Introduction

RHYNS syndrome (OMIM 602152) was defined in 1997 by the acronym of Retinitis pigmentosa, HYpopituitarism, Nephronophthisis and mild Skeletal dysplasia in a 17-year-old man [1]. A closely resembling phenotype with liver fibrosis has been previously described in a boy [2], while Hedera and collaborators [3] reported a similar condition in a family with two affected brothers. The observation of affected males only and recurrence in a sibling suggested either an X-linked or autosomal recessive inheritance pattern. Yet, the underlying genetic basis has remained unexplained, although the pattern of associated clinical features was compatible with a hereditary ciliary disorder.

In this study, we reported the 20-year follow-up of the original patient with RHYNS syndrome. Whole-exome sequencing led to the identification of a compound heterozygous variant in the gene TMEM67. The assignment of RYHNS syndrome as part of the wide spectrum of TMEM67-related ciliopathies is discussed.

Subjects and methods

Patient

The index male patient was re-evaluated at the age of 38 years, still in search of a molecular diagnosis after receiving a clinical diagnosis of RHYNS syndrome at young age [1]. In brief, he was born with congenital palsy of the III and IV cranial nerves on the right resulting in complete ophthalmoplegia and upper eyelid ptosis and congenital palsy of the VI cranial nerve on the left with exotropia. At the age of 4 years, he measured 90 cm (−4.9SD) and showed delayed bone age. Growth hormone and thyreotropin-releasing hormone deficiency were diagnosed and treated with replacement therapy until young adulthood. At this age, radiological examination showed mild signs of skeletal dysplasia consisting of osteopenia, thin tubular bones, epiphyseal hypoplasia and hypoplastic iliac bones with irregular acetabular margins. At age 11, retinitis pigmentosa and left sensorineural hearing loss were first diagnosed. Abnormal renal function was evident by age 12 years, when a renal biopsy demonstrated a histological pattern consistent with nephronophthisis (NPHP). A first kidney transplantation from a deceased donor was performed at age 29 but it was rejected. He was started on hemodialysis and, at age 34, a second renal transplantation was performed. Since then, his clinical condition remained stable.

We have evaluated the patient at age 38 years, when he measured 152 cm of height with weight 63.5 kg. Generalized and severe osteoporosis was diagnosed by DEXA examination (femoral head: T-Score −3.5, Z-Score −3.0, BMD 0.458 g/cm2; lumbar region: T-Score −3.1, Z-Score −3.0, BMD 0.752 g/cm2). A novel skeletal survey detected moderately shortened long bones, bowed radii, short femoral neck, brachydactyly at hands and feet with more severe involvement of middle phalanges, distal phalanx of the thumbs and metacarpals, moderately thickened calvarium, and rotoscoliosis (Fig. 1d). Diffuse reduction of the bone density with thinning of the diaphyseal cortex, was evident, particularly on hands (Fig. 1b, c).

Fig. 1
figure 1

Radiological skeletal survey of the proband at age 38 years. a Thickened calvarium. b Short and bowed radius (black open arrow). c Brachydactyly at hands with more severe involvement of middle phalanges, distal phalanx of the thumbs and metacarpals. Generalized reduction of bone density with thinning of the diaphyseal cortex is observed. d Rotoscoliosis. e Short femoral necks (open white arrow)

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Hormonal dosage showed increased parathyroid hormone levels in the blood (164 pg/ml, reference range 14–72 pg/ml), despite having received kidney transplantation, whereas all other hormonal levels were normal, as were liver enzymes and dedicated liver ultrasound.

Audiometry showed pantonal left-sided moderate-to-severe sensorineural hearing loss. Ophthalmologic examination confirmed no residual visual acuity and complete extinguishment of the electroretinogram in both eyes.

Neuropsychological evaluation excluded functional deficits, whereas brain imaging was normal. He obtained the chartered accountant qualification and he was completely self-sufficient in all daily life activities, although he did not have any current employment.

Whole-exome sequencing

In order to determine the genetic etiology of the RHYNS syndrome, whole-exome sequencing was performed hypothesizing an-underlying autosomal recessive inheritance pattern. Detailed procedures are available in the Supplementary Methods file.

Mini-gene splicing assay

Detailed methods are described in the Supplementary Methods file and illustrated in Supplementary Figure 1a.

Results

Two variants in TMEM67 (NM_153704.5; NP_714915.3; MIM#609884) were identified in the proband, each inherited from a heterozygous parent, consistent with compound heterozygosity and autosomal recessive inheritance. The two identified variants were confirmed in the proband and his parents by Sanger sequencing: the nonsense variant c.622A > T, p.(Arg208*) in exon 6 was inherited form the father, while the missense c.1289A > G variant, p.(Asp430Gly), near the splice acceptor site of exon 13 was present in the mother (exons are numbered like in NG_009190.1), resulting in a c.[622A > T];[1289A > G] genotype. The two healthy brothers were carriers of the nonsense variant only (Fig. 2). The missense variant was absent from the 1000 Genomes Project, the Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org) and the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org); conversely, the nonsense variant was present at extremely low frequency in the most comprehensive variant database with 49 out of 277,178 alleles reported in gnomAD.

