BACKGROUND

The story of gene discovery in the neurodegeneration with brain iron accumulation (NBIA) disorders is the story of recognizing and delineating each individual disorder. The field began with the observation of a shared pathologic finding, that of basal ganglia iron accumulation. This feature led to the ‘bulk’ diagnosis of what was formerly called Hallervorden-Spatz syndrome, an eponym that has been discarded and is now understood to include multiple distinct disorders. A critical factor that drove most of the NBIA gene discoveries, many of which occurred in parallel with advances in the Human Genome Project, was the collection of clinical data and biological samples by a small number of investigators. These important resources allowed for a growing clinical acumen and provided the data needed to recognize distinct phenotypes. Once a homogeneous phenotype was recognized, sufficient sample numbers were available to enable gene discovery, mutation testing, and confirmation in the era before whole genome analyses were performed.

Knowledge about the pathophysiology of most Mendelian disorders can be divided into the periods that precede and follow discovery of their genetic basis. Before a genetic defect is known, understanding of a disorder is typically based on what can be observed clinically and pathologically. With such constrained knowledge, inferences about disease pathophysiology and ideas for treatment are limited. With discovery of the disease gene, everything changes. Instantly, the encoded protein’s specific cellular function and role in defined pathways, if already known, can spark new hypotheses of pathogenesis. Powerful genetic tools to manipulate biological systems can then be used to test such hypotheses and refine and retest them, with the goal of accurately delineating the earliest molecular changes of the disease. The purpose of gaining insight into early changes is to reveal potential therapeutic targets that are proximate to the primary defect. This therapeutic approach has the potential to halt a disease process in that critical phase before cells respond irreparably to the primary defect. The power of single-gene disorders is that the first steps in the cascade of cellular changes can be identified and temporally sequenced, laying the foundation for high impact interventions. The essential element is knowledge of the mutated gene.

FROM ONE DISORDER TO MANY

The NBIA disorders are now known as a group of distinct single-gene disorders that share common neuropathologic features [1]. Their original grouping was also founded on this basis, with the common features of postmortem basal ganglia iron accumulation and axonal swellings or spheroids [2-4]. As their genetic bases have been elucidated, their delineation as specific disorders became possible. For nearly all NBIA disorders, the defective proteins have no known role in iron homeostasis. Instead, each disorder arises as a result of a primary pathway defect that then leads to secondary or later sequelae, including iron accumulation. Loss or mis-sensing of bioavailable iron is a common theme, leading to miscommunication between organelles and aberrant signaling for increased uptake, a phenomenon that is hypothesized to explain iron accumulation in many neurodegenerative disorders [5-10]. Is there still value in the NBIA grouping? While many common neurodegenerative disorders also lead to brain iron accumulation, including Parkinson disease, Alzheimer disease, and multiple sclerosis, in the NBIA disorders, iron accumulates specifically in the basal ganglia. This distinct pattern may indicate a final common pathway in their pathogenesis [11,12]. Beyond iron, their broad pathological overlap lends credence to the hypothesis of a shared pathophysiology, at least for some subset of the changes observed. Moreover, the community of people affected by one of the NBIA disorders and their families have joined together as one advocacy community to address their needs. For these reasons, the grouping has value and therefore is likely to endure.

Before the discovery of the first NBIA gene, pantothenate kinase-associated neurodegeneration (PKAN) was known for its main pathological features of brain iron accumulation and axonal dystrophy. Originally described a century ago, Hallervorden and Spatz [13] reported a family with five of nine siblings affected by a progressive neurological disorder manifesting with abnormal movements and loss of vision. Their key finding on postmortem gross examination of the fixed, unstained brain was that the globus pallidus appeared rust brown in color. Their examination concluded that the staining was from high iron content in the globus pallidus and substantia nigra. A contemporary examination of this century-old report reveals distinct pathological features that we now recognize to be specific to and virtually diagnostic of PKAN (Randy Woltjer, personal communication in 2019). The original Hallervorden-Spatz syndrome was PKAN.

