A De novo Mutation in Dystrophin Causing Muscular Dystrophy in a Female Patient.

BACKGROUND: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular diseases resulting from dystrophin (DMD) gene mutations. It has been known that the carrier of DMD mutations may also have symptoms of the disease. While de novo mutation is quite common in BMD/DMD patients, it is rarely reported in the female carriers.
METHODS: Two sporadic Chinese patients with progressive muscular dystrophy and their familial members were recruited. The targeted next-generation sequencing (NGS) and the multiplex ligation-dependent probe analysis (MLPA) were performed in the proband. Blood tests, electrocardiography, echocardiography, and electromyography were also evaluated.
RESULTS: Two novel mutations of DMD gene were identified, c.7318C>T (p.Q2440*) in the male proband and c.4983dupA (p.A1662Sfs*24) in the female carrier. The MLPA analysis did not detect any large rearrangements. The haplotype analysis indicated that the two mutations were derived from de novo mutagenesis.
CONCLUSIONS: We identified two novel de novo mutations of DMD gene in two Chinese pedigrees, one of which caused a female patient with muscular dystrophy. The mutational analysis is important for DMD patients and carriers in the absence of a family history. The NGS can help detect the mutations in MLPA-negative patients.

INTRODUCTION

Mutations in the dystrophin (Duchenne muscular dystrophy [DMD]) gene, which encodes a protein connecting the cytoskeleton of muscle fibers, result in X-linked recessive dystrophinopathy, including DMD and Becker muscular dystrophy (BMD).[1] DMD is the most common type of muscular dystrophy, affects 1:3500 to 6000 live male births, and is characterized by weakness of pelvic and shoulder muscles starting in early childhood.[2] DMD is thought to be caused by the mutations causing totally nonfunctional dystrophin protein.[3] In comparison, a reduced amount or shortened dystrophin was thought to lead in BMD, which has a milder clinical manifestation and better prognosis. The first symptoms of BMD start with a mean age at 11 years, and the average clinical course can be more than 45 years.[4]

In general, the heterozygous female carriers of DMD mutations are asymptomatic, as long as the gene function is compensated by the other normal allele. However, 2.5–22.0% of these carriers can develop symptoms which varied from mild muscular weakness to severe clinical complications, which are defined as manifesting or symptomatic carriers.[567]

Although the de novo mutation is quite common in BMD/DMD patients,[8] it is rarely reported in the female carriers.[910] In this study, we identified two novel de novo DMD mutations in two Chinese pedigrees, including one in a manifesting female carrier. To our knowledge, this is the first report of a de novo DMD mutation in a Chinese female carrier.

METHODS

Ethical approval

This study was approved by the Ethics Committee for Human Research in Second Affiliated Hospital, Zhejiang University School of Medicine. Informed consents were obtained from all participants before enrollment in the study.

Subjects and ethics statement

Two sporadic Chinese patients with progressive muscular dystrophy and their familial members were recruited. Before the mutation analysis, both patients performed the routine blood tests, electrocardiography, and electromyography. The blood test included a full blood count, liver and renal function, electrolytes, thyroid function, serum cortisol, glucose, lactate, myocardial enzymes, erythrocyte sedimentation rate, antinuclear antibody, antinuclear cytoplasmic antibody, and tumor markers. The clinical diagnosis was based on progressive muscular weakness, muscle strength, elevated levels of creatinine kinase (CK), and myogenic changes on electromyography. Two hundred individuals without a history of muscular dystrophy were recruited as controls for mutation analysis.

