Diagnosis of ALD

Authors: 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 adrenoleukodystrophy 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, in coordination, 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 adrenal 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 adrenoleukodystrophy 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 T2 signal in the parieto-occipital region with contrast enhancement at the advancing margin.

Very long-chain fatty acids

Males: The most used laboratory assay world-wide for diagnosing ALD 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 adrenoleukodystrophy 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 females [Valianpour et al. 2003].
Analysis of VLCFA is extremely specialized and therefore it is performed only in a few laboratories worldwide.

Important comment from Dr. Ann Moser: Lorenzo’s oil, a mixture of erucic and oleic acids, is still used in some countries to normalize VLCFA levels. The Peroxisome Disease laboratory at the Kennedy Krieger Institute in Baltimore routinely reports erucic acid (C22:1) levels when measuring plasma VLCFA. Certain oils used in cooking, such as mustard seed oil, have naturally high levels of erucic acid and, thus, can lead to an elevation similar to that observed during Lorenzo oil therapy.

Females with ALD: Increased concentration of VLCFA in plasma and/or cultured skin fibroblasts is present in approximately 85% of females with ALD; 15-20% of known females with ALD have normal plasma concentration of VLCFA. The average plasma VLCFA results obtained from females with ALD are shown in Table 1. The discriminant function reported in Moser AB et al (1999) is not able to distinguish all females with ALD from the normal control range (see the Figure). Women should be tested genetically when adrenoleukodystrophy is suspected and VLCFA concentrations are normal.

C26:0-lysophosphatidylcholine (C26:0-lysoPC)

The VLCFA containing C26:0-lysoPC is elevated in all ALD men and women and is used in ALD newborn screening. It has been demonstrated that women with ALD with plasma VLCFA levels in the normal range have elevated levels of C26:0-lysoPC in dried blood spots and plasma [Jaspers et al 2020]. Thus, C26:0-lysoPC outperforms VLCFA analysis as an ALD diagnostic biomarker.

Genetic testing

The ABCD1 gene is the only gene associated with adrenoleukodystrophy. More than 940 different (likely) pathogenic variants have been identified in ABCD1 [The ALD Variant Database]. Many adrenoleukodystrophy kindred’s have a unique pathogenic variant. All proven pathogenic variants identified in the ABCD1 gene are catalogued on this website.

Boehm and colleagues have developed and validated a robust DNA diagnostic test for adrenoleukodystrophy involving non-nested genomic amplification of the adrenoleukodystrophy 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] and (see also the sequencing page). This protocol provides a highly reliable means of determining heterozygous status in women at risk for transmitting adrenoleukodystrophy and is applicable to a clinical diagnostic laboratory. This method has become the diagnostic sequence-based analysis of choice for many laboratories worldwide.

Genetic counseling

Adrenoleukodystrophy is inherited in an X-linked manner. The ABCD1 gene is the only gene associated with adrenoleukodystrophy.
Parents of a male or female patient: About 93% of index cases have inherited the genetic defect from one of the parents, which often makes it likely that other family members, such as biological siblings and cousins, are also affected. However, in ~7% of cases the infant underwent a spontaneous mutation, also called “de novo“. In these cases there will not be other family members with the mutated gene. It is appropriate to measure plasma VLCFA levels in the mothers of both affected males and affected females and in the fathers of affected females (Figure below). When the pathogenic variant has been identified in an affected family member, genetic testing of the ABCD1 gene can be used in the evaluation of the parents and for extended family screening.
Sibs of an index patient (proband): The risk to sibs depends upon the genetic status of the parents, which can be clarified by pedigree analysis, VLCFA measurement, and molecular genetic testing.
If the proband’s mother has a pathogenic variant in the ABCD1 gene (heterozygote), the chance of transmitting the pathogenic variant in each pregnancy is 50%. Male and female sibs who inherit the pathogenic variant will be affected.
If the proband’s father has a pathogenic variant in the ABCD1 gene, all of the female sibs will be affected and none of the male sibs will be affected.
If neither parent is a has a pathogenic variant in the ABCD1 gene, the risk to sibs of a proband is low.
Offspring of a proband: Affected males transmit the ABCD1 variant to all of their daughters and none of their sons.
Affected females have a 50% chance of transmitting the ABCD1 variant in each pregnancy. Sons who inherit the pathogenic variant will be affected; daughters who inherit the pathogenic variant are carriers for the pathogenic variant and will most likely develop symptoms in adulthood.

