Identification of a novel WFS1 mutation (AFF344-345ins) in Japanese patients with Wolfram syndrome

Diabetes Research and Clinical Practice 69 (2005) 136–141 www.elsevier.com/locate/diabres

Identification of a novel WFS1 mutation (AFF344-345ins) in Japanese patients with Wolfram syndrome Kouichi Inukai *, Takuya Awata, Kiyoaki Inoue, Susumu Kurihara, Youhei Nakashima, Masaki Watanabe, Takahiro Sawa, Nobuki Takata, Shigehiro Katayama Division of Endocrinology and Diabetes, Department of Medicine, Saitama Medical School, Morohongo 38, Moroyama, Iruma-gun, Saitama 350-0495, Japan Received 24 June 2004; received in revised form 2 December 2004; accepted 4 January 2005 Available online 24 February 2005

Abstract Wolfram syndrome (WFS) is an autosomal recessive disorder characterized by early onset diabetes mellitus, progressive optic atrophy, sensorineural deafness and diabetes insipidus. Affected individuals may also have renal tract abnormalities as well as neurogical and psychiatric syndromes. WFS1 encoding a transmembrane protein was identified as the gene responsible for WFS. We report herein a Japanese family, of which two members had this syndrome. In the WFS1 gene of these patients, we identified a novel mutation, a nine nucleotide insertion (AFF344-345ins). In addition, one of these patients had preclinical hypopituitarism, which is an unusual feature of WFS. As only the two family members homozygous for the mutation showed WFS, these data support the notion that this mutation is the cause of WFS. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Wolfram syndrome; Hypopituitarism; WFS1

1. Introduction Wolfram syndrome (WFS), or DIDMOAD (diabetes insipidus, DM, optic atrophy and deafness), is a rare disorder with autosomal recessive inheritance [1]. Prevalence is estimated to be 1 in 100,000–770,000 [2,3]. The minimum criteria for diagnosis are diabetes mellitus and optic atrophy. WFS is also known to be a progressive neurodegenerative disorder, and many * Corresponding author. Fax: +81 492 76 1430. E-mail address: [email protected] (K. Inukai).

patients develop additional symptoms such as urinary-tract atony, ataxia, peripheral neuropathy and psychiatric illnesses [4–6]. As mitochondrial defects have been implicated in WFS [7], this disease was hypothesized to be a mitochondrion-mediated disorder. Subsequently, Inoue et al. succeeded in identifying the gene responsible for WFS, which was termed as WFS1 [8]. Biochemical studies indicated WFS1 protein to be an integral, endoglycosidase H-sensitive membrane glycoprotein localized predominantly in the endoplasmic reticulum [9]. In addition, overexpression of WFS1 reportedly increases calcium levels in oocytes,

0168-8227/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2005.01.002

K. Inukai et al. / Diabetes Research and Clinical Practice 69 (2005) 136–141

suggesting that WFS1 may be important in the regulation of intracellular Ca2+ homeostasis [10]. Though the precise function of WFS1 is presently unknown, it is certain that WFS1 mutations cause WFS. In the present study, we report a novel homozygous WFS1 mutation (AFF344-345ins). This mutation is located in the putative transmembrane domain of the protein, presumably resulting in WFS1 loss of function. The proband, in addition to various symptoms characterized by WFS, had preclinical hypopituitarism. Though a broad spectrum of the neuronal dysfunctions are possible in this neurodegenerative disorder, we found this case to have a neuronal finding not previously reported in WFS.

2. Case report The proband, 41 years of age at the time of investigation, was a woman born in Niigata prefecture, Japan. She developed diabetes mellitus at age 9 and diabetes insipidus at age 32 years. She is currently

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being treated with desmopressin and insulin. Bilateral optic atrophy was first detected at 32 years of age. As her past medical history suggested WFS, she was referred to our University Hospital for further examinations. We first evaluated her diabetes insipidus with a water deprivation test. She showed almost no increase in urine osmolality. Subsequently, a pitressin stimulation test showed a marked rise (70%) in urine osmolality. These data indicated that she had pituitary diabetes insipidus. On funduscopic examination, she had bilateral optic atrophy, confirmed by the presence of white papilla with well-demarcated borders, not attributable to any other disease. Her visual acuities were 0.3 (right eye)/0.2 (left eye). Genomic DNA was extracted from peripheral blood samples from individual family members. HLAgenotyping revealed the patient to be positive for HLA-DR2 (HLA typing; A24(9) B7 B52(5) Cw7 DR1 DR2), which is frequently observed in WFS [11]. Insulin secretion appeared to be completely absent (urinal C-peptide; <1 mg/day), though anti-GAD antibody and ICA were negative. An audiogram

