Co-Occurrence of Neurofibromatosis Type 1 and Polycystic Liver Disease: A Case of Hypertension with Variant

Introduction

Neurofibromatosis type 1 (NF1) is a common autosomal disorder (estimated prevalence at 1: 3000 live births) which has highly heterogeneous clinical and molecular manifestations [1]. The risk of developing a pheochromocytoma or paraganglioma (PHEO/PGL) is higher in patients with NF1, with a 0.1–5.7% lifetime risk of PHEO/PGL [2]. The median age of diagnosis of PHEO/PGL was 46 years (range 16–82 years), and most patients had high metanephrine and normetanephrine levels in plasma and urine [3,4]. Because of the low frequency of PHEO/PGL in individuals with NF-1, other causes of secondary hypertension (HT), especially renovascular disease, should also be excluded. Moreover, atypical phenotypes that do not fit with known features of NF1 syndrome should be investigated to exclude the co-occurrence of other genetic disorders.

The availability of whole-exome sequencing (WES) by a targeted next-generation sequencing (NGS) approach in routine clinical service transformed our ability to identify disease-causing variants, sometimes leading to the co-segregation of various pathogenic variants. WES provides affected individuals and their families with an unequivocal diagnosis to establish the correct molecular diagnosis and avoid unnecessary diagnostic and therapeutic efforts. Herein, we present a case of a young NF1 patient with HT and incidental findings of polycystic liver disease from the co-occurrence of NF1 and heterozygotic polycystic kidney and hepatic disease 1 (PKHD1) variants. To the best of our knowledge, this is the first reported case of a patient diagnosed with co-occurrence of NF1 and PKHD1 variants.

Case Report

A 37-year-old Thai woman with clinically diagnosed NF1 came to our endocrine clinic for investigation of the cause of her hypertension. She was first made aware of her elevated blood pressure (140/90 mmHg) during a routine annual influenza vaccination 2 years earlier, but did not seek medical attention until home blood pressure monitoring revealed recent worsening of persistent high blood pressure from 150/90 to 180/110 mmHg. She had no adrenergic symptoms and denied use of any medications or herbal supplements. She was the first child of non-consanguineous parents, with unremarkable family history. There was no family history of NF1. She was born at term after an uneventful pregnancy and had normal developmental milestones. At age 20 years, she noticed the presence of multiple café-au-lait spots on her trunk and a small plexiform neurofibroma on the right frank. She visited a skin clinic at another hospital and the clinical diagnosis of NF1 was established based on clinical criteria without genetic confirmation. She had always been lean – her height was 153 cm, weight was 51 kgs, and body mass index was 21.7 kg/m2. On physical examination, axillary freckling, multiple café-au-lait spots ≥5 mm in size, and small nodules (1–2 cm) spread over the skin of the trunk were demonstrated, as shown in Figure 1. No Lisch nodules were identified on slit-lamp examination and no hypertensive retinopathy was found from fundoscopy.

Biochemical investigations for secondary HT revealed that renal and liver function tests, blood cell counts, urinalysis, plasma electrolytes, plasma levels of renin and aldosterone, and plasma levels of metanephrine and normetanephrine were all within normal ranges. Plasma renin level was 2.03 ng/ mL/hour (reference range 1.50–5.70 ng/mL/hour) and plasma aldosterone level was 6.6 ng/Dl (reference range 4.0–31.0 ng/ dL) measured in the standing position. Further endocrine assessments including thyroid function tests and plasma morning cortisol revealed normal results. Cardiac assessments revealed normal electrocardiography (EKG) without evidence of regarding left ventricular hypertrophy and a zero coronary artery calcium score. No echocardiography was performed due to normal EKG result. Cerebrovascular evaluation including magnetic resonance imaging and magnetic resonance angiography also showed normal results. Abdominal computed tomography (CT) did not show any adrenal or abdominal mass but revealed innumerable liver cysts up to 2.0 cm scattered in both hepatic lobes, as revealed in Figure 2. A simple renal cyst (sized about 0.9 cm) was also found at the lower pole of the left kidney. Renal Doppler sonography showed no renal artery stenosis.

Given the presence of polycystic liver disease, which has never been reported as a clinical manifestation of NF1, WES was performed to detect NF1 variant and variants of other candidate genes for polycystic liver disease, including ALG8, ALG9, DNAJB11, DZIP1L, GANAB, LRP5, PKD1, PKD2, PKHD1, PRKCSH, SEC61B, and SEC63 genes [5]. A heterozygous variants c.5268+1G>A (also known as c.5205+1G>A or IVS28+1G>A) of NF1 gene was found and classified as a pathogenic variant from a previous report [6] and in silico analysis to predict abnormal splicing due to loss of donor site of intron 37. A heterozygous variant of the c.7594_7597del of PKHD1 gene was also found and classified as a pathogenic variant; it is absent from the population database (gnomAD 4.0), and this variant creates frameshift at codon 2532, with a premature translational stop codon after 46 amino acids. The Sanger sequencing confirmed both variants (Figure 3). According to the Combined Annotation Dependent Depletion (CADD) score used to rank pathogenicity of nucleotide variants as C-scores ranging 1–99, both variants demonstrated the highest score, at more than 30 (ENST00000358273.9: c.5268+1G>A: 33 and ENST00000371117.8: c.7594_7597del: 35). This variant was only reported once in a subject, but without phenotype provided (ClinVar: VCV002021566.2) [7]. However, the clinical course and abdominal CT in this patient did not align with autosomal recessive polycystic kidney disease (ARPKD), and only single heterozygous variant in PKHD1 gene was found. Therefore, this patient was a carrier of ARPKD, which predispositions to polycystic liver disease [8]. A more detailed examination in this patient showed no increased medullary echogenicity from renal ultrasonography.