While the family in that report manifested a presumed genetic disorder, cases reported later represented a clinically and pathologically heterogeneous group of disorders with iron accumulation and axonal dystrophy. All were referred to as ‘Hallervorden-Spatz syndrome’. In 1974, Dooling et al. [14] reviewed 64 cases from the medical literature, including two from a family in their care. They rightly concluded that several distinct diagnoses were likely to be represented within the group, and they sought to cluster them into subsets based on specific clinical features. Interestingly, the sisters that prompted Dooling’s work almost certainly did not have PKAN based on the presence of neuropathological features not found in PKAN, including Lewy bodies. More likely, they were affected with mitochondrial membrane protein-associated neurodegeneration (MPAN) or phospholipase A2-associated neurodegeneration (PLAN).

Since a diagnosis of Hallervorden-Spatz syndrome at the time relied on postmortem findings of iron accumulation, premortem diagnosis was possible only with the introduction of brain magnetic resonance imaging (MRI) into routine clinical care. Sethi et al. [15] described a distinctive pattern on brain MRI that they called the ‘eye-of-the-tiger’ sign, which they noted was not present in all patients with neuroimaging evidence of high basal ganglia iron. Brain MRI became more commonplace during the 1990s, making the premortem diagnosis of Hallervorden-Spatz syndrome possible. As new NBIA disorders were identified, their specific MRI patterns assisted in their clinical diagnosis.

As a whole, the NBIA disorders are clinically and radiographically distinct. Therefore, unlike disorders that are clinically indistinguishable, such as the ataxias and spastic paraplegias, the NBIA disorders are not genetic subtypes of a common phenotype. Instead, they are distinct clinical disorders that share common pathologic features. As such, each should be referred to as an NBIA disorder and not as a subtype or form of NBIA.

DISCOVERY OF THE GENE FOR PKAN

As the Human Genome Project advanced, work to identify the gene for Hallervorden-Spatz syndrome was underway. At least three laboratories independently pursued this goal, including the groups of Meitinger in Munich, Germany; Seidman in Boston, Massachusetts, USA; and Hayflick in Portland, Oregon, USA. In 1996, the gene for Hallervorden-Spatz syndrome was mapped to chromosome 20p12.3-p13 using linkage analysis in a large Amish pedigree [16]. History guided the naming of the locus during the particular interval of time after the disease gene was mapped but before its identity was known.

In 1992, a publication in the medical literature revealed the objectionable role that Hallervorden played in active ‘euthanasia’ programs in Nazi Germany [17]. Spatz was also a member of the Nazi party and engaged in the study of brains that had been obtained by killing people deemed to be ‘mentally defective’ and thus unworthy of living. In bringing this information to light, Shevell [17] implored the medical community to abandon the eponym ‘Hallervorden-Spatz syndrome’. Mapping of the disease gene locus provided just such an opportunity by requiring that a temporary name be applied to the region in which the gene was located but before the identity of the gene was known. The designation ‘NBIA’, for ‘neurodegeneration with brain iron accumulation’, was proposed by Hayflick and accepted by the Human Genome Nomenclature committee in 1996 as the term for the Hallervorden-Spatz syndrome disease gene region on chromosome 20.

The first NBIA gene was discovered in 2001 by the Gitschier and Hayflick groups using positional cloning [18]. The first mutation was found in the original Amish HS1 family in a gene encoding a novel pantothenate kinase homolog called PANK2. Their DNA had been used for screening candidate genes in the 20p13 region [16], and the homozygous seven-nucleotide exonic deletion leading to a frameshift and stop codon in the gene named ‘PANK2’ was immediately recognized to be pathogenic. The discovery, by Bing Zhou and Barbara Levinson [18] in Jane Gitschier’s Lab at UCSF, was heralded in an email to Hayflick late in the afternoon with the subject line reading: “Hold onto your hat!”. The high likelihood of pathogenicity of the original HS1 mutation gave both groups confidence in the disease gene discovery finding and led to further mutation screening in unrelated families to confirm the finding. Mutations in PANK2 were found in 32 of 38 individuals with a clinical diagnosis of an NBIA disorder [18].