Fig. 1. (A) Pedigree of the patient’s family with each individual’s status designated (solid symbols: affected, grey: diabetes mellitus without WFS, open: unaffected, arrow: proband). The numbers correspond to the lane numbers in C. (B) Sequence chromatograms of the region of exon 8 showing the 9-bp insertion (upper panel) in a patient homozygous for the mutation, along with a normal control (lower panel). (C) AFF344345ins in the family, showing the two affected individuals (proband-lane 2 and her younger brother-lane 4) to be homozygous for the mutation. The proband’s mother and younger sister (lanes 1 and 3, respectively) are heterozygous (normal control-lane 5). Markers indicate a 50 bp ladder.

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showed bilateral high-frequency hearing loss. Both urinary tract dilatation and neurogenic bladder were also observed (data not shown). Based on these findings, she was diagnosed as having WFS. Her parent’s marriage was not consanguineous, though both parents were born in the same district in Niigata. Among first-degree relatives (father, mother, younger sister and younger brother), only her younger brother appeared to be affected (Fig. 1A). He had developed diabetes mellitus at age 7 and was immediately put on insulin therapy. At 29 years of age, visual deterioration associated with optic atrophy was diagnosed. He had all major manifestations of DIDMOAD except DI. Mutation screening was performed for each family member by direct sequencing. PCR primers were prepared as previously described [8]. PCR products were directly sequenced, on both strands if necessary, using a dRhodamine Terminator Cycle Sequencing Kit (Applied Biosystems Japan, Tokyo, Japan). We amplified and directly sequenced eight exons and the flanking region of the WFS1 gene from the proband’s genomic DNA. A novel 9 base-pair (GCCTTCTTC) in-frame insertion was detected between codons 344 and 345 in exon 8, resulting in an Ala–Phe–Phe insertion (Fig. 1B). The

proband was homozygous for this insertion. Amino acid residues 334–348 in exon 8 have been evolutionarily conserved from mice to humans. Genotyping of the mutated WFS1 gene was carried out by analyzing PCR fragments including the mutated region, using an upstream primer, 50 catcagcaacctcaccatcgac-30 , and a downstream primer, 50 -ccatggagatgaaggacaggta-30 . PCR products were electrophoresed on 4% agarose gel and visualized by ethidium bromide staining. All family members provided informed consent to participate in the study. The same homozygous mutation was observed in the younger brother’s genomic DNA, while the mother and younger sister were revealed to be heterozygous for this mutation (Fig. 1C). The father, probably heterozygous for the mutation, had died of prostatic cancer at age 57. The mutation was not detected in 100 healthy Japanese subjects (200 chromosomes). These data suggested the mutation to be the cause of disease in this family, and support the notion that the WFS1 gene is responsible for WFS. As WFS is a neurodegenerative disorder, we examined systemic neuronal functions in detail. As shown in Table 1, pituitary hormone studies revealed anterior in addition to the expected posterior (diabetes

Table 1 Results of pituitary hormone studies A

0 min

15 min

30 min

60 min

90 min

120 min

TRH (500 ng) loading test

TSH (mU/ml) Prolactin (ng/ml)

0.34 27.0

– –

2.7 62.6

2.0 55.6

1.7 46.5

1.3 41.9

CRH (100 mg) loading test

ACTH (pg/ml) Cortisol (mg/dl)

28 7.6

63 12.7

60 14.7

30 12.7

23 10.4

16 8.3

LHRH (100 mg) loading test

LH (mlU/ml) FSH (mlU/ml)

5.5 2.6

7.6 2.5

11.2 3.1

14.7 3.2

– –

19.8 4.0

GRH (100 mg) loading test

GH (ng/ml)

1.2

19.3

24.2

25.9

24.3

13.0

B TSH (mU/ml) Free T3 (pg/ml) Free T4 (ng/dl)

0.27 2.6 0.91

ACTH (pg/ml) Cortisol (mg/dl) Urine 17-OHCS (mg)

21 7.1 2.2

ADH (pg/ml) Posm (mOsm/kg) Uosm (mOsm/kg)

1.0 318 137

C

6 a.m.

12 a.m.

6 p.m.

12 p.m.

ACTH (pg/ml) Cortisol (mg/dl)

21 7.1

13 4.1

23 6.3

33 7.5

(A) Pituitary hormone-releasing tests. LH–RH loading test was performed in the follicular phase; (B) basal plasma hormone levels; (C) daily fluctuation of free ACTH and cortisol excretion.