Targeted next-generation sequencing

Genomic DNA was extracted from peripheral blood by QIAamp Blood Genomic Extraction Kits (Qiagen, Hilden, Germany). Targeted capture, library preparation, and sequence amplification of 43 related genes of muscular dystrophy were then performed [Table 1]. The sequencing was performed on the Illumina HiSeq2000 platform (Genergy Biotechnology Co. Ltd., Shanghai, China), and the detailed information of targeted next-generation sequencing (NGS) can be found in our previously reported studies.[1112]

Table 1

List of genes responsible for related muscular dystrophy

Number	Disease	Gene	OMIM gene	Locus
1	DMD, BMD	DMD	300377	Xp21.2-p21.1
2	EDMD1	EMD	300384	Xq28
3	EDMD4	SYNE1	608441	6q25.1-q25.2
4	EDMD5	SYNE2	608442	14q23.2
5	EDMD6	FHL1	300163	Xq26.3
6	EDMD7	TMEM43	612048	3p25.1
7	LGMD1A, MFM3	MYOT	604103	5q31.2
8	LGMD1B, EDMD2/3	LMNA	150330	1q22
9	LGMD1C	CAV3	601253	3p25.3
10	LGMD1E	DNAJB6	611332	7q36.3
11	LGMD1F	TNPO3	610032	7q32.1
12	LGMD1G	HNRPDL	607137	4q21
13	LGMD2A	CAPN3	114240	15q15.1
14	LGMD2B, MMD1	DYSF	603009	2p13.2
15	LGMD2C	SGCG	608896	13q12.12
16	LGMD2C1/2K	POMT1	607423	9q34.13
17	LGMD2C14/2T	GMPPB	615320	3p21.31
18	LGMD2C2/2N	POMT2	607439	14q24.3
19	LGMD2C3/2O	POMGNT1	606822	1p34.1
20	LGMD2C4/2M	FKTN	607440	9q31.2
21	LGMD2C5/2I	FKRP	606596	19q13.32
22	LGMD2C7/2U	ISPD	614631	7p21.2
23	LGMD2C9/2P	DAG1	128239	3p21.31
24	LGMD2D	SGCA	600119	17q21.33
25	LGMD2E	SGCB	600900	4q12
26	LGMD2F	SGCD	601411	5q33.2-q33.3
27	LGMD2G	TCAP	604488	17q12
28	LGMD2H	TRIM32	602290	9q33.1
29	LGMD2J	TTN	188840	2q31.2
30	LGMD2L, MMD3	ANO5	608662	11p14.3
31	LGMD2Q	PLEC	601282	8q24.3
32	LGMD2S	TRAPPC11	614138	4q35.1
33	LGMD2V	GAA	606800	17q25.3
34	LGMD2W	LIMS2	607908	2q14
35	MFM1, LGMD2R	DES	125660	2q35
36	MFM2	CRYAB	123590	11q23.1
37	MFM4	LDB3	605906	10q23.2
38	MFM5	FLNC	102565	7q32.1
39	MFM6	BAG3	603883	10q26.11
40	UCMD1, BTHLM1	COL6A1	120220	21q22.3
41	UCMD1, BTHLM1	COL6A2	120240	21q22.3
42	UCMD1, BTHLM1	COL6A3	120250	2q37.3
43	UCMD2, BTHLM2	COL12A1	120320	6q13-q14

DMD: Duchenne muscular dystrophy; BMD: Becker muscular dystrophy; EDMD: Emery-Dreifussmuscular dystrophy; LGMD: Limb-girdle muscular dystrophy; MFM: Myofibrillar myopathy; MMD: Multi-minicore disease; UCMD: Ullrich congenital muscular dystrophy; BTHLM: Bethlem myopathy.

Sanger sequencing

Sanger sequencing was performed on ABI 3500xL Dx DNA Genetic Analyzer (Thermo Fisher Scientific, USA) with a procedure described previously.[13] All familial members and 200 controls were sequenced to confirm the identified mutations. The results were mapped and analyzed according to the standard DMD reference sequence (GenBank transcript ID: NM_004006.2).

Multiplex ligation-dependent probe analysis

Multiplex ligation-dependent probe analysis (MLPA) was used to detect the large rearrangements in the DMD gene.[8] MLPA was performed using the SALSA MLPA kit P034/P035 DMD (MRC-Holland, The Netherlands) according to the manufacturer's protocol. The cutoff values for duplication and deletion were set as >1.2 and <0.7, respectively.

