Two Adjacent Mutations on Chromosome 16 Discovered in a Patient Presenting with Generalized Convulsions After Influenza a Virus Infection

□ CASE REPORT □

Two Adjacent Mutations on Chromosome 16 Discovered in a Patient Presenting with Generalized Convulsions after Influenza A Virus Infection

Yorihiro Iwasaki 1,4, Makio Takahashi 2, Kandai Nozu 3, Sadayuki Matsumoto 2 and Hiroyuki Koshiyama 1

Abstract

A 49-year-old otherwise healthy man was admitted to our hospital because of repeated generalized convulsions after influenza A virus infection. His family history was notable for consanguinity of parents. Initial laboratory tests revealed metabolic alkalosis with hypomagnesemia, as well as an elevated high density lipoprotein cholesterol level. He was diagnosed with Gitelman’s syndrome and cholesteryl ester transfer protein deficiency by identifying homozygous mutations of causative genes, SLC12A3 and CETP, respectively. These two genes are located in the vicinity on chromosome 16, suggesting the possibility of autozygosity. This is the first case report highlighting the co-existence of these genetic disorders.

Key words: Gitelman’s syndrome, CETP deficiency, influenza, autozygosity

(Intern Med 50: 2179-2183, 2011) (DOI: 10.2169/internalmedicine.50.5515)

Introduction Case Report

Gitelman’s syndrome is an autosomal recessive renal tu- A 49-year-old Japanese male presented to a hospital com- bular disorder characterized by salt wasting, metabolic alka- plaining of fever, chills and arthralgia. Diagnosis of influ- losis, hypokalemia and hypomagnesemia (1). On the other enza A was made based on the positive result of the viral hand, cholesteryl ester transfer protein (CETP) deficiency is antigen. Oseltamivir phosphate and acetaminophen were pre- an autosomal recessive disorder known as a major cause of scribed. On the following day, he experienced repeated elevated high density lipoprotein (HDL) cholesterol level in tonic-clonic convulsions, and was transferred to our hospital the Japanese population (2). Although the causative genes of for further evaluation and treatment. His past medical his- these disorders, SLC12A3 and CETP, are located in the vi- tory was unremarkable, and family history was notable for cinity on the long arm of chromosome 16, the co-existence his parents’ consanguineous marriage (Fig. 1). Careful his- of them is currently unknown. Here, we report the first case tory taking did not reveal any remarkable episodes in his of Gitelman’s syndrome with CETP deficiency, whose initial siblings. He had lived in a remote island in Japan before he presentation is convulsions provoked by influenza A virus moved to an urban area several years ago. At the time of infection. presentation, he was taking no medication except for oseltamivir phosphate and acetaminophen. On admission, he was alert, body temperature was 37.2℃ and blood pressure was 102/43 mmHg. Physical examina-

１Center for Diabetes & Endocrinology, The Tazuke Kofukai Foundation Medical Research Institute Kitano Hospital, Japan, ２Center for Neurol- ogy, The Tazuke Kofukai Foundation Medical Research Institute Kitano Hospital, Japan, ３Department of Pediatrics, Kobe University Graduate School of Medicine, Japan and ４Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Japan Received for publication March 16, 2011; Accepted for publication June 14, 2011 Correspondence to Dr. Yorihiro Iwasaki, [email protected]

2179 Intern Med 50: 2179-2183, 2011 DOI: 10.2169/internalmedicine.50.5515 tion showed no abnormal findings. Magnetic resonance im- magnesemia, metabolic alkalosis and elevated HDL choles- aging of the brain, electroencephalogram and cerebrospinal terol level (Table 1). Chemical analysis of spot urine indi- fluid analysis did not show any abnormal results. Initial cated inappropriately increased excretion of chloride in the laboratory tests revealed hypochloremia, hypokalemia, hypo- presence of metabolic alkalosis, while he was not taking diuretics. Twenty-four-hour urine collection showed increased excretion of sodium (317 mEq/24h), chloride (320 mEq/24h), and decreased excretion of calcium (73.9 mg/ 24h). Plasma renin activity and plasma aldosterone concen- tration were elevated, at 9.0 ng/mL/hr and at 320 pg/mL, respectively. We concluded that electrolyte abnormalities, especially hypomagnesemia, were primary causes of repeated convulsions, since other causes of convulsions including influenza- associated encephalopathy were ruled out based on the above-mentioned results. Therefore, treatment was started with intravenous magnesium sulfate and potassium chloride to correct electrolyte and acid-base abnormalities. On day 2, serum levels of potassium and magnesium were maintained around the lower limit of normal range, and thereafter, his convulsions ceased. There was no muscle weakness during hospitalization. After he was discharged on the eighth day, oral potassium chloride and magnesium supplements were administered (Fig. 2). His systolic blood pressure remained Figure 1. The family pedigree of the patient. low, around 90-100 mmHg. Since the patient was prone to