Figure 2: (Left) If a woman is a carrier for the defective adrenoleukodystrophy gene she has the following possible outcomes with each newborn: when the child is a daughter, there is a 50% chance that the daughter receives the defective adrenoleukodystrophy gene and a 50% chance that the daughter is unaffected. In case the child is a boy, there is a 50% chance that the son has adrenoleukodystrophy and a 50% chance that he will be unaffected. (Right) For an X-linked disorder, such as adrenoleukodystrophy, if an affected man has children, then all of his sons will be free of the disease, since the father always passes his Y-chromosome on to his sons. However, all of his daughters will inherit the defective adrenoleukodystrophy gene (he always passes his only (affected) X-chromosome on to his daughter).

Heterozygote 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 affected. Because around 15% of females with ALD have normal plasma concentration of VLCFA, molecular genetic testing should be used to test those females with a normal concentration. This is easier if the disease-causing ABCD1 genetic variant has already been identified in the family. Alternatively, in 2020 it was demonstrated that plasma C26:0-lysoPC analysis outperforms plasma VLCFA analysis; women with ALD – but normal plasma VLCFA levels – had elevated C26:0-lysoPC levels [Jaspers et al 2020].

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 diagnosis to detect a possible affected male fetus can be offered to women whose carrier status has been clearly confirmed by genetic analysis of the ABCD1 gene. In some countries, noninvasive prenatal determination of fetal sex using cell-free fetal DNA present in maternal blood may be performed at 7 weeks of pregnancy. It relies upon the detection of Y chromosome sequences by PCR techniques. If the fetus is a male, ABCD1 genetic testing can be performed on a fresh chorionic villus sample (CVS) at 11–13 weeks of pregnancy. Alternatively, the determination of fetal sex can be performed by conventional cytogenetic techniques on a CVS sample at 11–13 weeks of pregnancy and then ABCD1 genetic testing performed if the fetus is a male. Genetic testing takes a few days. Prenatal diagnosis can also be done on amniotic cells at 15–18 weeks of gestation. This approach, however, requires at least an additional 2–3 weeks of amniotic cell culture in order to generate enough cell material for molecular analysis. In case the ABCD1 variant has not yet been identified in the family, but the carrier status of the female has been clearly established biochemically by the demonstration of increased VLCFA levels in her plasma, prenatal diagnosis of a male fetus has to be done by the measurement of VLCFA levels in cultured CVS cells or amniotic cells. Preimplantation genetic diagnosis can also be offered in some countries for selected cases. Usually this is done when an affected female has had at least two prenatal diagnoses leading to pregnancy interruption because the fetus was an adrenoleukodystrophy male. A significant number of women with adrenoleukodystrophy develop a myelopathy in adulthood and prenatal diagnosis of a female fetus to establish whether the fetus has adrenoleukodystrophy may be offered on an individual basis. This situation occurs more and more frequently when either the father or the pregnant mother has severe clinical symptoms.

Newborn screening

Early diagnosis of adrenoleukodystrophy is the key to saving lives, because newborn screening allows prospective monitoring for adrenal function and the onset of cerebral ALD. A newborn screening test has been developed. It detects elevated VLCFA levels (as C26:0-lysoPC) in bloodspots. On December 30, 2013, the state of New York initiated screening for adrenoleukodystrophy in newborns. In February 2016, adrenoleukodystrophy was added to the United States Recommended Uniform Screening Panel (RUSP). Since then other states and countries have started newborn screening programs, or have initiated processes intended to add adrenoleukodystrophy to their existing newborn screening program. Detailed and up-to-date information on adrenoleukodystrophy newborn screening can be found at the “newborn screening” page.

Last modified | 2022-10-03