Haplotype analysis

To verify that the family members were genetically related, we performed haplotype analysis based on 15 single-nucleotide polymorphisms (SNPs), including the SNPs within the DMD and the regions flanked DMD [Table 2]. The SNPs which had a linkage disequilibrium with r2 >0.8 were rule out using Haploview 4.2 software (Broad Institute, MA, USA).[14] The allele frequency was referred to the frequency of correspondent SNPs in Eastern Asian from Exome Aggregation Consortium (ExAC) variants database. The likelihood ratio for coparentage was calculated for each SNP and multiplied together to acquire a cumulative coparentage index (CPI).[15] The probability of the proband being the biological child of the alleged parents was calculated as P = CPI/(1 + CPI).

Table 2

Haplotype analysis based on 15 SNPs

Number	SNP	Position	Gene	Alleles	AF (1)	Family 1			Family 2
						Gi	Gm	LCP	Gi	Gm	Gp	LCP
1	rs3815049	13753457	OFD1	1(A)/2(G)	0.4479	1	1/2	0.91	1/2	1/1	2	2.02
2	rs2229137	19357664	PDHA1	1(A)/2(C)	0.2537	1	1/1	1.34	1/2	1/2	2	1.32
3	rs1800280	31478233	DMD	1(G)/2(A)	0.9234	1	1/1	13.05	2/2	2/2	2	1.17
4	rs2270672	31657979	DMD	1(C)/2(T)	0.3047	1	1/1	1.44	1/2	1/1	2	2.36
5	rs2222852	32035545	DMD	1(C)/2(G)	0.3523	1	1/1	1.54	1/2	1/2	1	1.10
6	rs7691	40600788	ATP6AP2	1(T)/2(C)	0.1773	1	1/1	1.22	2/2	1/2	2	15.91
7	rs145478020	47846285	ZNF81	1(C)/2(T)	0.1685	1	1/1	1.20	1/2	1/2	1	1.78
8	rs41305391	70330248	KIF4A	1(T)/2(C)	0.1480	1	1/1	1.17	2/2	1/2	2	22.83
9	rs72630048	72495283	HDAC8	1(C)/2(T)	0.7711	2	2/2	1.30	2/2	2/2	2	1.68
10	rs4148837	75114852	ABCB7	1(A)/2(G)	0.9297	1	1/2	7.11	2/2	2/2	2	1.16
11	rs2227291	78013005	ATP7A	1(G)/2(C)	0.2759	1	1/1	1.38	1/2	1/2	1	1.25
12	rs1126707	103787953	PLP1	1(T)/2(G)	0.2500	2	2/2	4.00	2/2	1/2	2	8.00
13	rs5973851	108174500	COL4A6	1(C)/2(T)	0.6116	1	1/1	2.57	2/2	2/2	2	2.67
14	rs5977104	129545053	OCRL	1(C)/2(G)	0.7680	2	2/2	1.30	1/2	1/2	2	1.40
15	rs2071134	153957384	HCFC1	1(C)/2(G)	0.7493	1	1/2	1.99	2/2	1/2	2	0.89

AF: Allele frequency; G: Genotypes for the tested individual (Gi), putative maternal (Gm), and paternal (Gp) parents; LCP: Likelihood ratio for coparentage; SNPs: Single-nucleotide polymorphisms.

RESULTS

Clinical manifestation

Patient 1 was an 18-year-old boy with progressive weakness of both legs for 8 years. His parents denied any family history. The neurological examination revealed a significantly weakened power in the neck flexors (2+/5). The power in the proximal upper extremities was 4+/5 (deltoids) and lower extremities was 4/5 (bilateral hip flexor, hip extensor), and the other muscles were within a relatively normal range. Muscle reflexes were decreased with no abnormalities in the sensation examination. The Babinski sign was negative. The Gower sign was not obvious, but pseudohypertrophy could be seen in the right calf.

