Peripheral neuropathy and 46XY gonadal dysgenesis: A heterogeneous entity

Neuromuscular Disorders 19 (2009) 172–175

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Case report

Peripheral neuropathy and 46XY gonadal dysgenesis: A heterogeneous entity Jonathan Baets a,c,1, Ines Dierick b,c,1, Chantal Ceuterick-de Groote d, Jenneke van den Ende e, Jean-Jacques Martin d, Karin Geens f, Wim Robberecht g, Eva Nelis a,c, Vincent Timmerman b,c, Peter De Jonghe a,c,h,* a

Neurogenetics group, VIB Department of Molecular Genetics, University of Antwerp, Universiteitsplein 1, B-2610 Antwerpen, Belgium Peripheral Neuropathy group, VIB, Department of Molecular Genetics, University of Antwerp, Antwerpen, Belgium c Laboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, Antwerpen, Belgium d Laboratory of Ultrastructural Neuropathology, Institute Born-Bunge, University of Antwerp, Antwerpen, Belgium e Center of Medical Genetics, University of Antwerpen, Belgium f Division of Neurology, Klina General Hospital, Brasschaat, Belgium g Division of Neurology, University Hospital of Leuven, Leuven, Belgium h Division of Neurology, University Hospital Antwerp, Antwerpen, Belgium b

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Article history: Received 11 August 2008 Received in revised form 27 October 2008 Accepted 14 November 2008

Keywords: Gonadal dysgenesis Peripheral neuropathy DHH Sural nerve biopsy Minifascicle formation

a b s t r a c t Gonadal dysgenesis with normal male karyotype (46XY) is a sexual differentiation disorder. So far three patients have been reported presenting the association of 46XY gonadal dysgenesis with peripheral neuropathy. Examination of sural nerves revealed minifascicle formation in two of them. In one patient, a mutation was found in desert hedgehog homolog (Drosophila), a gene important in gonadal differentiation and peripheral nerve development. We studied neuropathological and molecular genetic aspects of a patient with 46XY gonadal dysgenesis and peripheral neuropathy. Examination of a sural nerve biopsy specimen revealed an axonal neuropathy with pronounced axonal loss, limited signs of axonal regeneration and no minifascicle formation. A normal male karyotype was found (46XY) without micro-deletions in the Y chromosome. No mutations were found in the sex determining region Y gene, peripheral myelin protein 22, Myelin Protein Zero, Gap-Junction protein Beta 1, Mitofusin 2 or desert hedgehog homolog. The absence of minifascicle formation and the absence of a mutation in desert hedgehog homolog in this patient with gonadal dysgenesis and peripheral neuropathy expand the clinical and genetic heterogeneity of this rare entity. Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction The sexual differentiation disorder gonadal dysgenesis (GD) with 46XY karyotype and a female phenotype is classified in (1) a pure form characterized by the presence of two streak gonads, and (2) a partial form characterized by one streak gonad and one testis [1]. The main genetic causes of 46XY GD are point mutations and micro-deletions in the SRY (sex determining region Y) gene, accounting for 15–20% of 46XY GD patients [1–3]. Mutations in other genes of the male sex-determinating pathway are predicted to be involved in the remainder of 46XY GD patients [1]. Three reports have demonstrated the association of 46XY GD with peripheral neuropathy [4– 6]. In the first two patients [4,5] sural nerve biopsy examination showed the unusual finding of minifascicle (MF) formation, consist-

ing of several axon-Schwann cell units surrounded by perineural cells [7]. In the patient with partial GD with peripheral neuropathy and MF-formation, a mutation was found in desert hedgehog homolog (Drosophila) (DHH) [8]. This member of the hedgehog signalling proteins plays an important role in male sex determination [9] and is essential for the structural and functional integrity of the peripheral nerve [10]. In the other two patients presenting a pure GD no mutations in DHH could be demonstrated [5,6]. Mutations in DHH were also found in patients with 46XY GD without obvious clinical signs of peripheral neuropathy, extending the clinical phenotypes associated with DHH mutations [11]. 2. Materials and methods 2.1. Patient

* Corresponding author. Address: Neurogenetics group, VIB, Department of Molecular Genetics, University of Antwerp, Universiteitsplein 1, B-2610 Antwerpen, Belgium. Tel.: +32 3 265 10 50; fax: +32 3 265 10 13. E-mail address: [email protected] (P.D. Jonghe). 1 Both authors contributed equally to this work. 0960-8966/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.nmd.2008.11.006

