Phenotype variability of infantile-onset multisystem neurologic, endocrine, and pancreatic disease IMNEPD

Infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD) has been recently linked to biallelic mutation of the peptidyl-tRNA hydrolase 2 gene PTRH2. Two index patients with IMNEPD in the original report had multiple neurological symptoms such as postnatal microcephaly, intellectual disability, developmental delay, sensorineural deafness, cerebellar atrophy, ataxia, and peripheral neuropathy. In addition, distal muscle weakness and abnormalities of thyroid, pancreas, and liver were found. Here, we report five further IMNEPD patients with a different homozygous PTRH2 mutation, broaden the phenotypic spectrum of the disease and differentiate common symptoms and interindividual variability in IMNEPD associated with a unique mutation. We thereby hope to better define IMNEPD and promote recognition and diagnosis of this novel disease entity. Electronic supplementary material The online version of this article (doi:10.1186/s13023-016-0433-z) contains supplementary material, which is available to authorized users.


Introduction
The infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD; MIM#616263) was recently reported by us as a novel disease entity in two individuals from a consanguineous family of Yazidian-Turkish descent [1]. We further demonstrated the association of a homozygous nonsense mutation in the PTRH2 gene (MIM*608625) to IMNEPD through functional and molecular data in human and mouse [1]. The two index patients in the original report presented with postnatal microcephaly, moderate intellectual disability, abnormal rhythmic rapid activity on EEG, sensorineural deafness, and delayed speech development. They suffered from distal muscle weakness and delayed motor milestones, and later developed progressive ataxia and progressive cerebellar atrophy. Peripheral demyelinating sensorimotor neuropathy and endocrine abnormalities with affection of the pancreas, thyroid, and liver were furthermore present [1]. Our single-family report was rapidly further supported by a second case without detailed analysis of the disease phenotype [2]. Here, we report five further IMNEPD patients from two consanguineous families with a PTRH2 missense mutation and discuss their phenotype, thereby illustrating both core and variable features of IMNEPD.

Material and methods
Informed consent was obtained from the parents of the patients for the molecular genetic analysis, the publication of clinical data, photos, magnetic resonance images (MRI) and studies on fibroblasts. DNA extraction from blood samples and Sanger sequencing was performed using standard protocols. Samples from patients and controls were used in this study with approval from the local ethics committees of the Charité (approval no. EA1/212/08). Quantitative real-time PCR (qPCR) and Western blot were performed with established methods reported previously [1]. Primer sequences are provided in the supplementary data (Additional file 1: Table S3).

Results
We report five IMNEPD patients from two consanguineous families of Tunisian and Saudi Arabian descent, all with the homozygous missense mutation c.254A > C in exon 2 of the PTRH2 gene (NM_016077.4; Fig. 1). This mutation causes an amino acid exchange of glutamate to proline (p.Q85P, NP_057161, Fig. 1a) and putatively affects structure, folding, and stability of PTRH2 by altering hydrogen bridge bonds within the protein [2]. In line with this, PTRH2 protein levels were strongly reduced in fibroblasts from patient II.1, family 2, while PTRH2 mRNA levels were unchanged (Additional file 2: Figure S1). The mutation segregates with the disease phenotype and is heterozygous in the healthy parents of the patients. Patient II.10, family 3, was previously reported, without detailed clinical information, in a large genetic screening study on consanguineous families with developmental delay [2].
Neurologic core features of (almost) all patients were intellectual disability, motor delay, severe speech delay, ataxia, and sensorineural hearing loss (Table 1, Additional file 3: Table S1). Furthermore, exocrine pancreatic insufficiency with reduced pancreas elastase levels was detected in almost all patients. It was partly associated with consecutive deficiency of lipophilic vitamins and failure to thrive in the first years of life ( Table 1). Insufficiency of the endocrine pancreas function in IMNEPD had already been suggested based on the marginal HbA1c elevation in an index patient of the original IMNEPD family [1] and is now supported by a clinically manifest diabetes mellitus of patient II.4, family 2 (Additional file 4: Table S2). In this patient abdominal sonography at age 9 had shown a hyperechogenic pancreas indicating lipomatosis, and pancreatic atrophy was now diagnosed by magnetic resonance imaging at the age of 17 years (Fig. 2). Hepatomegaly had already been detected in the index patients of the first IMNEPD family [1], but it had remained unclear whether hepatomegaly resulted from fibrosis or steatosis. A liver biopsy performed in patient II.4, family 2, for hepatomegaly and slightly increased transaminase values now revealed hepatic micro-and macrosteatosis (Fig. 1). Slightly increased transaminase values and lactate dehydrogenase activities in further patients could indicate a mild liver (and/or muscle disease) (Additional file 4: Table S2). Despite the identification of the same PTRH2 mutation in all five patients, phenotypic variability could be observed. For instance, facial palsy, distal muscle weakness, truncal hypotonia, peripheral demyelinating neuropathy, and cerebellar atrophy were only occasionally present (Table 1). Further minor features were skeletal anomalies, especially anomalies of the fingers, and mild facial abnormalities such as exotropia, ptosis, and thin upper lip vermilion.