The blood tests were all grossly normal apart from elevated muscle enzymes, including CK (7866 U/L, normal reference: <145 U/L), CK isoenzyme (CK-MB) (117 U/L, normal reference: <24 U/L), alanine transaminase (166 U/L, normal reference: <45 U/L), aspartate transaminase (AST) (102 U/L, normal reference: <34 U/L), and lactate dehydrogenase (LDH) (566 U/L, normal reference: <248 U/L). Electrocardiography showed a sinus arrhythmia with high voltage of left ventricle. The echocardiography showed an enlarged left ventricle with decreased function (ejection fraction: 48.7%). An electromyography study revealed a myogenic damage with short duration and low-amplitude polyphasic potentials in voluntary contraction. The patient had been initially considered as BMD clinically; however, the screening of DMD gene by MLPA failed to detect any duplication/deletion.

Patient 2 was a 45-year-old woman with 10 years of progressive weakness of the muscles in legs. On direct questioning, she reported no disturbance of bladder or bowel function or of sensation, no voice changing, choking or swallowing difficulty, no double vision, skin rash or dryness of the mouth, and no fatigable element to the weakness. She denied any family history and had one son without similar symptoms. On the neurological examination, the cranial nerves were normal. The power in the proximal lower extremities was 4/5 (bilateral hip flexor, hip extensor, knee flexor, and knee extensor), and the other muscles were within a relatively normal range. Muscle tone and reflexes were normal and symmetrical, and there were no abnormalities in sensation tests. The Babinski sign was negative. The Gower sign, calf pseudohypertrophy, or fasciculation was not observed, and there was no tenderness or discomfort on palpation.

The CK was mildly elevated to 1826 U/L. The CK-MB, AST, and LDH were 34 U/L, 39 U/L, and 307 U/L, respectively. Other blood tests were normal. Electrocardiography showed mild T wave changes and suspicious Q waves in the lateral wall leads (I, aVL, V5, V6-). The echocardiography showed a decreased diastolic function of the left ventricular. An electromyography study revealed a mild myogenic damage with short-duration polyphasic potentials in voluntary contraction. The magnetic resonance imaging of thighs revealed the muscle atrophy and fatty infiltration. As limb-girdle muscular dystrophy was taken into the clinical consideration first, the targeted NGS was performed for the further diagnosis.

Mutation analysis

After sequencing of two sporadic cases, two novel DMD variants were identified [Figure 1]. Patient 1 carried a novel nonsense variant, NM_004006.2: c.7318C>T (p.Q2440*), resulting in the substitution of a new stop codon termination for the glutamine. This variant was not found in 200 controls, 1000 Genomes Project (1000G), and ExAC database. According to the American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines,[16] the variant was classified as “pathogenic (Ia)” (PVS1 + PS2 + PM2). After the mutation was identified, the patient was finally diagnosed with BMD.

Figure 1

Left: Pedigree of the family 1 (a) and family 2 (c) with mutations in Duchenne muscular dystrophy gene. The empty symbols indicate clinically unaffected individuals. The black symbol filled indicates an affected patient. The symbol filled only half way with black color indicates a manifesting carrier. Right: Chromatogram of the Duchenne muscular dystrophy mutations in family 1 (b) and family 2 (d). The upper panel is a normal sequence, whereas the lower panel represents the mutated one.

Patient 2 carried a novel frameshift variant, NM_004006.2: c.4983dupA (p.A1662Sfs*24), resulting in the substitution of serine for the previous alanine and the addition of 23 additional new amino acid residues prior to the stop codon termination within the new reading frame. Similarly, this heterozygous variant was not found in 200 controls, 1000G, and ExAC database. According to the ACMG Standards and Guidelines,[16] this variant was classified as “pathogenic (Ia)” (PVS1+PS2+PM2). In addition, the MLPA did not detect any large duplication/deletion in the DMD gene. Therefore, the female patient was diagnosed as a manifesting DMD carrier.