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Fig. 2. Scan from T1 weighted magnetic resonance imaging of the brain of a 41-year-old woman with Wolfram syndrome. Left panel: sagittal, right panel: frontal.

insipidus) hypopituitarism. On CRH, TRH and LHRH stimulation tests, ACTH, TSH and FSH responses were imparied, while the GH response to GRH loading was within normal range (Table 1A). Furthermore, LH release in response to LH–RH was delayed. Basal plasma GH, ACTH and TSH levels were slightly below normal, while basal cortisol and thyroid hormone levels were in low normal ranges (Table 1B), suggesting impaired secretions of pituitary ACTH and TSH. Examination of the 24-h free ACTHcortisol excretion rate revealed loss of the daily fluctuation of ACTH secretion resulting in decreased cortisol excretion (Table 1C), which was confirmed by a relatively low level of the urinary 17-OHCS excretion. Despite these impaired pituitary functions, the patient had none of the clinical manifestations often seen in hypopituitarism, such as amenorrhea, fatigue and decreased appetite. Damage to the anterior pituitary usually results from a pituitary adenoma or postpartum infarction, both of which were ruled out in this case, based on the normal pituitary and hypothalamic MRI findings (Fig. 2). Though loss of signals from the hypothalamus and posterior pituitary was previously observed on MRI of diabetes insipidus patients [12], there was neither signal loss nor brainstem atrophy in our case. Given that posterior pituitary signals correspond to ADH-containing granules, we can speculate that the diabetes insipidus in our case might have been caused by impairment of ADH excretion rather than of ADH production in the posterior pituitary. Taking these findings together, this

patient apparently had preclinical panhypopituitarism, suggesting that this disorder might be related to the neurodegeneration which occurs in WFS.

3. Discussion Wolfram syndrome is best defined as a neurodegenerative disorder involving the central nervous system, peripheral nerves and neuroendocrine tissue. Various homozygous or compound heterozygous mutations, including nonsense, frameshift, deletion, insertion and missense mutations, have been identified to date in many of the families studied [8,13–16]. We present the previously reported locations of the mutations found in WFS patients in Fig. 3. The WFS1 protein consists of nine predicted helical transmembrane domains. Herein, we identified a novel in-frame WFS1 mutation, a 9 base-insertion in the putative first transmembrane domain of WFS1. In the present and earlier studies, both deletion and insertion frameshift-mutations have been identified in much of the WFS1 gene, while in-frame mutations, such as the one in our present case, are located exclusively in transmembrane or juxtatransmembrane regions. Given that proper membrane-insertion of WFS1 is essential for its function, it is reasonable to speculate that these in-frame mutations are likely to produce critical defects in the WFS1 protein, which prevent it being properly inserted into the membrane. Very recently, WFS1 knock out mice were established

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Fig. 3. Locations of previously reported and presently known mutations found in WFS patients. Predicted transmembrane domains are underlined.

[17]. These mice exhibited progressive b-cell loss and increased apoptosis, leading to impaired glucose homeostasis. Further study is needed to clarify the mechanism by which the WFS1-encoded transmembrane protein functions in b-cell maintenance. Our patient exhibited preclinical panhypopituitarism in addition to the loss of posterior pituitary function, which is a newly described feature of WFS. The functional anterior pituitary impairment previously reported in WFS patients was early-onset hypogonadism [4]. Very recently, anterior pituitary dysfunction was reported [18]. In their report, abnormal secretion of one or more pituitary hormones was observed in 75% of WFS patients, suggesting that pituitary dysfunction may be a common feature in WFS. Notably, the hypopituitarism seen in WFS does not produce clinical symptoms, which may explain the lack of pituitary examinations in previous studies. The present case indicates that pituitary hormone examinations should be performed when evaluating

WFS even if the patient has no clinical features of anterior hypopituitarism. Although WFS phenotypes are highly variable, no distinct clinical subgroups have been described to date. The proband’s younger brother, who had WFS and was homozygous for the same mutation as his sister, had no manifestations of either anterior or posterior hypopituitarism. Based on these observations, the insertion (AFF344-345ins) may not be directly related to anterior pituitary function, but we suspect that our patient’s impaired anterior pituitary function is attributable to this WFS1 mutation. Her brother will be followed closely for the development of panhypopituitarism. In conclusion, we performed a mutation analysis of the WFS1 gene and identified a novel homozygous WFS1 mutation (AFF344-345ins) in Japanese WFS patients. This mutation is located in the putative first transmembrane domain of WFS1. It is noteworthy that an in-frame insertion of only three amino acids can result in loss of WFS1 function.

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