible for both DCM/
LVNC and CFTD in other patients cannot be conclusively
Myosin, the molecular motor responsible for muscle
contraction, exists in multiple forms, which dictate muscle
properties, such as shortening velocity and contractile
force. The majority of MYH genes known to be present
in mammals are associated in two highly conserved gene
clusters [29,30]. MYH6 and MYH7 are two tandemly
arrayed genes located on human chromosome 14, which
code for the cardiac myosins, - and -MYH; -MYH is
also expressed in slow skeletal muscles. Another gene
cluster, located on human chromosome 17, codes for
the six skeletal myosins, including the adult fast 2A-,
2Xand 2B-MYH, the developmental embryonic and neonatal/
perinatal isoforms, and MYH13, an isoform expressed
Figure 8 Secondary structures prediction of ITGA7. (A) Secondary structures predictions for the ITGA7-882E (left) and ITGA7-882 K (right)
proteins. The program also performed an accurate prediction of the real-valued relative solvent accessibility. The 882 E to K amino acid
substitution changes the beta strands (green arrows) and alpha helix (red lines) structures of the ITGA7 protein. All changes are indicated with
black arrows. (B) Enlarged area with the region containing the mutation highlighted.
specifically in extraocular muscles. Three additional genes
coding for sarcomeric MYHs, i.e., MYH7B, MYH15 and
MYH16, have been discovered recently. We showed that
MYH7B is expressed in the adult human heart and
muscle; moreover, Warkman and coworkers recently
determined that MYH7B is expressed in the myocardium
. Developmental analysis showed Myh7b expression
in cardiac and skeletal muscles of Xenopus, chick and
mouse embryos and in smooth muscle tissues during
the later stages of mouse embryogenesis . Heterozygous
mutations in eight sarcomere proteins (MYH7, ACTC1,
TNNT2, TNNI3, MYL2, MYL3, MYBPC3 and TPM1) have
been identified in a significant proportion of patients with
LVNC in adults and children [14-16,32,33]. Approximately
20% of LVNC patients carried a mutation in MYH7, which
was the most prevalent LVNC disease gene; missense
mutations were the most prevalent types of mutations
in all sarcomere proteins analyzed [16,32]. LVNC is
characterized by a trabecular meshwork and deep intertrabecular
myocardial recesses communicating with the left
ventricular (LV) cavity . Clinical features range from a
non-penetrant disease in adult carriers to heart failure,
arrhythmia and thromboembolism [34,35]. The penetrance
of a mutation is defined as the percentage of mutation
carriers expressing a phenotype, and most
autosomaldominant cardiomyopathies are characterized by
incomplete penetrance or more age-related penetrance .
There is also variable expressivity in cardiomyopathies,
and there can even be large differences among relatives
of the same family (intrafamilial variability) who carry
the same mutation. In our family, high phenotypic
variability and reduced penetrance were observed.
This is the first indication that a mutation in the MYH7B
gene causes LVNC cardiomyopathy. We demonstrated that
the arginine at position 890 of the MYH7B gene is highly
conserved in all species; this region is also conserved in the
MYH7 gene, which, when mutated, causes LVNC. These
data further support the concept that sarcomere genes are
associated with LVNC.
Integrin 71 is a specific cellular receptor for the
basement membrane protein laminin-1 and for the laminin
isoforms 2 and 4 [37,38]. The 7 subunit is expressed
mainly in skeletal and cardiac muscles and has been
suggested to be involved in differentiation and
migration processes during myogenesis [39-41].
Mice homozygous for a null allele of the Itga7 gene
are viable and fertile, indicating that the 71 integrin is
not essential for myogenesis. However, a histological
analysis of skeletal muscle revealed typical symptoms of
a progressive muscular dystrophy starting soon after birth,
but with a distinct variability in different muscle types
. The knock-down of zebrafish Itga7 results in muscle
fiber detachments similar to those observed in lama2
and lama4-deficient embryos . The human deficiency
in integrin 7 causes a mild disorder that is best
characterized as congenital myopathy. Three patients with
mutations in the ITGA7 gene have been described. One patient
had splice mutations on both alleles: one mutation caused
a 21-bp insertion in the conserved cysteine-rich region,
and the other caused a 98-bp deletion. A second patient
was a compound heterozygote for the same 98-bp deletion
and had a 1-bp frame-shift deletion in the other allele.
The third patient showed a marked deficiency in the
ITGA7 mRNA, but no mutations in the coding region
were described. In muscle biopsies, patients 1 and 3
showed a poorly defined congenital myopathy, which was
associated with mental retardation in patient 1. Patient 2
presented a clinical and pathological picture typical of
muscular dystrophy, with substantial fatty acid
replacement and fiber size variation (MIM 613204) .
Our proband (V-4) harbored a homozygous missense
mutation in a highly conserved region of the protein.
The typical pattern of CFTD was observed in the muscle
biopsy, characterized by the predominance of type 1 fibers
with smaller calibers than type 2 fibers, with no evidence
of either congenital muscular dystrophy or muscular
dystrophy. Clinically, she had no symptoms of a delay in
mental development, which occurs only in a few cases
of CFTD. Our proband and the above-mentioned cases,
showing muscle disorders present from birth, all
support the important role of ITGA7 in myogenesis. The
differences in their phenotypes may be related to their
diverse patterns of gene mutation.
This study identifies two novel disease genes. Mutation
in MYH7B causes a classical LVNC phenotype, whereas
mutation in ITGA7 causes CFTD. The synergic effect of
these two mutations causes the severe phenotype observed
in the proband. This study provides new insights into the
genetics of cardiomyopathy and congenital myopathy.
TE, DF, FN and FG performed the sequencing and expression analyses. SS,
GL, AV, DD, OF GP, and GDI recruited the family described herein and
collected the clinical data. TE, SS, GL, FG and GDI oversaw all aspects of the
research. TE and GDI initiated, planned and coordinated the study. TE, SS, GL
and GDI wrote the manuscript. All authors read, edited and approved the
final version of the manuscript.
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