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 situations:

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

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

• All men with primary adrenal insufficiency, with or without evidence of neurologic abnormalities.

• Middle-aged or older women with progressive paraparesis, abnormal sphincter control, and sensory disturbances primarily in the legs. The diagnosis of adrenoleukodystrophy may be difficult to establish in a woman with a negative family history. Diagnosis is based on clinical features, most commonly progressive spastic paraparesis, and a panel of laboratory tests.

Neuroimaging

Brain MRI is always abnormal in neurologically symptomatic men and often provides the first diagnostic clue. In approximately 85% of affected individuals, MRI shows a characteristic pattern of symmetrically enhanced T2 signal in the parieto-occipital region with contrast enhancement at the advancing margin.

Very long-chain fatty acids

Men: The most commonly used laboratory test worldwide for the diagnosis of ALD is the measurement of plasma very long-chain fatty acid (VLCFA) concentrations. 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.
VLCFA analysis is highly specialized and is performed in only a few laboratories worldwide.
Important note 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 may cause an elevation similar to that seen with Lorenzo oil therapy.

Women with ALD: Increased levels of VLCFA in plasma and/or cultured skin fibroblasts is present in approximately 85% of women with ALD; 15-20% of known women with ALD have normal plasma levels of VLCFA. Females should be genetically tested if adrenoleukodystrophy is suspected and VLCFA concentrations are normal.

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

VLCFA containing C26:0-lysoPC is elevated in all men and >99% women with ALD and is used in newborn screening for ALD. It has been shown 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 a diagnostic biomarker for ALD.

Genetic testing

The ABCD1 gene is the only gene associated with adrenoleukodystrophy. More than 1200 different (likely) pathogenic variants have been identified in ABCD1 [The ALD Variant Database]. Many adrenoleukodystrophy kindreds 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 pattern. The ABCD1 gene is the only gene associated with adrenoleukodystrophy.
Parents of a male or female patient: Approximately 93% of index cases have inherited the genetic defect from one parent, which often makes it likely that other family members, such as biological siblings and cousins, are also affected. However, in ~7% of cases the child has undergone a spontaneous mutation, also called “de novo“. In these cases, there are no other family members with the mutated gene. It is appropriate to measure plasma VLCFA levels in the mothers of both affected males and females and in the fathers of affected females (Figure below). Once the pathogenic variant has been identified in an affected family member, genetic testing of the ABCD1 gene can be used to evaluate the parents to screen the for extended family.
Siblings of an index patient (proband): The risk to siblings depends on the genetic status of the parents, which can be determined by pedigree analysis, VLCFA measurement, and molecular genetic testing.
If the proband’s mother has a pathogenic variant in the ABCD1 gene (heterozygote), there is a 50% chance of transmitting the pathogenic variant in each pregnancy. Male and female siblings who inherit the pathogenic variant will be affected.
If the proband’s father has a pathogenic variant in the ABCD1 gene, all female siblings will be affected and none of the male siblings will be affected.
If neither parent has a pathogenic variant in the ABCD1 gene, the risk to the proband’s siblings is low.
Offspring of a proband: Affected males will 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 are affected; daughters who inherit the pathogenic variant are carriers of 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: If the child is a daughter, there is a 50% chance that the daughter will inherit the defective adrenoleukodystrophy gene and a 50% chance that the daughter will be unaffected. If the child is a boy, there is a 50% chance that the son will have adrenoleukodystrophy and a 50% chance that he will be unaffected. (Right) In an X-linked disease such as adrenoleukodystrophy, if an affected man has children, all of his sons will be free of the disease because the father always passes on his Y chromosome on to his sons. However, all of his daughters will inherit the defective adrenoleukodystrophy gene (he always passes on his affected X chromosome on to his daughter).

Heterozygote identification

Testing at-risk female relatives for carrier status is a two-step process. First, the plasma concentration of VLCFA is measured; if abnormal, the female is affected. Because approximately 15% of women with ALD have normal plasma concentrations of VLCFA, molecular genetic testing should be used to test those women with normal concentrations. This is easier if the disease-causing ABCD1 genetic variant has already been identified in the family. Alternatively, it was shown in 2020 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 on 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 affected.

Evaluation of at-risk family members is important for management and genetic counseling, but it is not always done adequately. Several factors may contribute to inadequate evaluation:

• The diagnosis of an affected individual with severe disabilities can be devastating to a family. Immediate concerns may overshadow timely testing of family members.
• Molecular genetic testing has recently become clinically available, but its availability may not be widely 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 may affect their ability to obtain or maintain health insurance coverage.
• Individuals may have incomplete knowledge of at-risk family members and may not want to inform them of 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, non-invasive prenatal determination of fetal sex using cell-free fetal DNA in maternal blood can be performed at 7 weeks of gestation. It is based on the detection of Y chromosome sequences by PCR techniques. If the fetus is male, ABCD1 genetic testing can be performed on a fresh chorionic villus sample (CVS) at 11 to 13 weeks of gestation. Alternatively, fetal sex can be determined using conventional cytogenetic techniques on a CVS sample at 11 to 13 weeks of gestation followed by ABCD1 genetic testing if the fetus is male. Genetic testing takes several days. Prenatal diagnosis can also be done on amniotic cells at 15 to 18 weeks of pregnancy. However, this approach requires at least an additional 2 to 3 weeks of amniotic cell culture to generate enough cell material for molecular analysis. If 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 elevated VLCFA levels in her plasma, prenatal diagnosis of a male fetus must be performed by measuring VLCFA levels in cultured CVS cells or amniotic cells. In some countries, preimplantation genetic diagnosis may also be offered in selected cases. This is usually done when an affected woman has had at least two prenatal diagnoses that resulted in pregnancy termination because the fetus was a male with adrenoleukodystrophy. A significant number of women with adrenoleukodystrophy develop myelopathy in adulthood, and prenatal diagnosis of a female fetus to determine whether the fetus has adrenoleukodystrophy may be offered on an individual basis. This situation is becoming more common when either the father or the pregnant mother has severe clinical symptoms.

Newborn screening

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

Last modified | 2024-06-22