That day in March was remarkable for the contrast of what was known at the start of the day versus what was known at the end. In the morning, iron and axonal spheroids were known to accumulate in the disorder, but its pathophysiology was otherwise entirely opaque. By the end of that day, we had identified a gene, a protein, a pathway, and a potential treatment (vitamin B5) for this devastating disease.

The disease associated with mutations in PANK2 was called ‘pantothenate kinase-associated neurodegeneration’ or PKAN [18], a name suggested by Barbara Levinson, as we sought to shift away from use of the objectionable eponym. The wider group of heterogeneous phenotypes associated with iron accumulation and axonal spheroids was collectively termed ‘NBIA’ [19]. Since then, many more NBIA disease genes have been identified, and the use of this umbrella term inclusive of all NBIA disorders has gained favor [20].

Discovery of the PANK2 gene enabled a more refined delineation of the NBIA disorders, which helped to drive the discoveries of further disease genes. The phenotypes associated with mutations in PANK2 were described, as was the strong association with a specific brain MRI pattern [21]. Years earlier, Sethi et al. [15] described the MRI changes seen in Hallervorden-Spatz syndrome and called them the ‘eye-of-the-tiger’ sign. This distinctive pattern on T2-weighted imaging of a central region of high signal intensity surrounded by an area of low signal intensity in the bilateral globus pallidus is readily recognizable and was subsequently shown to be virtually pathognomonic for PKAN [21]. As a corollary, we could now identify individuals without mutations in PANK2 and without an ‘eye-of-the-tiger’ sign. These formed the cohorts that we and others used for further disease gene discovery, an effort that was greatly enabled by our international biobank of NBIA samples and clinical data.

DISCOVERY OF THE GENE FOR PLAN

The registry and biosample collection begun by the Hayflick group in 1993 had grown to more than 200 families by 2001, largely through the dedication of neurologists from around the world. With key samples and phenotype data contributed by Nardocci in Milan and many others, we localized the gene for infantile neuroaxonal dystrophy (INAD) to chromosome 22q [22]. Concurrently, Eamonn Maher’s group in the UK mapped the gene to a partially overlapping region using a large inbred Pakistani family [22]. Our lab groups joined efforts, and Neil Morgan in Eamonn Maher’s group at the University of Birmingham School of Medicine, UK discovered the first mutations in PLA2G6 in early 2006 [22]. As with PANK2, we screened our cohort for mutations in PLA2G6 and confirmed its identity as the underlying disease gene [22]. As with PKAN, better delineation of the phenotype followed, including a description of its full spectrum [23-25].

With the discovery of the PLA2G6 gene, a naming convention was adopted for each of the NBIA disorders. NBIA would continue to refer to the composite group, and each genetic disorder would be given a name that referenced the specific gene or protein defect. The intent was to link a rational name to the underlying cause and to create an acronym that was easy to remember. Although I initially resisted Maher’s idea of calling INAD ‘phospholipase A2-associated neurodegeneration’ or ‘PLAN’, he was right in seeing the value of applying this convention [23]. The term ‘INAD’ is still in use; however, the eponym has been largely abandoned given Seitelberger’s support of the Third Reich [26]. ‘PLAN’ is now a widely used term, with distinction by age at onset into the infantile, juvenile and adult forms of PLAN [27].

DISCOVERY OF THE GENE FOR MPAN

In 2011, the Prokisch and Meitinger groups in Munich reported mutations in C19orf12 in a large Eastern European cohort with a new NBIA disorder [28]. This marked the discovery of the gene that they named ‘mitochondrial membrane proteinassociated neurodegeneration’, or ‘MPAN’. A founder mutation was the basis for this disorder being much more common in Eastern Europe than elsewhere. Similar to PKAN and PLAN, MPAN was first reported as an autosomal recessive disorder. Gregory et al. [29] subsequently reported the observation that single mutant alleles in some cases could cause an autosomal dominant form of MPAN, with clinical features similar to those of recessive MPAN.

