Undiagnosed Disease Network collaborative approach in diagnosing rare disease in a patient with a mosaic CACNA1D variant

The Undiagnosed Disease Network (UDN) is comprised of clinical and research experts collaborating to diagnose rare disease. The UDN is funded by the National Institutes of Health and includes 12 different clinical sites (About Us, 2022). Here we highlight the success of collaborative efforts within the UDN Clinical Site at Vanderbilt University Medical Center (VUMC) in utilizing a cohort of experts in bioinformatics, structural biology, and genetics specialists in diagnosing rare disease. Our UDN team identified a de novo mosaic CACNA1D variant c.2299T>C in a 5‐year‐old female with a history of global developmental delay, dystonia, dyskinesis, and seizures. Using a collaborative multidisciplinary approach, our VUMC UDN team diagnosed the participant with Primary Aldosteronism, Seizures, and Neurologic abnormalities (PASNA) OMIM: 615474 due to a rare mosaic CACNA1D variant (O'Neill, 2013). Interestingly, this patient was mosaic, a phenotypic trait previously unreported in PASNA cases. This report highlights the importance of a multidisciplinary approach in diagnosing rare disease.


| INTRODUCTION
The Undiagnosed Disease Network (UDN) is a collaborative effort funded by the National Institutes of Health seeking to diagnose rare disease (About Us, 2022).The UDN includes 12 clinical sites distributed across the United States as well as a coordinating center, a biorepository, and core laboratories (sequencing, model organism, and metabolomics) (Tinker et al., 2023).Applicants to the UDN have experienced a diagnostic odyssey in which thorough clinical evaluations, often spanning months or years, have failed to find a unifying diagnosis for their constellation of symptoms.Once accepted to the UDN, participants consent to IRB protocol and undergo evaluation and testing.As of November 9, 2023, 2305 participants have been evaluated by the UDN and 697 who have completed their UDN evaluation received a diagnosis (Facts and Figures, n.d.).This case highlights the success of the UDN multidisciplinary approach to precision medicine that was required to diagnose rare disease found in our participant.
The UDN participant in this case was seen at the Vanderbilt University Medical Center (VUMC) UDN clinical site and was diagnosed with Primary Aldosteronism, Seizures, and Neurologic abnormalities (PASNA) syndrome (OMIM# 615474) (O'Neill, 2013).Clinical presentations of PASNA syndrome include primary hyperaldosteronism causing marked hypertension and hypokalemia as well as neurologic symptoms including seizure, cerebral palsy, developmental delay, cortical blindness, and movement dysfunction such as athetosis (Scholl et al., 2013).Not all patients with pathogenic CACNA1D variants have hypertension, hyperaldosteronism, or evidence of hyperinsulinemia, demonstrating variable expressivity in the phenotype (Alzahrani et al., 2023).
PASNA syndrome is caused by heterozygous variants in the CAC-NA1D gene, which encodes a α 1 subunit of L-type voltage-gated calcium channel (Ca v 1.3) (Azizan et al., 2013).Ca v 1.3 channels are highly expressed in adrenal glands and play an important role in regulation of aldosterone production, which helps to control blood pressure and electrolytes.Somatic variants in the CACNA1D gene are associated with aldosterone-producing adenoma.Ca v 1.3 channels are also expressed in various tissues, including brain, sinoatrial and atrioventricular node, cochlea, and pancreatic β cells (Koschak et al., 2001;Scharinger et al., 2015;Schlick et al., 2010;Yang & Berggren, 2006).

| UDN synergism
The VUMC UDN team relies on a multidisciplinary approach in evaluation of patients.Participants at our site who have consented to IRB protocol undergo comprehensive medical record review, genomic sequencing (GS), genome data board review, and clinical evaluation (Figure 1).The UDN GS data is meticulously analyzed by a team of experts in medical genetics, structural biology, bioinformatics, genetic counseling, neurology, endocrinology, internal medicine, and nurse practitioners.The candidate variants generated from GS data are narrowed down by phenotyping, metabolomic testing, RNA sequencing, or functional studies.

| BioVU
BioVU is Vanderbilt's biorepository of DNA extracted from surplus from routine clinical blood test samples that are used in research to discover genotype/phenotype correlation (BioVU j Department of Biomedical Informatics, n.d.).Data were drawn from the Research Derivative, a copy of VUMC's EHR stored in the Observational Medical Outcomes Partnership common data model that includes demographics, clinical notes, International Classification of Disease, and Current Procedural Terminology codes used in this project.Our team relies on this biorepository as we compare genotype/phenotype with our proband.In this case, the candidate research variants were searched among the biorepository with respect to our patient's phenotype of global developmental delay, dystonia, chorea, and seizures.