Discussion
Ptrh2 is an evolutionarily well conserved mitochondrial protein, which prevents the accumulation of dissociated peptidyl-tRNAs and thus an inhibition of protein synthesis [3]. As part of an integrin signaling complex, Ptrh2 regulates the fine balance between cell survival and apoptosis; it also has a role in cell size control [1,[4][5][6][7]. We recently highlighted the role of PTRH2 for human development by linking a homozygous PTRH2 gene nonsense mutation (c.269_270delCT, p.A90fs) to the disease infantile multisystem neurologic, endocrine, and pancreatic disease (IMNEPD) [1]. Since the original description, we have identified five further patients of three consanguineous families of Tunisian and Saudi Arabian descent with an IMNEPD phenotype and a homozygous missense mutation of PTRH2 (c.254A > C, p.Q85P; Fig. 1). The mutation causes a strong downregulation and a predicted dysfunction of the PTRH2 protein (Additional file 2: Figure S1) [2]. Comparison of the phenotype of the original and the newly identified families illustrates both core features and phenotypic variability of this novel disease entity ( Table 1). The core phenotype, mutual to almost all affected patients, comprises intellectual disability, motor and severe speech delay, ataxia, sensorineural hearing loss, and pancreatic insufficiency ( Table 1, Additional file 3: Table S1). Progressive cerebellar atrophy and ataxia imposed as key features of IMNEPD in the index family with a homozygous nonsense mutation of PTRH2. This was further underlined given the phenotype of mutant Ptrh2 mice with microcephaly and severe cerebellar atrophy [1]. However, in the light of PTRH2 missense mutationspresumably with higher residual PTRH2 levels -progressive cerebellar atrophy was present in only one patient. The lack of cerebellar atrophy in some patients with a homozygous PTRH2 missense mutation and the lack of microcephaly in all patients with a homozygous PTRH2 missense mutation may be attributed to interindividual variability and/or correlate with the quantity or residual function of PTRH2. Ataxia was present in almost all IMNEPD patients, also in those without cerebellar hypotrophy/atrophy, suggesting that ataxia may not be attributed exclusively to progressive cerebellar atrophy, but could also result from demyelinating peripheral neuropathy detected in several patients (Table 1). Apart from variations in the neurologic phenotype, both patients with the nonsense mutation had hypothyroidism, while thyroxine values in all patients with the missense mutation were normal. However, two patients with a missense mutation presented with elevated thyroxine stimulating hormones (TSH), likely indicating latent thyroid insufficiency. Hepatomegaly and/or abnormal liver parenchyma morphology on ultrasound were present in both patients with a nonsense mutation and one patient with a missense mutation of PTRH2. We had speculated before that hepatomegaly and abnormal liver echogenicity were due to fibrosis or steatosis, and we can now demonstrate diffuse and extensive micro-and macrosteatosis in a liver biopsy specimen of patient II.4, family 2. Pancreatic insufficiency is a feature present in most IMNEPD patients: exocrine insufficiency was identified in both patients with a nonsense mutation and four patients with a missense mutation; signs of endocrine insufficiency were found in both patients with the nonsense mutation (borderline HbA1c elevation) and in one patient with the missense mutation (insulin-dependent diabetes mellitus). In the latter