This 51 year old patient was born from a non-consanguineous marriage after a normal pregnancy and delivery. She was raised as a female. Apart from a mild cognitive impairment developmen-

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tal milestones were normal. Because of primary amenorrhea she was examined at 17 years of age. On clinical examination no secondary sexual characteristics were found, gynaecological examination revealed poorly developed female genitalia with a blinded vagina and absence of a uterus. An exploratory laparoscopy revealed bilateral streak gonads and confirmed the absence of a uterus. Endocrinological studies were compatible with hypergonadotropic hypogonadism. Routine blood analysis revealed no abnormalities. At the age of 43 the patient developed progressive distal weakness and sensory abnormalities in legs and arms. Neurological examination showed moderate distal weakness and sensory disturbances in all limbs. Tendon reflexes were normal in the upper limb and weak to absent in the lower limbs. Obesity and a mild cognitive impairment were noted but without other clinical abnormalities. CT-imaging of the brain and EEG were normal. There was no family history of peripheral neuropathy nor was there exposure to neurotoxic substances or other obvious causes of acquired neuropathy. Thyroid, liver and kidney function as well as vitamin B12 and folic acid levels were normal. Serology for Borrelia burgdorferi and HIV was negative, protein electrophoresis did not reveal monoclonal bands, no Bence–Jones proteins were found. A screen for systemic auto-immune disease was normal. An electrophysiological study was performed. Median motor nerve conduction velocity (NCV) was 41.4 m/s, the amplitude of the compound muscle action potential (CMAP) was 2.8 mV. Ulnar motor NCV was 32.5 m/s with, CMAP amplitude 1.7 mV. Common peroneal nerve motor NCV was 27.3 m/s with a CMAP amplitude of 0.9 mV. The sensory NCV of the ulnar nerve was 20.8 m/s, the amplitude of the sensory nerve action potential (SNAP) was 6.2 lV. No SNAP’s were detected for the median nerve. Electromyography revealed chronic neurogenic changes.

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Fig. 1. Semithin resin transverse section of a sural nerve fascicle showing normal peri- and endoneurium without MF-formations. (A) Widespread loss of myelinated fibres. Magnification: 444. (B) Presence of a sporadic regenerating cluster (arrow), thinly myelinated fibre (arrowhead) and denervated band (thick arrow). Magnification: 900. Araldite-embedded sections stained with Toluidine blue.

2.2. Sural nerve biopsy A biopsy was performed on the left sural nerve at the age of 47 years. Examination was performed according to conventional techniques for light and electron microscopy. 2.3. Cytogenetic and molecular genetic analysis Chromosomal analysis was performed on metaphases from cultured peripheral blood lymphocytes from two independent blood samples, using standard G-banding techniques. Fluorescent in situ hybridization (FISH) DNA-probes were used to exclude micro-deletions in the Yq11-region, the Yq12, SHOX and SRY region. Genomic DNA was isolated from the patient’s blood leukocytes using standard procedures. Duplication or deletion of PMP22 (peripheral myelin protein 22) was excluded by microsatellite analysis [12] and coding regions of PMP22, MPZ (myelin protein zero), GJB1 (gap junction protein, beta 1, 32 kDa), MFN2 (mitofusin 2), SRY and DHH were PCR-amplified and direct sequenced. 3. Results 3.1. Sural nerve biopsy Light microscopic examination revealed 13 nerve fascicles without MF-formation. Arrays of grouped Schwann cells were bordered by endoneural connective tissue but there were no surrounding perineurial cells. Loss of large myelinated fibres was the main finding (Fig. 1A). Only rare clusters (Fig. 1B) of two small myelinated axons were observed. Several thinly myelinated fibres were present.

