Unexpected DMD Gene Mutations Detected by CMA and CNV-seq in amniotic Fluid and Aborted Fetus Samples

Qiuhua Wu Northwest Women’s and Children’s Hospital Lihui Yang Northwest Women’s and Children’s Hospital Qiujie Jin Northwest Women’s and Children’s Hospital Rui Wang Northwest Women’s and Children’s Hospital Wen Zhai Northwest Women’s and Children’s Hospital Yating Wang Northwest Women’s and Children’s Hospital Fengrui Shi Northwest Women’s and Children’s Hospital Wenjing Cheng Northwest Women’s and Children’s Hospital Bo Cai Northwest Women’s and Children’s Hospital Rong Qiang Northwest Women’s and Children’s Hospital Chao Lou (  louchao811@126.com ) Northwest Women’s and Children’s Hospital


Introduction
Dystrophin gene(DMD gene) is located in chromosome Xp21.1, mutations in this gene can cause DMD or BMD [1]. Most of the patients were male, DMD incidence among live-born boys is nearly 1/3500, the symptom of walking disability may emerge at 3~5 years old, getting worse and worse step by step when he grow up until losing ability to walk, died at about 30 years old because of heart and lung failure [2]. BMD incidence among live-born boys is about 1/20000, clinical manifestations are milder than DMD, generally have late onset age and longer life cycles. One of DMD/BMD problems is that patients with no family history can not usually be found before birth, but after the new technologies such as genome-wide high resolution microarray analysis (CMA) , single nucleotide polymorphism (SNP)and high-throughput next generation sequencing technologies (CNV-seq) are widely used in prenatal diagnosis [3][4][5], more and more minor structural abnormalities such as chromosome microdeletions and microduplications have been detected. There may even be some unexpected ndings that do not totally match the patient's clinical indications,such as DMD/BMD. We found seven DMD gene mutations cases by CMA and CNV-seq accidentally, and then used MLPA (Multiplex ligation-dependent probe ampli cation) to con rm the mutations and found new mutations ( Fig.1,Fig2).. The mutations may lead to fetal DMD or mild BMD after mutations analysis.

Samples
Patients with abnormal serological Down screening, B ultrasound abnormality or high NIPT(Non-invasive Prenatal Testing) risk came to our department for invasive prenatal diagnosis. The spontaneous abortion sample was send from the Department of Gynaecology and Obstetrics, Northwest Women's and Children's Hospital, Xi'an, China.( Tab.1). All methods of prenatal diagnosis were introduced to patients, consents were signed by patients, and the tests were selected by patients voluntarily. aCGH test Getting 10 ml amniotic uid by amniocentesis, cells in amniotic uid were collected by centrifugation and divided into two 1.5 ml EP tubes, one tube was used for experiments, the other was stored at -20°c. Using PureGene tissue extraction kit (Qiagen) to extract genomic DNA. Marking, purifying, hybridizing, washing and scanning the DNA according to Agilent 8×60 K chip detection process. Uploading the scanning data to Cytogenomics software (Agilent Technologies) and analyzing the scanning data preliminarily. At the same time, the scanning data was converted into .zip le and uploaded to Genoglyphix (Perkin Elmer), using public and laboratory databases such as DGV DECIPHER ISCA OMIM ClinVar PubMed to analyze pathogenicity of CNVs fragment ≥100 kb. The CNVs were classi ed into three categories according to the 2015 ACMG guidelines :(1) Pathogenic CNVs; (2) Variant Unknown Signi cance (VUS) CNVs; (3) Benign CNVs.

CNV-seq
Getting amniotic uid was described as in CMA test. The extraction of gnomic DNA from amniotic uid and skin of aborted fetus was taken by DNeasy Tissue Kit (69506) (Qiagen) [6]. Using Chromosome CNV Detection Kit KR0040 (Berry Genomics Corporation, Beijing, China) to detect CNV-seq. The procedure was performed as described before [7]: Nearly 50 ng DNA was hydrolyzed by restriction enzymes to obtain DNA fragments at an average size of 200 bp and established DNA libraries by small DNA fragments end lling, adapter ligation, and PCR ampli cation. To determine all sequences on the NextSeq 500 high-throughput sequencing platform ( Illumina USA). Compared the sequences with h19 Human Genome Database by parallel alignment software and analyzed pathogenicity of CNVs fragment ≥100 kb as described in CMA test.
MLPA test DMD Gene Detection Kit was SALSA MLPA kit DMD P034/P035( MRC company, Holland) ABI3500DX Genetic Analyzer(AB Applied Biosystems) was used for capillary electrophoresis, and Coffalyser NET software was used for data analysis.

Discussion
Mutations in DMD genes can lead to muscular dystrophy (DMD or BMD). If the deletion or duplication mutations in DMD gene destroys the reading frame and result in the anti-myatrophin protein content less than 3% or can't produce myoatrophin, patients show DMD. If deletion or duplication mutations keep the reading frame intact or less destoryed , it leads to the anti-myojiatrophy protein keeping part of the function, patients show BMD. But there are also a few patients who DMD gene deletion or duplication did not alter the reading frame , it still show DMD. [8] The main DMD gene mutation type of is partial deletion or repetition of 79 gene exons, nearly account for 60%~70% of all mutation types. The rest are point mutations, minor insertion or deletion mutations|. Despite many studies on gene therapy in DMD, such as exon hopping, readout of nonsense stop codon or injection of vectors with full DMD gene, gene therapy still faces great challenges and dilemmas [9]. There is still no effective treatment for DMD/BMD patients until now [10,11], early diagnosis and early treatment can prolong the survival age and improve the quality of life of DMD patients, but can not be cured. DMD patients with no family history of hereditary diseases can not usually be found before birth [12][13][14][15][16]. A majority of DMD patients develop clinical symptoms around age 3. But the results of serum creatine kinase level, neuroelectrical trophysiological examination and DMD gene detection were clearly diagnosed until 5 years old, so genetic counseling and screening for DMD genes prior to or in early pregnancy are particularly important, prenatal diagnosis is an important approach to prevent the birth of the patient with a family history of DMD/BMD or with no history.
High-resolution genomic CMA and high-throughput next generation sequencing CNV were used in prenatal diagnosis not only detect chromosomal microdeletion and microduplication syndrome, but also helped to detect single gene disease caused by gene deletion or duplication, can improve the diagnosis rate of disease. In this study, the pregnancy of all male fetuses were terminated. Two female fetuses were born: Case6 is normal; but Case 3 showed upward bent legs and heavy hair, WES did not nd problems. One female fetus (Case 5) was aborted.,( data of 7 patients showed in Tab 1).We compared the DMD gene mutations between fetus and mother, found some mutations inherited from mother (Fig 1) and some didn't (Fig 2), it also reveals the importance of prenatal diagnosis because the mother is carrier or healthy woman, and with no family history of DMD. So it is bene cial to the correct evaluation of fetal prognosis in prenatal clinical consultation, and provide a more comprehensive advice to decide if the pregnancy should continue or be terminated to prevent the birth of children with defects. Availability of data and materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request

Competing interests
The authors declare that they have no competing interests