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Exome sequencing identifies mutations in ABCD1 and DACH2in two brothers with a distinct phenotype
© Zhang et al.; licensee BioMed Central Ltd. 2014
Received: 2 September 2013
Accepted: 5 September 2014
Published: 19 September 2014
We report on two brothers with a distinct syndromic phenotype and explore the potential pathogenic cause.
Cytogenetic tests and exome sequencing were performed on the two brothers and their parents. Variants detected by exome sequencing were validated by Sanger sequencing.
The main phenotype of the two brothers included congenital language disorder, growth retardation, intellectual disability, difficulty in standing and walking, and urinary and fecal incontinence. To the best of our knowledge, no similar phenotype has been reported previously. No abnormalities were detected by G-banding chromosome analysis or array comparative genomic hybridization. However, exome sequencing revealed novel mutations in the ATP-binding cassette, sub-family D member 1 (ABCD1) and Dachshund homolog 2 (DACH2) genes in both brothers. The ABCD1 mutation was a missense mutation c.1126G > C in exon 3 leading to a p.E376Q substitution. The DACH2 mutation was also a missense mutation c.1069A > T in exon 6, leading to a p.S357C substitution. The mother was an asymptomatic heterozygous carrier. Plasma levels of very-long-chain fatty acids were increased in both brothers, suggesting a diagnosis of adrenoleukodystrophy (ALD); however, their phenotype was not compatible with any reported forms of ALD. DACH2 plays an important role in the regulation of brain and limb development, suggesting that this mutation may be involved in the phenotype of the two brothers.
The distinct phenotype demonstrated by these two brothers might represent a new form of ALD or a new syndrome. The combination of mutations in ABCD1 and DACH2 provides a plausible mechanism for this phenotype.
Adrenoleukodystrophy (ALD; OMIM#300100) is a serious progressive, genetic disorder that affects the adrenal glands, the spinal cord and the white matter of the nervous system. It is thought to be caused by genetic defects in the ATP-binding cassette, sub-family D member 1 (ABCD1) (OMIM*300371) gene ,. ALD is characterized by variations in phenotypic expression; seven ALD forms have been reported so far, including childhood cerebral, adolescent cerebral, adult cerebral, adrenomyeloneuropathy (AMN), Addison-only, asymptomatic or presymptomatic, and olivo-ponto-cerebellar ALD ,. However, there is no exact genotype-phenotype correlation between ABCD1 defects and clinical phenotype. Furthermore, the combination of defects in ABCD1 with defects in other genes, such as the hemophilia A gene  or DXS1357E, can complicate and worsen the clinical phenotype.
We present two brothers with a distinct phenotype including congenital language disorder, growth retardation, severe intellectual disability, inattention, dysphoria, drooling, heterophony, difficulty in walking and standing without aid, standing on tiptoe with aid, urinary incontinence, and fecal incontinence. To the best of our knowledge, no cases with a similar phenotype have been reported previously. We performed cytogenetic tests and exome sequencing in the two brothers and their parents to screen for potential pathogenic causes.
Two brothers and their parents, as well as 500 unaffected individuals (250 males and 250 females) of matched geographical ancestry, were enrolled in this study. The study was approved by the Shenzhen People's Hospital Ethics Committee, which abides by the Helsinki Declaration on ethical principles for medical research involving human subjects. Written informed consent was obtained from all the participants or their guardians.
Genomic DNA was obtained from peripheral blood lymphocytes from all individuals, using standard procedures .
G-banding chromosome analysis (~850 bands) was performed on cultures of peripheral blood lymphocytes from the two brothers and their parents, according to standard techniques .
Array comparative genomic hybridization
Array comparative genomic hybridization (array-CGH) was performed using Agilent Technologies' Array CGH Kits (Santa Clara, CA, USA). This platform is 60-mer oligonucleotide-based microarray that allows genome-wide survey and molecular profiling of genomic aberrations with a resolution of ~20 kb (Kit 244A). DNAs were labeled by random priming (Agilent Technologies) for 2 h using Cy5-dUTP for test DNAs and Cy3-dUTP for reference DNAs. Labeled products were column-purified. After probe denaturation and pre-annealing with 50 μg of Cot-1 DNA, hybridization was performed at 65°C with rotation for 40 h. After two washing steps, the arrays were analyzed using an Agilent scanner and Feature Extraction 10.5.0.1 software. The data were analyzed using CGH Analytics 4.0 software (Agilent Technologies). The Aberration Detection Method 2 algorithm was used to identify aberrant intervals.
