Identification of two novel mutations of ABCD1 gene in pedigrees with X - linked adrenoleukodystrophy and review of the literatures

: Background: X-linked adrenoleukodysrophy (ALD) is an inherited peroxisomal metabolism disorder, results from the loss-of-function mutation of ATP-binding cassette protein subfamily D1 ( ABCD1 ) gene. The dysfunction of ALD protein, a peroxisomal ATP-binding cassette transporter, results in the excessive saturated very long chain fatty acids (VLCFAs) accumulation in organs including brain, spine and adrenal cortex. X-ALD is characterized as the childhood, adolescent, adult cerebral ALD, adrenomyeloneuropathy (AMN), adrenal insufficiency, and asymptomatic phenotypes, exhibiting a high variety of clinical neurological manifestations with or without adrenocortical insufficiency. Results: In this study, we reported two cases of X-ALD, which were firstly diagnosed as adrenal insufficiency (Addison’s disease) and treated with adrenocortical supplement. However, both of the cases progressed as neurological symptoms and signs after decades. Elevated VLCFAs level, brain MRI scan and genetic analysis confirmed final diagnosis. In addition, we identified two novel mutations of ABCD1 gene, c.874_876delGAG (p.Glu292del) and c.96_97delCT (p.Tyr33Profs*161) in exon 1 of ABCD1 gene. Sanger sequencing confirmed that the proband’s mother of the first case was hemizygous carrying the same variant. Adrenal insufficiency-only type is very rare, however, it may be the starting performance of X-ALD. In addition, we summarized reported mutation sites and clinical manifestations to investigate the correlationship of phenotype-genotype of X-ALD. Conclusions: The early warning manifestations should be noticed, and the probability of X-ALD should be considered. This report could be beneficial for the early diagnosis and genetic counseling for patients with X-ALD.

Some individuals were diagnosed as Addison's disease without symptoms of cerebral or myelopathy ALD for decades. However, the early-onset Addison's disease may gradually progress to exhibit neuropsychiatric symptoms, showing a time-dependent fellow-up [9].
In this study, we reported two pedigrees of X-ALD patients firstly diagnosed as Addison's disease for decades, and gradually exhibited variable clinical neuropsychiatric manifestations in their late twenty's, and showed rapid progression and poor prognosis. In addition, we identified two novel mutations of ABCD1 gene, c.874_876delGAG (p.Glu292del) and c.96_97delCT (p.Tyr33Profs*161), in the two pedigrees of X-ALD. Further study of genotype-phenotype correlation analysis is needed to provide deeper insights into X-ALD.

Patients' recruitment
The diagnosis of X-ALD depends on clinical symptoms, biochemical parameters, neurological imaging and genetic analysis. This study was approved by the ethics committee of the Affiliated Hospital of Qingdao University.

Genetic analysis and Prediction of pathogenicity
Genomic DNA was extracted from peripheral blood sample using the QIAamp and Mutation Taster (http://www.mutationtaster.org) to predict the possible impacts of amino acid substitution on the structure and function. The PROVEAN score represented the normalized probability that the amino acid change is deleterious. The cutoff of PROVEAN score was -2.5. The PROVEAN score <-2.5 was predicted to be deleterious. In addition, the following forty genes associated with congenital adrenal hypoplasia or glucocorticoid deficiency were investigated for case 2, including MC2R,

Three-dimensional structure prediction of protein and the potential dysfunction
We identified the alteration of the ABCD1 encoded ALD protein structure and potential dysfunction using the SWISS-MODEL work-space (http://swissmodel.expasy.org).

Case presentations and biochemistry results:
Case 1: A 27-year old male was presented in hospital with weakness, fatigue, poor academic performance, exhibiting skin and mucous membrane pigmentation, especially in areolae, joints and axilla for two decades. The cortisol at 8am was reduced 3.58 nmol/L (range 171-536 nmol/L), and adrenocorticotropic hormone (ACTH) was increased 499.30 pg/mL (range 7.2-63.6 pg/mL). After diagnosed as primary adrenal insufficiency (Addison's disease), he was administrated with glucocorticoid replacement (hydrocortisone 20mg per day) for one year, and the skin pigmentation and weakness were improved. In recently half a year, the patient acted mild cognitive impairment, slurred speech, ataxia, and rapidly progressed as walking instability, incontinence, and paroxysmal blurring vision. Physical examination detected scanty scalp hair, Vgrade muscle strength, hypomyotonia, tendon hyperreflexia (+++), positive Barbinski's sign and Gordon's sign. The light and deep reflex were normal.
The heel-to-knee test, alternating movement test, and Romberg's test were negative.
The respiratory, cardiovascular, and abdominal examinations were unremarkable. Brain MRI showed symmetrical distribution of hyperintensity long T1 and long T2 signal in of bilateral splenium of corpus callosum, and widened fissures and sulci ( Figure 1A).
Serum VLCFA levels were significantly increased in the proband, and also elevated in the proband's mother (Table 1, Figure 2A II-10). Until now, his mother did not show manifestations of cerebral disorders, myeleterosis, or adrenal insufficiency. His maternal grandmother (Figure 2A, I-2) died at her 55-year-old due to encephalopathy.
However, family members were unable to provide valid information, and no blood samples could be obtained for testing. Other siblings of his mother did not showed neuropsychiatric manifestations. Genetic analysis revealed variants c.874_876delGAG of ABCD1 gene in exon 1. According to the clinical manifestations and genetic analysis, a pedigree of X-ALD was constructed. The patient was administrated with hydrocortisone 20mg per day, mecobalamine, and compound vitamin B. During the fellow-up, the proband showed rapidly progressed neuropsychiatric manifestations including blurred vision, instability of gait, and incontinence within six months. Thus, the pedigree of X-ALD was suspected. During the fellow-up, the patient was treated with hydrocortisone and fludrocortisone, and serum sodium level was maintained in normal range. However, he showed rapidly progressed symptoms of incontinence and consciousness dysfunction.