DISCOVERY OF THE GENE FOR BPAN

Still unidentified among the idiopathic NBIA disorders at that time was the disorder that would eventually turn out to be the most prevalent in most populations. The BPAN phenotype of childhood developmental disability and seizures with aggressive parkinsonism in early adulthood was recognized several years before the disease gene was discovered [30]. Insightful observations from years of data collection enabled the coalescence of a clinically homogeneous cohort for whole exome sequencing, the first time this strategy was employed for gene discovery in an NBIA disorder. In a partnership between the Prokisch and Hayflick groups, mutations in WDR45 were found in a group of twenty affected individuals [31]. This effort also marked the first time after the initial mutations were found that the larger NBIA scientific community came together to mine the individual biobanks for additional cases. WDR45, or WIPI4, is a beta-propeller protein important for enabling the interactions of key autophagic proteins. This led to the name ‘betapropeller protein-associated neurodegeneration’ or ‘BPAN’. The fact that WDR45 localizes to the X chromosome was missed despite an obviously skewed gender balance in the initial cohort (3 males and 17 females). Had we recognized this, the gene hunt would have been greatly simplified. Whole exome sequencing yielded an answer in just a few weeks. BPAN is of course now known to be an X-linked dominant NBIA disorder arising mostly from de novo mutations.

OTHER NBIA DISORDERS

PKAN, PLAN, MPAN and BPAN account for the vast majority of NBIA disorders in all populations. Subsequent discoveries of the FAHN and CoPAN genes largely completed the task of identifying the genes underlying this heterogeneous group. ‘Fatty acid hydroxylase-associated neurodegeneration’ or ‘FAHN’, was discovered in a collaboration by the Hardy and Hayflick groups [32], with the choice of names endorsed prepublication by Stan Fahn (personal communication in 2009), one of the giants in the field of movement disorders. Mutations in the COASY gene, encoding coenzyme A (CoA) synthase and responsible for CoPAN, were found by the Tiranti group and represented an important discovery as the second NBIA disease gene in the CoA synthesis pathway [33]. This was the second time the larger NBIA scientific collective was invited to join forces by screening for mutations to contribute to the discovery. Together, more than 95% of NBIA disorders can be attributed to mutations in PANK2, PLA2G6, C19orf12, WDR45, FA2H, and COASY. Neuroferritinopathy and aceruloplasminemia, caused by mutations in FTL and CP, respectively, are often included among the NBIA disorders but are distinguished by a known role for these proteins in iron homeostasis.

WHAT CONSTITUTES AN NBIA DISORDER?

Other genes have been proposed as new NBIA disease genes, and some may reasonably fit. Moreover, more NBIA genes are likely to be discovered, albeit accounting for only a small fraction of cases. What are the criteria that define an NBIA disorder? Specific criteria are lacking but could include the following: 1) single-gene disorder; 2) increased iron in the globus pallidus with or without increased iron in the substantia nigra; and 3) neurodegeneration. These criteria will certainly spark debate, but more important than delineating specific inclusion/exclusion criteria is answering the questions ‘What is the value of such criteria?’ and ‘Does it matter what is included/excluded?’ I would offer that it does not matter and that there is limited value in seeking to define what is and is not an NBIA disorder.

LOOKING AHEAD

Most NBIA disease genes were found during what was arguably the heyday of human disease gene discovery. This rich period coincided with the Human Genome Project, which of course enabled and accelerated these findings. For most NBIA disorders, the focus has now turned to advancing the knowledge of their pathophysiologies and developing rational therapeutics, including gene therapies. These diseases all begin at the gene level, and for some, perhaps they will end there too.

Notes

Conflicts of Interest

The author has no financial conflicts of interest.

Funding Statement

This work was funded by NIH grants from the NEI, NINDS, and NICHD and by the NBIA Disorders Association, Hoffnungsbaum e.V., and AISNAF.

Acknowledgments

I am grateful to the families and the worldwide scientific community for their contributions, collaboration, and support.

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