By electron microscopy loss of myelinated axons (Fig. 2A) was confirmed and an increase of collagen fibres without perineurial infiltration. Most myelinated axons had a diameter from 1.7 to 5 lm and myelin sheaths showed a normal periodicity. Axonal degeneration was found occasionally. There were rare myelin ovoids (Fig. 2B) and sporadic denervated Schwann cell bands. Regenerating clusters, with rare myelinated axons (Fig. 2A), demonstrated several unmyelinated axonal sprouts. Occasional small onion bulb formations (OBF) were noted (not illustrated). Few fibroblasts were seen at the border of Schwann cell complexes (Fig. 2B) which were associated with unmyelinated axons or infrequently, small myelinated axons. These complexes were not circumscribed by perineurial cells and they were different from OBF. 3.2. Cytogenetic and molecular genetic analysis Cytogenetic analysis showed a normal male karyotype (46XY). FISH analysis did not reveal micro-deletions in the Yq11, Yq12, SRY and SHOX region and gene analysis excluded coding mutations in the SRY gene ruling out the major molecular causes of GD. We excluded the PMP22 duplication/deletion and mutations in the coding region of genes that are involved in the most common forms of hereditary motor and sensory neuropathy (HMSN) by direct DNA-sequencing; these are, PMP22, MPZ, GJB1 and MFN2. In addition coding mutations were excluded in DHH. 4. Discussion The patient presented a 46XY pure GD with impaired mental capacities. No mutations or micro-deletions were found in the SRY gene. Of interest, our patient developed a motor and sensory

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mutation in DHH was found in one of these patients [8]. Furthermore, the presence of MF-formations in dhh-null mice, have demonstrated a key role for DHH in perineurial formation [14]. The association between MF neuropathy and DHH mutations is not exclusive, as no DHH mutation was found in the second patient with pure 46XY GD and a peripheral neuropathy [5]. In addition, MF-formation can be found in (acquired) conditions of the peripheral nerve in the absence of GD [15,16]. In contrast no MF-formation was found in the most recently published patient [6]. Therefore, one might criticise that the association between the GD and the peripheral neuropathy in this particular patient is coincidental, as there is no clear common clinical or neuropathological factor linking this neuropathy with the GD as was the case in the first two patients [4,5]. However, our findings were very similar to this most recent report. This second patient makes it highly unlikely that the association between GD and a neuropathy without MF-formation is merely coincidental. Until now only one DHH mutation is reported to be associated with this combination of GD and peripheral neuropathy [8] making the genetic contribution of DHH uncertain. This suggests that different molecular defects may be associated with the combination of abnormal gonadal and perineural differentiation. There is increasing evidence that genes implicated in neurodevelopment and sex determination are dosage-sensitive (for review see [1,17]). Therefore, altered gene dosage, resulting from molecular defects in regulatory or coding regions of DHH or other genes involved in testicular determination and peripheral nerve development could explain the clinical picture in these patients. Another clinical feature of our patient is impaired mental function, pointing to a more syndromic form of GD. The involvement of the central nervous system in this complex phenotype was previously also reported [4]. This report demonstrates now with more certainty that GD is associated with pathologically diverse neuropathies underlining the clinical, pathological and also genetic heterogeneity of this rare entity. Fig. 2. Electron micrograph images of a transverse section of a sural nerve fascicle. (A) Reduced density of myelinated fibres, small clusters (arrows), and fibroblasts (F) surrounding normal areas without minifascicle formations. Magnification: 2240. (B) Myelin ovoid (M) and a Schwann cell complex (arrows) with closely packed Schwann cell processes. Magnification: 3240. Standard electron microscopic techniques, fixation in glutaraldehyde, postfixation in osmium tetroxide, embedding in araldite, staining with uranyl acetate and lead citrate.

neuropathy at the age of 43 years. NCV studies revealed a peripheral neuropathy that could be defined as intermediate when using a median motor NCV of 38 m/s as the classical cut-off for demyelinating neuropathies [13]. Examination of sural nerve demonstrated an axonal neuropathy. The extensive loss of fastconduction large caliber myelinated axons might explain the reduction of several NCVs within range of demyelinating neuropathies. No MF-formation was observed in our patient. This peripheral neuropathy was not associated with a PMP22 duplication/ deletion or mutations in PMP22, MPZ, MFN2 or GJB1, major genetic defects associated with HMSN. The clinical entity of GD and peripheral neuropathy is uncommon and has been described in only three other reports [4–6]. Electrophysiology in two of these patients [4,5] showed similar intermediate NCVs as in our patient, electrophysiology in the recently published third patient suggested an axonal motor–sensory neuropathy [6]. Examination of sural nerves in the first two patients revealed MF-formation, variable loss of myelinated axons, limited signs of demyelination and axonal regeneration [4,5]. The formation of these MF’s was first thought to be an indirect consequence of reduced DHH signalling, since a loss-of-function

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