Genetic analysis and Prediction of pathogenicity
Genetic sequencing of ABCD1 gene was performed on the probands of two cases and the familial relatives of the proband in case one. In Case one, genetic sequencing detected heterozygous mutation of c.874_876delGAG (p.Glu292del) in ABCD1 gene Exon 1 (Figure 2A and 2B), leading to the deletion of translation product glutamate and accumulation of VLCFAs (Table 1). Sanger sequencing of ABCD1 gene was performed on his mother, and confirmed the hemizygous point mutation for the same variant. In case two, gene sequencing showed a heterozygous deletion mutation c.96_97delCT in ABCD1 gene ( Figure 2C and 2D), leading to the frameshift and premature transcription termination of amino acid p.Tyr33Profs*161. His immediate family members (parents and his sister) refused to perform genetic sequencing.
The frequency of those variants was examined in the reference database. Both novel mutations were not detected in the database including ESP6500, 1000 Genomic, or dbSNP ( Table 2). The potential pathogenicity of both mutations were investigated using prediction bioinformatics, suggesting both mutations were possible to be disease causing ( Table 2).

Three-dimensional structure prediction of protein and the potential dysfunction
The ABCD1 gene encoded ALD protein contains transmembrane domains and three segments on the peroxisome side. We identified the predicted alteration of the ALD protein structure and its potential dysfunction induced by mutations c.874_876delGAG (p.Glu292del) and c.96_97delCT (p.Tyr33Profs*161) ( Figure 3).
The amino acid changes in ALD protein due to those deletion mutations of ABCD1 gene might affect function, leading to the impaired degradation of VLCFAs and inflammation environment.