Fig. 2
figure 2

Pedigree of the RHYNS family and TMEM67 electropherograms. a Family tree showing the proband (filled square symbol) and two healthy sibs. Circles and squares indicate females and males, respectively. b Genomic sequence electropherograms demonstrate a nonsense variant (c.622A > T, p.Arg208*) in the father (I-1) and all 3 sons (II-1, II-2, II-3) and a missense variant (c.1289A > G, p.Asp430Gly) in the affected son (II-1) and his mother (I-2). Arrows indicate a compound heterozygous variant in the affected son and heterozygous changes in all other individuals

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Our hypothesis that the close proximity of the c.1289A > G variant potentially interferes with splicing was investigated with a mini-gene assay utilizing the pSPL3 vector system. Transfection with the wild-type construct resulted in the trapping of exon 13 (387 bp PCR product), whereas the mutant c.1289A > G construct and the empty vector control lack exon 13 (263 bp PCR product) (Supplementary Figure 1b). Accordingly, one predicted possible outcome of the c.1289A > G variant is exon 13 skipping, r.(1289_1412)del, and, in this case, the formation of a frameshifted protein, p.(Asp430Serfs*9).

The identified variants affecting TMEM67 were submitted to the LOVD database (URL: https://databases.lovd.nl/shared/genes/TMEM67; patient ID: # 00155014).

Discussion

In this study, we report the identification of biallelic variants in the TMEM67 gene as the underlying genetic defect causative of RHYNS syndrome. These findings extend the spectrum of phenotypes resulting from TMEM67 variants to now eight distinguishable ciliopathies (Table 1). Their clinical manifestations display a wide range of presentations ranging from lethal phenotypes to patients with organ-specific involvement. The pleiotropic effect of TMEM67 reflects the expression in different tissues during development. In particular, in situ hybridization studies in human embryos have shown that at early gestational age, in addition to discrete brain structures, TMEM67 transcripts were particularly abundant in cartilage primordia, kidney, stomach, and large intrahepatic bile ducts of the liver. At 51–53 days, intense expression was seen in the outer layer of the retina and, later, in cartilage primordia including those of ribs and upper limbs (especially the digits) [4].

Table 1 TMEM67-related phenotypes and distinctive clinical manifestations
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Initially, variants in TMEM67 were identified in Meckel syndrome (MKS), a lethal disorder displaying central nervous system malformations, typically occipital encephalocele, multicystic kidneys, ductal plate dysplasia with congenital hepatic fibrosis (CHF) and postaxial polydactyly [5]. Subsequently, Baala and collaborators [6] identified TMEM67 variants in three patients with pure (isolated) Joubert syndrome (JS). Indeed, different subtypes of JS were associated to TMEM67 variants with distinct genotype–phenotype correlations within the spectrum of JS-related disorders (JSRDs), a group of pleiotropic ciliopathies, which share in common the Molar Tooth Sign (MTS) at brain imaging [7]. The strongest correlation was defined with JS and CHF, with ~ 70% patients affected by so-called COACH syndrome (Cerebellar vermis hypo/aplasia, Oligophrenia, congenital Ataxia, ocular Coloboma, and Hepatic fibrosis) carrying biallelic TMEM67 variants [8].

In addition to MKS and JSRDs, ~ 10% of patients affected by NPHP and CHF without neurological involvement and normal brain imaging (NPHP11; MIM #613550) had TMEM67 variants [9]. Interestingly, the same gene was also mutated in three children with a unique association of polycystic kidney (mimicking autosomal recessive polycystic kidney disease–ARPKD), NPHP, CHF and midbrain-hindbrain abnormalities within the MTS spectrum [10]. Of note, the spectrum of kidney disease in TMEM67 is broad including NPHP, an overlap phenotype of autosomal recessive polycystic kidney disease/NPHP and prenatally enlarged cystic kidneys such as in MKS [11]. More recently, Tarailo-Graovac et al. [12]. described a young adult patient with two variants in TMEM67, who displayed, in addition to the MTS and cerebellar atrophy at brain imaging, mild intellectual disability, adolescent-onset dementia, vertical gaze palsy, ataxia, and progressive hepatic fibrosis, overlapping Niemann-Pick type C manifestations. At last, TMEM67 was mutated in an otherwise healthy adult patient affected by isolated congenital hepatic fibrosis, which represented so far, the mildest end of the TMEM67-related spectrum [13].

The allelic spectrum of TMEM67 includes missense, truncating and splice site variants, as well as rare multiexon deletions. Two truncating variants (either frameshift, nonsense, or splice site variants) occur with high frequency in MKS and are not reported in non-lethal phenotypes [14]. Conversely, two missense variants or a combination of truncating/splicing and missense variants are prevalent in less-severe phenotypes within the JSRD spectrum, i.e., JS and COACH [8]. Hypomorphic variants in TMEM67 are associated with NPHP and liver fibrosis (NPHP11), whereas more than half of these patients display ocular involvement [9]. Interestingly, our patient carried one truncating and one splicing variant and his phenotype was mainly characterized by retinitis pigmentosa, NPHP without any neurologic involvement or liver fibrosis. The absence of either neurologic or hepatic involvement is surprising since these are major manifestations of TMEM67 variants (Table 1). Among the two variants identified in our patient, only the c.622A > T, p.(Arg208*) was previously reported in three cases of MKS, two in combination with another truncating variant and one with c.755 T > C p.(Met252Thr) missense change [15]. Altogether these observations emphasize the role of, yet unidentified, modifier factors in other genes modulating the penetrance of clinical manifestations. Of note, variants at different loci interacting epistatically under a “multiallelic” inheritance have been proposed as a model for disease penetrance in ciliopathies such as NPHP [16].

In addition, our patient had hypopituitarism without structural abnormalities of the pituitary gland on brain magnetic resonance imaging. Interestingly, growth hormone deficiency was frequently reported in JS caused by variants in two distinct genes (KIAA0753 and CELSR2) in association to absent, small or ectopic pituitary gland, strengthening the importance of ciliary function also in the development of the hypophysis and its function [17, 18].

In conclusion, our data place RHYNS syndrome within the wide spectrum of TMEM67-related ciliopathies, ranging from early lethality to adults with liver fibrosis. More studies are encouraged to decipher modifier factors influencing clinical variability.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.