Exon capture and sequencing, read mapping and single nucleotide polymorphism detection
Targeted capture and massive parallel sequencing of approximately 201,904 coding exons from genomic DNA from the two patients and their mother were performed using the Agilent SureSelect Human All Exon kit, following the manufacturer's protocols. Briefly, genomic DNA was sheared by sonication and the DNA fragments were then purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The fragment ends were repaired and adaptors were ligated to the fragments (NEBNext DNA sample prep, New England Biolabs). The adapter-ligated templates were purified using Agencourt AMPure SPRI beads and fragments with an insert size of about 250 bp were excised. Extracted DNA was amplified by ligation-mediated polymerase chain reaction, purified, and hybridized to the SureSelect Biotinylated RNA Library `baits' (Agilent) for enrichment. Each captured library was then loaded on a HiSeq 2000 platform for sequencing. Raw image files were processed using Illumina Pipeline (v1.6) for base-calling with default parameters. SOAPaligner (v2.01) was used to align the sequencing reads to the NCBI human genome reference assembly (build 36.3). Reads that aligned to the designed target region were collected for single nucleotide polymorphism (SNP) identification and subsequent analysis. The consensus sequence and quality of each allele was calculated by SOAPsnp.
Detection of insertions and deletions
Insertions and deletions (Indels) in the exome regions were identified by de novo assembly of the sequencing reads. The reads were assembled using SOAPdenovo with the 31-mer option enabled and the assembled consensus sequences were then aligned to the reference genome by LASTZ. The alignment result was passed to axtBest to separate orthologous from paralogous alignments. Finally, breakpoints in the alignment were identified and the genotypes of Indels were annotated.
To distinguish between potentially pathogenic mutations and other variants, we only focused on non-synonymous (NS) variants, splice acceptor and donor site mutations (SS), and short coding Indels, anticipating that synonymous variants would less likely to be pathogenic. The variants were compared and filtered using public databases, including dbSNP (v129), 1000 Genome Project (20100208 release), eight HapMap exomes, and YH genome. A novel variant was defined as one that did not exist in these datasets. Only recessive models of inheritance (autosomal recessive model and X-linked recessive model) were considered because of the normal phenotypes of the parents.
Variants detected by exome sequencing were validated by Sanger sequencing.
The hormones detection results of two brothers
1.3 ~ 3.1 nmol/L
66 ~ 181 nmol/L
Free triiodothyronine (FT3)
3.10 ~ 6.80 pmol/L
Free thyroxine (FT4)
12 ~ 22 pmol/L
Thyrotropic-stimulating hormone (TSH)
0.27 ~ 4.20 uIU/ml
Luteinizing hormone (LH)
1.70 ~ 8.60 mIU/ml
Follicle-stimulating hormone (FSH)
1.5 ~ 12.4 mIU/ml
98 ~ 456 uIU/ml
49.2 ~ 218 pmol/L
9.90 ~ 27.80 nmol/L
0.70 ~ 4.30 nmol/L
171 ~ 536 nmol/L
Adrenocorticotropic hormone (ACTH)
2.2 ~ 16.6 pmol/L
The karyotypes of the two brothers and their parents were normal according to G-banding chromosome analysis. No submicroscopic chromosome aberrations were detected by array-CGH.
Summary of original exome sequencing data
Map bases (Gb)
Map bases rate (%)
Exon map bases (Gb)
Exon map bases rate (%)
Exon length (Mb)
Covered length (Mb)
Mean sequencing depth
Identification of the candidate genes for two brothers by exome sequencing
Not in dbSNP129, 1000 Genome Project, eight HapMap exomes, and YH genome
Comparing to the mother
Shared by two cases
The exome sequencing and sanger sequencing results of the 3 candidate genes
We report on two brothers who presented with a distinct phenotype. The potential pathogenic cause was investigated using G-banding chromosome analysis and high-resolution array-CGH in the two brothers and their parents, but no abnormalities were detected. Exome sequencing was therefore performed on the brothers and their mother, to detect genetic variations. The small sample and uncertain pathogenesis of the disease meant that both autosomal recessive and X-linked recessive models were possible. The rarity of the disorder makes it unlikely that causative variants would be present in the general population, and we therefore compared our detected variants with those in dbSNP129, 1000 Genomes Project, eight HapMap exomes and YH genome to eliminate shared variants. Sequencing and comparison of the coding region from the affected brothers and their unaffected mother, and filtering out of the benign changes using public databases, led to the identification of three candidate genes: one autosomal gene GNAQ and two X chromosomal genes ABCD1 and DACH2. Subsequent Sanger sequencing showed that the mutations in GNAQ were false positives.
The ABCD1 mutation was a novel missense mutation, c.1126G > C transition (E376Q), in exon 3. Several lines of evidence support a causative role for this mutation in the brothers' phenotype: 1) ABCD1 mutation is consistent with an X-linked recessive model, which was one of the two possible inherited models; 2) the ABCD1 mutation is not present in the public databases, including dbSNP129, 1000 Genomes Project, eight HapMap exomes and YH genome, and was not found in 500 normal ethnicity-matched controls, excluding the possibility of an amino acid substitution polymorphism; and 3) comparative analyses of ABCD1 in other species show that E376 is conserved among primates, rodents, and other vertebrate species.
ABCD1 encodes an integral peroxisomal membrane protein (ALD protein) that belongs to the ATP-binding cassette-transporter superfamily . The peroxisomal ATP-binding cassette transporter is involved in the import of VLCFA into the peroxisome. Defects in ABCD1 have been shown to be associated with impaired peroxisomalociated with impaired peroxisomal β-oxidation and accumulation of saturated VLCFA in all tissues of the body, and are considered to be the underlying cause of ALD ,.
