Diagnosis of X-ALD

Hugo W Moser, M.D. Ann B Moser, B.A. Steven J Steinberg, Ph.D. and Stephan Kemp Ph.D.

Clinical diagnosis

The diagnosis of X-ALD should be considered in four distinct clinical settings:

• Boys with symptoms of attention deficit disorder, who in addition show signs of dementia, progressive behavioral disturbance, vision loss, difficulty in understanding spoken language, worsening handwriting, incoordination, or other neurological disturbances.

• Young or middle-aged men with progressive gait disorders, leg stiffness or weakness, abnormalities of sphincter control and sexual dysfunction, with or without adrenal insufficiency or cognitive or behavioral deficits

• All males with primary adrenocortical insufficiency, with or without evidence of neurological abnormality

• Middle-aged or older women with progressive paraparesis, abnormalities of sphincter control, and sensory disturbances mainly affecting the legs. It may be difficult to establish the diagnosis of X-ALD in a female with a negative family history. Diagnosis is based upon clinical features, most commonly progressive spastic paraparesis, and a panel of laboratory tests.

Neuroimaging Brain MRI is always abnormal in neurologically symptomatic males and often provides the first diagnostic lead. In approximately 85% of affected individuals, MRI shows a characteristic pattern of symmetrical enhanced T-2 signal in the parieto-occipital region with contrast enhancement at the advancing margin. Brain MRI is abnormal in fewer than 10% of individuals who are heterozygous for the ABCD1 gene.

Very long-chain fatty acids

Males: The most important laboratory assay is the measurement of the concentration of very long chain fatty acids (VLCFA) in plasma. VLCFA levels are elevated in 99.9% of males with X-ALD of all ages regardless of the presence or absence of clinical symptoms. The three parameters analyzed are: the concentration of C26:0, the ratio of C24:0/C22:0, and the ratio of C26:0/C22:0. Table 1 shows the mean results for normal controls, affected males, and carrier females [F Valianpour et al., 2003].
Analysis of VLCFA is extremely specialized and therefore it is performed only in a few laboratories worldwide.

Females: Increased concentration of VLCFA in plasma and/or cultured skin fibroblasts is present in approximately 85% of females; 15% of known carriers have normal plasma concentration of VLCFA. The average plasma VLCFA results obtained from obligate heterozygotes are shown in Table 1. The discriminant function reported in Moser AB et al (1999) is not able to distinguish all carriers from the normal control range. Women should be tested genetically when X-ALD is suspected and VLCFA concentrations are normal.

Mutation analysis

The ABCD1 is the only gene associated with X-ALD. More than 500 different mutations have been identified in ABCD1 [Kemp et al 2001]. Most X-ALD kindreds have a unique mutation. The mutations in the ABCD1 gene are catalogued on this website.

Boehm and colleagues have developed and validated a robust DNA diagnostic test for X-ALD involving non-nested genomic amplification of the X-ALD gene, followed by fluorescent dye-primer sequencing and analysis. The method covers all coding exons and the flanking intron-exon junctions in 10 separate amplicons [Boehm et al., 1999]. This protocol provides a highly reliable means of determining carrier status in women at risk for transmitting X-ALD and is applicable to a clinical diagnostic laboratory. This method has become the diagnostic sequence-based analysis of choice for many laboratories world wide.

Carrier identification

Testing of at-risk female relatives for carrier status is a two-step process. Measurement of plasma concentration of VLCFA is performed first; if abnormal, the female is a carrier. Because 15% of female carriers have normal plasma concentration of VLCFA, molecular genetic testing should be used to test those females with a normal concentration if the disease-causing ABCD1 mutation has been identified in the family. In one laboratory, the mutations in 97% of obligate carriers were fully identified (n=29).

Extended family testing

Depending upon their gender, family relationship, and the carrier status of the proband's parents, the proband's aunts and uncles and their offspring may be at risk of being carriers or of being affected.

Evaluation of at-risk family members is important for management and genetic counseling but is often implemented insufficiently. Several factors may contribute to insufficient evaluation:

• Establishing the diagnosis in an affected individual with severe disability may be devastating to a family. Immediate concerns may overshadow the timely testing of family members.

• Molecular genetic testing has been clinically available for a short time; but its availability may not yet be generally known.

• Insurance companies may not cover the cost of testing at-risk family members.

• Some at-risk family members may choose not to be tested because they fear that a positive result might impair their ability to obtain or retain medical insurance coverage.

• Individuals may have incomplete knowledge about at-risk family members and may not wish to inform them about the risk.

Prenatal testing

Prenatal testing is possible for pregnancies of women who are carriers in whom the risk of having an affected male is 25% (or 50% if the fetus is known to be male). The usual procedure is to determine sex by karyotyping fetal cells obtained by chorionic villus sampling (CVS) at about 10-12 weeks' gestation (gestational age is expressed as menstrual weeks calculated either from the first day of the last normal menstrual period or by ultrasound measurements) or by amniocentesis at 16-18 weeks' gestation. If the karyotype is 46,XY (male) and if the disease-causing mutation has been identified in a family member, DNA from fetal cells can be analyzed for the known disease-causing mutation.

If mutation analysis is not possible, very long chain fatty acids (VLCFA) can be measured in cultured amniocytes or cultured chorionic villus cells [Wanders et al 1998, Moser AB et al 1999]. False negative test results with this latter approach have been reported, but may have been related to technical factors.

Protein analysis

The ABCD1 gene product, ALDP, is not detectable by means of immunofluorescence analysis in about 70% of affected individuals. For reasons that are not well understood, the gene product may be absent even in individuals who have missense mutations. The principal biochemical abnormality is the accumulation of saturated very long chain fatty acids, particularly hexacosanoic (C26:0) and tetracosanoic (C24:0) fatty acids, as a result of the impaired capacity to degrade these substances, a function that normally takes place in the peroxisome. It is not yet known how the defect in ALDP leads to the accumulation of very long chain fatty acids. The protein may be required for the transport of these fatty acids into the peroxisome.

Detection of ALDP using immunofluorescence: (left) fibroblasts from a control show punctate staining indicating the normal presence of ALDP in peroxisomes; (middle) fibroblasts from a male patient with X-ALD and a mutation that affects ALDP stability. Note that there is no punctate staining; (right) fibroblasts from a carrier with X-ALD from the same family as the male patient. The ABCD1 gene is located on the X-chromosome. Females have two X chromosomes. However, in each cell only 1 X-chromosome is active. The cells that show punctate staining are those that have an active copy of the normal ABCD1 gene, while those that do not show punctate staining are the cells the have an active ABCD1 gene harboring the mutation. For more details follow this link. Images were made by Merel Ebberink

Last update: 19-Nov-2009