The final diagnosis of co-occurrence of NF1 and HT-related heterozygotic PKHD1 variants with polycystic liver disease was then made based on previous reports of clinical manifestations caused by single heterozygous variants in the PKHD1 gene and no identified other causes of HT. The patient was treated with angiotensin II receptor blocker (azilsartan 40 mg per day) and her blood pressure improved with less than 130/80 mmHg after 1 week. Blood pressure was well-controlled during 1-year follow-up. Long-term surveillances of potential clinical features of NF1 are planned.

Discussion

NF1 is a common neurocutaneous syndrome with variable expression, causing a higher risk of various benign and malignant tumors from hyperactivation of the Ras signaling pathway as a sequalae of the lack of neurofibromin [9]. The RAS pathway is involved in multiple cell types, including those in the central and peripheral nervous system, skin, retina, blood vessels, bones, and skeletal tissue. Imaging plays a vital role in the diagnosis and assessment of individuals with NF1 [10]. The spectrum of pathologic findings, including gastrointestinal stromal tumors, PHEO/PGL, urogenital neurofibromas, and renal artery stenosis, could be a part of NF1 syndrome. The presence of polycystic liver disease in this patient poses a diagnostic challenge from its non-NF1 phenotype. Although rare, 2 or more pathogenic germline variants could exist in the same patient. To the best of our knowledge, this is the first report of co-occurrence of NF1 and polycystic liver disease with HT.

The most common causes of hypertension in NF1 are essential hypertension, PHEO/PGL, and renal artery stenosis [1]. PHEO/PG was thought to occur in few individuals with NF1 [3]. However, it needs to be excluded when patients with NF1 develop HT or require surgery under general anesthesia. Among children with NF1, renal artery stenosis, which occurs at the ostia from arterial mesodermal dysplasia, is the most common cause of secondary HT [11]. In our patient, both conditions had been excluded, and essential HT was unlikely due to her normal body mass index. Therefore, further genetic investigations with WES could elucidate the atypical phenotype found in this patient.

The PKHD1 gene is one of the largest human genes, containing 67 exons, and codes fibrocystin/polyductin protein, which has been implicated in early kidney disease development, cell signaling, and proliferation [12]. Homozygous or compound heterozygous variants in PKHD1 gene causes autosomal recessive polycystic kidney disease (ARPKD), which is characterized by progressive cystic degeneration of the kidneys and congenital hepatic fibrosis [13]. In contrast to the more common autosomal dominant polycystic kidney disease (ADPKD), with an estimated prevalence of about 1: 1000, ARPKD is much rarer, with an estimated incidence of 1: 20 000 to 1: 40 000 [12]. While liver cysts occur only rarely in ARPKD patients, 10% of people who are carriers of PKHD1 variants have liver cysts [8,14]. Although early-onset HT is very common in homozygous PKHD1 variants (ARPKD) from intrarenal renin-angiotensin system upregulation [15], early hypertension seen in patients with heterozygous variants in PKHD1 gene has not been reported, but increased medullary echogenicity in the kidneys consistent with nephrocalcinosis had been detected in some individuals with heterozygous PKHD1 variants [8]. Data from a mouse model showed that heterozygous PKHD1 carriers can develop ARPKD-associated liver cysts only later in life, after the occurrence of a “second hit” affecting the PKHD1 wild-type allele in somatic tissues [16]. Moreover, a recent study in ADPKD variant carriers showed that the development of HT preceded the diagnosis of chronic kidney disease by an average of 11 years [17]. Therefore, more data are needed to better assess the risk of early-onset HT and sporadic manifestation of ARPKD in individuals with heterozygous PKHD1 pathogenic variants. Given that vascular hypertension occurs in 15–20% of NF1 patients through mechanisms that are not fully understood, the causal relationship between the PKHD1 variant and hypertension should be further studied with more accumulated cases and possible mechanisms.

Few case reports of co-occurrence of ADPKD and NF1 in a single individual have been reported [18–20]. It is unclear if these associations with NF1 are coincidental or shared underlying molecular function of the neurofibromin and fibrocystin/polyductin proteins. A recent report of co-occurrence of pathogenic PKD2 and NF1 variants postulated that loss of neurofibromin function reduces control of Ras and aberrant activation of the serine/threonine kinase mammalian target of rapamycin (mTOR) pathway, leading to increased cell growth, and could have a critical role in the pathogenesis of ADPKD [20,21]. In ARPKD, activation of the AKT/mTOR signaling in nephrectomy specimens of children with genetically proven ARPKD has been found [22]. Nevertheless, further molecular investigations are required to understand the synergistic relationship of co-existing NF1 and PKHD1 variants to define a common molecular pathway. Moreover, WES cannot detect large insertions, deletions, or copy number variation (CNV), and further techniques with animal models are required for more clarification of atypical features observed in this case. Clinically, this patient requires lifelong surveillance to monitor growth of polycystic liver disease and other complications from NF1 syndrome.

Conclusions

In summary, our case highlights the diagnostic challenges of atypical phenotypes among individuals with NF1, which might depend on the background of other genes. This case describes the first reported case of a young patient diagnosed with NF 1 with polycystic liver disease and hypertension as a co-occurrence of NF1 and PKHD1 variants. With increasing affordability of WES, its utility in uncovering the possibility of being affected by 2 inherited genetic conditions should be considered when findings are incompatible with the primary disease. While the polycystic liver disease in this patient could be attributed to the PKHD1 variant, the development of early-onset hypertension could be a manifestation of NF1 or other unidentified etiology.