Phenotypic comparison between reported male ALD forms and two brothers
Onset at 3-10 years of age with a peak at seven years. This form virtually never occurs before three years of age. Affected boys present with progressive behavioural and cognitive neurological deficits, such as inattention, hyperactivity, deterioration in handwriting skills, diminishing school performance; difficulty in understanding speech, spatial orientation; clumsiness; visual disturbances; and aggressive behavior. Brain MRI examination can be strikingly abnormal even when symptoms are relatively mild. Most individuals have impaired adrenocortical function at the time that neurological disturbances are first noted. Total disability often within 3 years.
Onset before 2 years of age. Presentation is not progressive, including congenital language disorder, intellectual disability, growth retardation, response retardation, dysphoria, drooling; difficulty in walking and standing; urinary incontinence and fecal incontinence; movement and sensory dysfunctions of lower limbs; normal brain MRI, normal adrenocortical function.
Onset at 28 ± 9 years, progressive stiffness and weakness of legs, abnormalities of sphincter control, sexual dysfunction, distal axonopathy, inflammation mild or absent, mainly spinal cord involvement, cerebral involvement later in 45% of cases.
Like childhood cerebral, but onset at 10-21 years of age. Somewhat slower progression.
Dementia, behavioral disturbances. Rapid inflammatory cerebral progression resembling the childhood form, without preceding AMN, onset after 21 years of age.
Primary adrenocortical insufficiency without neurological abnormalities, including unexplained vomiting, weakness, coma, onset before 7.5 years of age.
Asymptomatic or presymptomatic
ALD gene abnormality without neurological or endocrine abnormalities, further studies can reveal subclinical adrenal insufficiency or mild AMN phenotype. This form is common in boys under 4 years of age.
Cerebral and brain stem involvement, onset between adolescence to adulthood.
The unique phenotype of the two brothers initially suggested a novel syndrome, rather than ALD. However, a missense mutation in ABCD1 was unexpectedly identified in both brothers and their mother by exome sequencing. Although PolyPhen-2 predicted that the biophysical consequences of the variant c.1126G > C (E376Q) of ABCD1 were likely to be functionally benign, other variants in the same exon, such as c.1114A > T (p.K372*) and c.1137C > G (p.S379R), have been reported to lead to ALD . Further laboratory tests showed elevated plasma VLCFA levels in the brothers, and no mutations were detected in genes associated with other peroxisomal diseases, such as Zellweger syndrome, acyl-CoA oxidase deficiency, D-bi-functional protein deficiency, and b-ketothiolase deficiency. The brothers should be therefore diagnosed with ALD.
Nevertheless, the phenotype exhibited by the two brothers was not consistent with any reported ALD forms. Their ages at onset would suggest childhood cerebral ALD (CCALD), but their conditions were not progressive, their brain MRI results were normal, and they had severe congenital language and motor disorders and intellectual disability. In contrast, cognitive and motor development are normal in CCALD prior to the onset of demyelinating lesions visible at brain MRI, suggesting that the brothers' phenotype differed from CCALD. In addition, both brothers had urinary and fecal incontinence, their ages at onset were younger than 2 years old, they had severe congenital intellectual disabilities, and were unable to walk and speak, indicating a phenotype incompatible with AMN and other known forms of ALD. The distinct phenotype displayed by the two brothers suggests that other pathogenetic factors, in addition to ABCD1 mutation, may have been responsible for their condition.
DACH2 is a homolog of dachshund. The dachshund/Dach gene family encodes transcriptional cofactors that are conserved between insects and vertebrate. Drosophila dachshund is a critical regulator of eye, brain, and limb formation, and null mutations in dachshund result in abnormal retinal, brain, genital, and limb development -. The vertebrate homologs Dach1 and Dach2 also play an important role in the development of the retina, brain and limbs . DACH2 encodes a transcription factor characterized by the presence of three conserved domains , which is involved in the regulation of organogenesis, myogenesis, brain and limb development ,. The first domain (DD1) at the N-terminus (amino acids 66-162) and the second domain (DD2) at the C-terminus (amino acids 452-543) are highly conserved in all members of the DACH protein family and appear to be involved in DNA binding and in the interaction with EYA proteins, respectively ,. A third domain DD3 is present in the central portion of the protein (amino acids 314-412) and is shared by all members of the DACH1 and DACH2 subfamilies, but its detailed function remains unknown. The variant c.1069A > T transition (p.S357C) of DACH2 detected in the present study was located in the DD3 domain and was predicted to be functionally damaging by PolyPhen-2, suggesting a potential role in two brothers- phenotype. The combined mutations in ABCD1 and DACH2 thus provide a plausible explanation for the abnormal phenotype observed in both brothers. However, further functional studies are needed to clarify the effects of these variants.
The distinct phenotype demonstrated by two brothers might represent a new form of ALD or a new syndrome. The combination of mutations in ABCD1 and DACH2 provides a plausible mechanism for this phenotype.
We gratefully acknowledge the participation of all the participants.
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