Discussion:
X-ALD is a demyelinating and neurodegenerative disorder, due to the loss-offunction mutation of ABCD1 gene. ABCD1 gene codes the peroxisome transporter protein ALDP, which plays an important role in regulating peroxisome oxidation and degradation of VLCFAs [5]. The dysfunction of ALDP, a fatty acid transporter, results in the impaired VLCFA oxidation and excessive VLCFAs accumulation in tissues [5,10]. Excessive VLCFAs in cytoplasm leads to mitochondrion dysfunction and ER stress, finally caused cell death. In addition, accumulated VLCFAs arouses inflammation, results in demyelinating and neurodegeneration.
In this study, we reported two Chinese X-ALD pedigrees. Both of the probands were diagnosed as adrenal insufficiency at beginning without neuropsychiatric symptoms for decades, but developed into neurological disorders at their late 20's, and showed rapidly progression and poor prognosis. Two novel mutations in exon 1 of ABCD1 gene were detected and suggested to be associated with X-ALD.
According to the previous reports, the majority is cerebral ALD among those seven types of ALD [11]. CCALD is early onset with a feature of rapidly progressed neuro-inflammatory of cerebral demyelination. Neurological symptoms of AMN commonly occur from the ages of 20s, with manifests as a chronic progressive paraparesis, accompanied with sensory and motor disturbances [12]. Due to the variable clinical manifestations, some are difficult to make the diagnose at early stage [13]. Adrenocortical insufficiency was the reported as the first clinical manifestation of 38% X-ALD, greater than the group of spinal cord 25% and cerebral disorders 14.5% [9]. To be noticed, some of those male patients diagnosed as adrenal insufficiency may develop with cerebral or myeloneuropathy ultimately, and show a rapid progressive neurological symptoms and poor prognosis. On the contrast, female ALD patients rarely show adrenocortical insufficiency or cerebral ALD. Most of female patients suffer from lately-onset myelopathy [10,14]. We reported two cases both were firstly diagnosed as Addison's disease, and then the therapy focused on the adrenocortical replacement. However, after the neurological symptoms and signs become apparent, both of the cases were rapidly progressed. The therapy including fatty acid restriction or neurotrophic treatment did not show therapeutic effect.
Therefore, at diagnosis of congenital adrenal insufficiency, the probability of ALD should be fully considered especially for male patients [8,15]. To make the early diagnosis, family history, MRI scan of brain or spinal cord, VLCFAs measurement, and genetic screening may be helpful for those patients with subtle early neurological manifestations.
We identified two novel point mutations in exon 1 of ABCD1 gene in the pedigree of first case (c.874_876delGAG, p.Glu292del) and in second case (c.96_97delCT, p.Tyr33Profs*161). The genetic sequencing demonstrated nucleotides deletion, resulting in those frameshift mutations. Based on the clinical symptoms and positive neurologic signs, family history, imaging, biochemical characteristics and the results of pathogenicity prediction using bioinformatics tools, we suggest those mutations as pathogenic variants. However, the biopsy of brain, spinal cord, and adrenal gland were not done. Based on the elevated plasma VLCFAs, the deleterious change of ALDP impaired the cellular beta-oxidation of fatty acid [16].
There are more than thousand mutations have been reported in ALD database (http://www.x-ald.nl/). Among them, 67.57% of them are pathogenic mutations, 9.65% are synonymous, 2.16% are benign, 18.65% are VUS (variant of undetermined significance), and 1.87% are status unknown mutations detected during the screening of newborns ( Figure 4A). More than 77.5% are point mutations, other types including 12.93% deletion, 1.97% del-insertion, 4.22% insertion, and 3.37% duplication ( Figure   4B). Most of the mutations locate in the exon (92.94%), others in the intron (6.18%), 5'UTR (0.61%) and 3'UTR (0.26%), respectively [4]. In the database, nearly half of the mutations were reported anchoring in exon 1 ( Figure 4C), which encodes the transmembrane domain (TMD) of the protein, contains ligand-specific binding sites and plays an important role in the localization of ALDP [17]. Another clustering of mutations occurs in exon 6, which encode the ATP binding domain of ALDP [18].
ALDP locates in the peroxisome membrane, to transport VLCFAs from cytosol into the peroxisome for degradation. Pathogenic ABCD1 mutations may induce defective the stability of ALDP transmembrane structure region and ATPase activity, to affect the localization of VLCFAs to the peroxisome correctly. As a result, VLCFAs cannot be transported into peroxisomes for β-oxidation, then accumulate mainly in the white matter, neuron axons of central nervous system, and adrenal cortex. Excess VLCFAs accumulation leads to destructive progression by triggering oxidative stress and mitochondrial dysfunction, chronic inflammation, lipid-induced neuron apoptosis, and degenerative changes in the nervous system, and finally leading to neurodegeneration and adrenal insufficiency [3,19]. Though some variations may not affect protein translation or function, they still represent polymorphisms. The genotype-phenotype correlationship have not been identified clearly [20][21]. To further investigated the relationship between genotype and phenotype, we searched the database of X-ALD Therapeutic strategies include adrenocortical replacement, some of the adrenal insufficiency patients may require both glucocorticoid and mineralocorticoid to maintain water electrolyte balance. For severe adrenal insufficiency, or even adrenal crisis, timely initiation of steroid replacement is essential. Nutrition intervention, for instance, low-fat diet and Lorenzo's Oil may reduce VLCFAs level [22]. In this way, the risk of development with cerebral disorders may be reduced. However, they lack the evidence to improve the symptoms and progression. Another efficiency approach is hematopoietic stem cell transplantation (HSCT), especially in the early stage of the disease [23]. Transplantation with autologous bone marrow transfected in vitro with ABCD1gene modification has been performed [24][25]. Bone marrow transplant has been reported in CCALD therapy, most of the patients achieved improved prognosis.
However, the pre-existed severe symptoms may influence the outcome after transplantation [26]. In animal experiments, Abcd1mice show reduction of the mitochondrial biogenesis driven by PGC-1α/PPARγ pathway [27]. Oxidative stress is involved in the physiopathological mechanism of neurodegenerative cascade and mitochondrial depletion [28]. Pioglitazone, a PPARγ/PGC-1α axis activator may modulate the anti-inflammatory and anti-oxidant response. Recently, pioglitazone is under phase II Clinical trials for adrenoleukodystrophy patients, reveals a potential for clinical translation [29]. Therapy options are limited, in addition, some therapeutic approaches relay on the early diagnosis and intervention. Therefore, gene sequencing analysis may be an important tool for newborn screening and genetic counseling.

Conclusions:
In this study, we reported two novel mutations of ABCD1 gene in men with X-ALD. These two cases were both considered as adrenal insufficiency at first, and finally diagnosed with X-ALD when neurological manifestations occurred after several years. In these two pedigrees, we identified two novel mutations of ABCD1 gene, c.874_876delGAG (p.Glu292del) and c.96_97delCT (p.Tyr33Profs*161).
Screening test for ALD, such as VLCFA concentration and brain MRI scan, should be considered in the early-onset Addison's disease patients with family history to screen X-ALD early.

Declarations:
Ethic approval and consent to participate: All procedures performed in this study involving human participants were in accordance with the ethical standards of the Affiliated Hospital of Qingdao University with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.