| Structural biology
Our structural biology specialists used the "Human L-type voltagegated calcium channel Ca V 1.3 at 3.0 Angstrom resolution" structure (PDB ID: 7UHG) for structural analyses (Yao et al., 2022).This PDB structure was refined using the Rosetta cryo-EM framework to optimize the protein interactions and to build the hydrogen atoms (Wang  et al., 2016).The resulting optimized Ca V 1.3 structure was used for the Rosetta ΔΔG calculations.The purpose of these calculations is to measure the change in the stability of the system caused by a variant by calculating the difference between a mutant and a native state.
Positive ΔΔG values indicate destabilization and negative ΔΔG values indicate stabilization by a variant.We investigated F767L and F767S variants using our method.F767S was included because it is a known pathogenic variant that causes gain-of-function activity, so structural and energetic comparisons with this variant were used to understand the extent of the effect of the F767L variant (Török et al., 2022).
Briefly, the corresponding in silico variant was introduced into the system with Rosetta, followed by a relax calculation to minimize the energy of the residue backbone and sidechains within 12 Å of residue 767 (Conway et al., 2014;Tyka et al., 2011).This was repeated with the F767F synonymous variant to cancel out the energy changes caused by the relax protocol itself.The energy differences between the F767L or F767S and F767F variants were calculated to obtain the final ΔΔG value.

| Case presentation
The proband was a 5-year-old female referred to the VUMC UDN site for esotropia, global developmental delay, dystonia, and dyskinesis most consistent with chorea.She also had a history of seizures, insomnia, Chiari type 1 malformation, syringomyelia, cortical visual impairment, and obstructive sleep apnea (OSA).She was the 6 lb. 8 oz.product of a full-term uncomplicated pregnancy and delivery.
Hypoglycemia was reported first 1-2 days of life in the nursery then improved.At 6 months of age concerns were first noted by her parents.Developmentally, she did not sit, stand, walk, or use any words at 5 years of age.There were no other similarly affected family members.
Prior to referral to the UDN the participant underwent a comprehensive workup seeking to identify the cause of her constellation of symptoms.Brain MRI demonstrated a type 1 malformation and subsequently two small syringomyelia.She had a normal chromosome microarray, Prader-Willi/Angelman syndrome DNA methylation tests, and exome sequencing, which did not identify a unifying diagnosis for her symptoms.

| UDN workup
A GS sequencing trio that included both parents and the participant was ordered as part of her UDN workup.Our UDN Sequencing Core Lab identified a de novo mosaic (27% variant allele fraction) CACNA1D variant NM_000720.4:c.2299T>C;p.Phe767Leu.A CADD score of 32 and GERP score of 6.47 further suggests a deleterious impact on gene function.This variant was confirmed by Sanger sequencing.This variant was absent from gnomAD exome and genome data with coverage of 79.4 and 34.8, respectively (Kopanos et al., 2019).Other testing during her UDN evaluation included normal chromosome breakage study, alpha-fetoprotein, and aldosterone to narrow down candidate variants.Renin was found to be low (<2.1) with reference range being 2.5-45.7 pg/mL.Low renin has been found in PASNA phenotype (Scholl et al., 2013).Physical exam by a geneticist noted shallow palmar creases and a broad nasal bridge, otherwise nondysmorphic.

| Structural biology
The ΔΔG value calculated for the F767L variant was 5.3 Rosetta Energy Units (REU), which indicates destabilization caused by the variant.Since REU is not an absolute metric and can only be understood in relative terms, we compared the F767L variant's effect with that of F767S.The latter value was 3.9 REU, which suggests that the F767L variant destabilizes the Ca V 1.3 at a higher extent than the known pathogenic variant F767S.A closer look at the variant site with UCSF Chimera shows that the Phe to Leu substitution causes loss of critical contacts with residues such as L677, L763, and I1137 (Figure 2), which may explain the destabilization caused by this variant (Pettersen et al., 2004).
The destabilization induced by the F767L variant found in our participant is thought to cause a gain-of-function that may be related to the effect of this variant on the state distribution of Ca V 1.3.By destabilizing the closed state of the receptor, F767L may push the equilibrium between the two states toward the open state, increasing the probability or length of Ca V 1.3 channel opening.

