New discovery improves understanding and diagnosis of primary ciliary dyskinesia

In a discovery that could reshape the diagnosis of a baffling rare lung disease, researchers at Harvard Medical School and University Children’s Hospital Münster have mapped, in stunning molecular detail, the structure of a largely neglected region at the base of cilia — the tiny, hair-like appendages that sweep mucus, fluid, and debris out of our airways. By combining state-of-the-art cryo-electron tomography with human genetics, the team has uncovered mutations in two previously unsuspected genes that break down the structural integrity of this region, known as the transition zone, and in doing so cause primary ciliary dyskinesia, a devastating genetic disorder that afflicts an estimated one in several thousand people worldwide.

The findings, published Sept. 10 in the journal Science, are significant not just for what they reveal about the architecture of the cilium, but also for what they promise for patients. Between 20 and 30 percent of people with primary ciliary dyskinesia, or PCD, currently receive a genetic diagnosis that leaves their disease unexplained — no known genetic mutation, no clear molecular culprit. This new work offers at least some of those patients, and their physicians, a long-sought answer, and points to fresh molecular targets for the development of therapies in a field where treatment options remain painfully limited.

Primary ciliary dyskinesia is a rare, inherited condition that arises when the cilia that line the respiratory tract, the inner ear, the reproductive organs, and other tissues fail to beat in the coordinated rhythm needed to move fluids and particles along. When these microscopic machinery components break down, mucus accumulates in the lungs and sinuses, fluid builds up in the middle ear, and infection follows infection, often leading to chronic respiratory illness, scarring, and permanent lung damage. Roughly half of patients also experience abnormal organ placement or incomplete organ formation during embryonic development, because motile cilia also play a crucial role in establishing the left-right asymmetry of the body plan. The disease is usually diagnosed in childhood, typically after parents and pediatricians notice a relentless cycle of congestion, coughing, and ear infections that fails to respond to conventional treatment.

To develop PCD, most people must inherit two copies of a disease-causing gene, one from each parent. More than 50 such genes have been identified to date, yet a substantial fraction of cases — as many as three in ten — remain genetically unexplained. For those patients, the new study offers a previously unexplored corner of the cilium as a place to look: the transition zone.

The transition zone sits at the base of the cilium, where the organelle emerges from the cell body. It is, in essence, a molecular gatekeeper — a structured region that controls what proteins and other cargo are allowed to pass from the cell into the cilium’s main shaft. Structural abnormalities in the transition zone had already been linked to ciliopathies involving non-motile cilia, but until now, the mutations known to cause PCD had been traced to a different part of the organelle, the axoneme, the beating core of the cilium. The transition zone, in the context of motile cilia, had largely escaped scrutiny.

That changed thanks to a technological marriage. Haixia Zhou, a research fellow in the laboratory of Alan Brown, professor of biological chemistry and molecular pharmacology at Harvard Medical School, applied a cutting-edge imaging technique called cryo-focused ion beam milling-assisted electron tomography, or cryo-FIB-ET, to cultured cells derived from the lining of the human respiratory tract. The technique is a sophisticated evolution of cryo-electron microscopy, or cryo-EM, in which biological samples are flash-frozen in a glassy, vitrified state that preserves them in near-native form. In conventional cryo-EM, samples must be extraordinarily thin — on the order of 100 to 250 nanometers — for the electron beam to penetrate them and produce interpretable images. Most biological structures, including cilia, are far too thick. Cryo-FIB milling solves this problem by using a focused beam of ions to carve the frozen sample into ultrathin slices, shaving away material until the region of interest is thin enough for the microscope’s electrons to pass through.

The researchers then captured high-resolution snapshots of the transition zone from multiple angles and used advanced computational reconstruction, augmented with artificial intelligence tools, to assemble them into three-dimensional atomic models. The resulting images resolved the transition zone down to the sub-nanometer scale — fine enough to identify individual proteins and trace how they interlock with one another.

What the team found was a remarkably intricate piece of molecular engineering. Nine distinct protein types populate the transition zone, and four of them assemble into complexes that physically link together the cilium’s major structural elements — long, tube-like scaffolds called doublet microtubules that run the length of the organelle and provide the rigid framework against which the ciliary beat is generated. These linker complexes, the researchers discovered, act as the cross-braces of the transition zone, holding its architecture together and preserving its function as a selective gate.

The genetic leap came from the clinic. Heymut Omran, co-senior author of the study and a pediatrician at University Children’s Hospital Münster who treats children with ciliopathies, brought to the collaboration patients whose PCD had resisted genetic explanation for years. Working with co-first author Lea Terbeck and colleagues in Münster, along with collaborators at the University of Geneva and Boston University, the team identified mutations in the genes encoding two of the transition-zone linker proteins: ECT2L and DZANK1. Neither gene had previously been implicated in PCD.

When the researchers examined cilia carrying these mutations, the consequences were unmistakable. The linker complexes that hold the transition zone together were disrupted, and the cilia adopted abnormal shapes — some developing bulbous, swollen tips. More critically, the cilia lost their ability to beat in synchrony. In the airways, where millions of cilia must pulse in coordinated waves to push mucus upward and out of the lungs, this loss of coordination is catastrophic. Mucus stagnates, microbes colonize, and the cycle of infection and inflammation that defines PCD begins.

The researchers suspect the underlying mechanism is subtle but profound: because the transition zone acts as a gate into the cilium’s main body, mutations in ECT2L and DZANK1 may allow the wrong proteins to enter — or prevent the right ones from doing so — corrupting the cilium’s internal composition and, with it, its capacity for coordinated motion. In this view, PCD arising from transition-zone defects is not simply a failure of the cilium’s motor machinery, but a failure of the molecular customs office that governs what that machinery is made of.

The work deepens fundamental understanding of how motile cilia are built and function, and it calls scientists’ attention to a region of the organelle that has been comparatively neglected in studies of motile-cilia disease. It also expands the roster of genes known to cause PCD, immediately improving the diagnostic landscape. For the 20 to 30 percent of patients whose PCD has been a genetic mystery, testing for mutations in ECT2L, DZANK1, and other transition-zone genes now becomes a concrete possibility — and with a genetic diagnosis often comes clarity about prognosis, guidance for reproductive counseling, and eligibility for emerging targeted therapies.

Beyond diagnosis, the newly identified proteins offer researchers concrete targets for drug development. Current treatments for PCD can only slow disease progression; nothing yet exists to halt or reverse it. Knowing precisely which molecular linkers fail, and seeing their structures at atomic resolution, gives structural biologists and medicinal chemists a starting point for designing interventions — whether small molecules that stabilize defective linkers, gene therapies that restore functional copies of the affected genes, or other strategies yet to be imagined.

For Alan Brown, the study exemplifies a broader methodological promise. “This is an example of how combining in situ structural biology with genetics can address fundamental questions and illuminate the causes of human disease,” he said. By imaging proteins in their native cellular environment rather than in isolation, and by anchoring those structural observations to real patients with real mutations, the team has demonstrated a template for future discoveries — one in which the transition zone, long a blind spot in motile-cilia biology, now stands revealed in all its molecular complexity, and in which a new chapter opens in the effort to understand, diagnose, and eventually treat one of medicine’s most stubborn rare diseases.

The research was conducted at the Harvard Cryo-EM Center for Structural Biology at HMS, which acquired cryo-FIB-ET capabilities in 2022, with contributions from the HMS Electron Microscopy Core Facility and the SBGrid Consortium. The authors also extended their thanks to the individuals with PCD and their families who participated in the study — the patients whose cells and genetic samples made this molecular portrait of the transition zone possible.