Surprisingly, the mutations of two patients were not found in their family members. Therefore, we performed the haplotype analysis in two families based on 15 SNPs within and flanking the DMD gene [Table 2 and Figure 2]. Using reverse parentage testing, we acquired a CPI value of 20,562 in family 1 and a value of 341,303 in family 2. The calculated probability that the proband was the biological child of the alleged parents was >99.99% both in family 1 and family 2, which indicated that the two novel mutations were all derived from de novo mutagenesis in the pedigrees.

Figure 2

Haplotype analysis of probands and the alleged parents in family 1 (a) and family 2 (b) based on 15 single-nucleotide polymorphisms. Numbers beside the symbols represent the genotypes of the 15 single-nucleotide polymorphisms for each individual (1 and 2 denote the specific allele for each single-nucleotide polymorphism).

DISCUSSION

Large rearrangements (large deletions/duplications) count for 77.7% of all DMD mutations.[17] Therefore, the MLPA turned to be a more preferable and faster method for mutation screening in DMD.[8] However, in our patients, the mutations were all small lesions, including nonsense and frameshift mutations, which could count for 8.9% and 7.1% of DMD mutations, respectively.[17] Therefore, the full-length sequencing is essential for diagnosis of dystrophinopathy as well as other myopathies when the MLPA result is negative.[1819]

The severity of the clinical manifestation in DMD/BMD patients generally depends on the occurrence of translation reading frame disruption and premature termination of protein synthesis.[20] In DMD patients, most mutations are null mutations which predict a truncated protein or mRNA with a premature stop codon. Nonsense and frameshift mutations account for up to 48% and 32% of all small lesions in DMD patients, while nonsense and frameshift mutations represent only 24% and 16% of them in BMD patients, respectively.[17] This reading frame rule holds true, respectively, for 96% and 93% of the mutations in DMD and BMD patients. As an exception, the milder phenotype in our patient carrying a nonsense mutation (exon 51) indicated some other modified factors in the genotype–phenotype correlation in DMD/BMD patients.

Although the clinical manifestation of female carriers vary significantly, the muscle weakness is usually mild and proximally distributed.[5] The pelvic girdle is more frequently and earlier affected than the shoulder girdle. Age of onset is also variable, ranging from the first to the fourth decade. The serum CK is an important marker for screening the patients. A CK levels >1000 U/L in isolated female cases of myopathy should put consideration of an underlying dystrophinopathy in the front burner. About 10% of the isolated cases of myopathy with hyperCKemia were proven to have a dystrophinopathy as the cause of their disease (manifesting DMD carriers).[21]

In DMD, BMD, and manifesting carriers, cardiomyopathy should always be considered. It is the leading cause of death in DMD and the main clinical complication in BMD and carriers of DMD mutations, which may not be accompanied with muscle weakness.[3] Up to 40% female carriers can exhibit cardiac involvement, including left ventricle dilatation, global or segmental wall motion abnormality, and dilated cardiomyopathy.[7] In our patients, the cardiac evaluation also detected the electrophysiological abnormality and decreased cardiac function at varying degrees. It was recently reported that the addition of eplerenone to background angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker therapy attenuates the progressive decline in the left ventricular systolic function in DMD patients with preserved ejection fraction.[22]

In summary, the female manifesting carrier of DMD mutation is a challenging condition for diagnosis which often relies on a clear X-linked family history of dystrophinopathy. Therefore, mutational analysis of the DMD gene is typically required for the suspected patients, particularly in the absence of a family history. Using the targeted NGS, we identified two novel mutations of DMD gene in two Chinese pedigrees, which broaden its mutation spectrum. Early diagnosis can motivate early treatment such as ACEIs or β-blockers to delay serious complications.

Financial support and sponsorship

This work was supported by grants from the National Natural Science Foundation of China (No. 81125009) and the research foundation for distinguished scholar of Zhejiang University (No. 188020-193810101/089).

Conflicts of interest

There are no conflicts of interest.