| Final diagnosis
We report here an atypical case of PASNA syndrome due to de novo mosaic CACNA1D variant (NM_000720.4:c.2299T>C;p.Phe767Leu) found in 27% of her reads.This is the first report of PASNA syndrome caused by a de novo mosaic CACNA1D variant.The p.Phe767Leu variant identified in our patient is listed under MANE select transcript (NM_001128840.3) as p.Phe747Leu found in somatic form in aldosterone producing adenomas (Fernandes-Rosa et al., 2014).This is the first report of p.Phe767Leu attributable to germline disease.A multidisciplinary approach using BioVu findings of genotype/phenotype matches, structural biology findings of destabilizing effect, and phenotyping were able to diagnoses PASNA in our participant (O'Neill, 2013).Her phenotype that overlaps with PASNA includes low renin, history of hypokalemia, cortical blindness, global developmental delay, seizures, and movement disorders.Heterozygosity for germline CACNA1D missense gain of function variants have been associated with primary aldosteronism.There is one report of two females presenting with primary aldosteronism and other findings (Scholl et al., 2013).The first had high aldosterone levels with low renin and elevated aldosterone to renin ratio, seizures, cortical blindness, global developmental delay, cerebral palsy, and spasticity.The second had cerebral palsy, spastic quadriplegia, athetosis, developmental delay, seizures and she later developed symptoms of aldosteronism with hypertension, hypokalemia, and elevated aldosterone.It is important to note the second patient reported in Scholl et al. (2013) had a traumatic birth which may have contributed to her spastic quadriplegia.

| Treatment implications
There is association between PASNA and left ventricular hypertrophy, biventricular hypertrophy, ventricular septal defect, patent foramen ovale, second-degree heart block, and pulmonary hypertension (O'Neill, 2013).Due to the association between PASNA and cardiac symptoms, we referred our patient to cardiology for baseline evaluation including EKG and echocardiogram.Additionally, studies below have shown dihydropyridine LTCC blockers may have therapeutic benefit in individuals with gain of function CACANA1D variants (Pinggera et al., 2015;Pinggera et al., 2017).We recommended to our cardiologist that they consider possible treatment with dihydropyridine LTCC blockers if clinically indicated as our participant's variant appears to be gain of function.The patient was also referred to endocrinology and nephrology to monitor glucose and aldosterone levels.

| UDN utility
Here  (Alzahrani et al., 2023).To our knowledge, there is no previous report of cases of PASNA syndrome presenting with mosaic etiology.Our participant's degree of mosaicism may explain why she does not have recognizable symptoms of aldosteronism at this time, but as previously reported symptoms of aldosteronism may develop over time (Scholl et al., 2013).
The collaborative efforts within the UDN site at VUMC were paramount in diagnosing rare disease in this case by use of gene match individuals from our BioVU cohort and structural biology interpretation.Access to our institutional biorepository is highly useful in interrogating variants of interest.Gene match individuals found in BioVU with overlapping phenotype of developmental delay, seizures, insomnia, OSA, and dystonia contributed to diagnosis discovery in our patient.Structural biology in-depth interpretation find that F767L variant destabilizes the Ca V 1.3 at a higher extent than the known pathogenic variant F767S.Such collaborative efforts of BioVU repository and structural biology in-depth analysis are not available in the clinical setting.The cumulative findings provided fruitful recommendations to the patient's clinical team regarding management and treatment.

| CONCLUSION
The collective efforts of a multidisciplinary UDN team approach were paramount in diagnosing a rare disease in an individual who was previously undiagnosed despite a thorough clinical evaluation.This case The residues within 3 Å of F767 shown for the F767F (left, beige) and F767L (right, blue) structures.
expands the phenotype of PASNA as no other cases have been reported with mosaic etiology to our knowledge.This case highlights the crucial role of such a team in addressing challenging cases and underscores the importance of a multidisciplinary approach in diagnosing rare disease.
The left side of the figure path indicates participants undergoing visit first pipeline.The right side of the figure path indicates sequencing first pipeline.UDN, Undiagnosed Disease Network.
BioVU analysis discovered two gene matched individuals previously evaluated at VUMC affected by pathogenic variants in CACNA1D.The first had a developmental delay, speech delay, autism, seizures, insomnia, OSA, and hydrocephalus.This patient harbored a presumed pathogenic variant CACNA1D NM_000720.3:c.1998dup(p.Met667Hisfs*31).The second BioVU case had a CACNA1D NM_000720.3:c.2686C>T(p.Arg896Cys) presumed pathogenic variant and developmental delay, concern for seizures, dystonia, tremors, delayed teeth eruption, and premature pubic hair.The genotype and phenotype matches of developmental delay, seizures, insomnia, OSA, and dystonia aided in the diagnosis of our patient.
we described a patient diagnosed with PASNA syndrome caused by de novo mosaic (27% variant allele fraction) c.2299T>C; p.-Phe767Leu variant in the CACNA1D gene that was predicted to dis- rupt gene function by GERP and CADD scores, absence from general population genomic databases, genotype/phenotype matches of two other patients in BioVU, and structural biology analysis.Similar to other patients reported in the literature, our patient presented with seizures, global developmental delay, insomnia, movement disorder including dystonia and dyskinesis, and cortical visual impairment.A broad spectrum of neurological phenotypes has been reported in individuals with CACNA1D missense gain of function variants, including intellectual disability, developmental delay autism spectrum disorder, seizures, aggressive and self-injurious behaviors, and sleep disorders