Table 1. Laboratory Data

On admission 2 months later Normal range Serum biochemistry

Sodium 133 141 136-147 (mEq/L) Potassium 2 2.8 3.6-5.0 (mEq/L) Chloride 85 91 98-108 (mEq/L) Calcium 8.9 10.9 8.5-10.3 (mg/dL) Phosphorus 3 4.7 2.5-4.5 (mg/dL) Magnesium 1.4 1.5 1.7-2.2 (mg/dL) Blood glucose 132 106 70-109 (mg/dL) Triglyceride 48 98 45-150 (mg/dL) HDL cholesterol 167 176 40-70 (mg/dL) LDL cholesterol 63 62 <140 (mg/dL) Blood urea nitrogen 14.4 18.1 8.0-20.0 (mg/dL) Creatinine 0.79 0.8 0.6-1.3 (mg/dL) Plasma Renin activity * 9 0.3-2.9 (ng/ml/hr) Aldosterone * 320 29.9-159 (pg/mL) Arterial blood gas analysis pH 7.568 Bicarbonate 34.7 22-28 (mEq/L) Base excess 10.9 -2- +2 (mEq/L)

Partial pressure of CO2 37.8 35-45 (mmHg)

Partial pressure of O2 134.2 80-100 (mmHg) Spot urine electrolyte levels Sodium 154 97 (mEq/L) Potassium 87.3 51.2 (mEq/L) Chloride 161 102 (mEq/L) Calcium 2.3 6.2 (mEq/L) Magnesium 5.4 11.9 (mEq/L) * Measured after 30 minutes of bed rest HDL: high density lipoprotein, LDL: low density lipoprotein

2180 Intern Med 50: 2179-2183, 2011 DOI: 10.2169/internalmedicine.50.5515

1 2 3 4 5 6 7 8 91011121314151617Day

IV KCKCll 2.72.7g/dayg/day IV KClKCl 1.5g/day1.5g/day OralOral KKClCl 2.1g2.1g/day/day

IV MMgSOgSO4 2.4g/day2.4g/day OralOral MMgOgO 00.99g/day.99g/day

Figure 2. Changes in serum potassium and magnesium levels. IV: intravenous, KCl: potassium

chloride, MgSO4: magnesium sulfate, MgO: magnesium oxide

hypokalemia and hypomagnesemia even two months later milder than Bartter’s syndrome (8). However, as an initial (Table 1), we encouraged him to regularly eat a sufficient symptom, a generalized convulsion in an adult after influ- amount of vegetables and seafood. enza virus infection has not yet been reported. In the present According to characteristic electrolytes and acid-base ab- case, biochemical abnormalities, including hypomagnesemia normalities, Gitelman’s syndrome was clinically suspected. and metabolic alkalosis, may predispose the patient to sei- Sequencing of the causative gene (3), SLC12A3, revealed a zures. According to the lifestyle history, it is speculated that homozygous missense mutation of T180K in exon 4 the patient remained asymptomatic until his middle ages be- (Fig. S1). Furthermore, the fact that the patient has an ex- cause he was eating a lot of seafood with salt daily in a re- tremely high HDL cholesterol level prompted us to analyze mote island to compensate for tubular salt wasting. After he CETP gene, which is located in the vicinity of SLC12A3 moved to an urban area, his chronic biochemical abnormali- gene. As a result of high through-put invader assay (4), a ties worsened, which could increase the risk for convulsions homozygous missense mutation of D442G in exon 15 was in case of influenza virus infection. identified. These results confirmed the co-existence of the CETP deficiency is the most frequent genetic cause of two genetic disorders associated with the long arm of chro- high HDL cholesterol level in Japanese (2, 9) and other mosome 16 (Fig. 3). Eastern Asian populations (10, 11), although it is rare among Caucasians (12). Because of impaired reverse choles- Discussion terol transport capacity, the disease may be associated with increased cardiovascular risk in spite of the high HDL cho- Gitelman’s syndrome was first reported by Gitelman et al lesterol level (2), although the risk remains controver- as a variant of Bartter’s syndrome (1). The most distinguish- sial (13, 14). There are two hot spots of point mutations in able clinical feature from Bartter’s syndrome is the presence CETP gene in Japanese; Intron 14+1 G to A and of hypocalciuria. Simon et al demonstrated complete linkage D442G (15), the latter of which is the mutation found in the of the syndrome to SLC12A3 gene locus encoding the present case. thiazide-sensitive Na-Cl cotransporter (5). Although it is Intriguingly, the locus responsible for Gitelman’s syn- often considered to be a rare disorder, a recent report sug- drome (16q13) is in the vicinity of that responsible for gests that the frequency of mutated alleles is relatively high CETP deficiency (16q21) on chromosome 16 (Fig. 3). De- in the Japanese population (6). The point mutation identified spite the relatively high prevalence of the two genetic disor- in the present case, T180K in exon 4, was previously re- ders in Japan, the frequency of co-existence is unknown. ported (7). According to the HapMap database [Hapmap Data Rel 27 It is not uncommon that the diagnosis of Gitelman’s syn- Phase II+III, Feb09 (16)], in a selected Japanese population, drome is made later in life since its symptoms are generally there are two linkage disequilibrium (LD) blocks in the fo-

2181 Intern Med 50: 2179-2183, 2011 DOI: 10.2169/internalmedicine.50.5515

16p13.3 16p13.2 NUP93

16p13.1 Gitelman’s syndrome 16p12 SLC12A3 Hypokalemia 16p11.2 rs2304480 16p11.1 T180K Hypomagnesemia rs2229209 16q11.1 Metabolic alkalosis 16q11.2

16q12.1 HERPUD1 16q12.2 122702bases 16q13 16q21 CETP CETP ĚĞĮĐŝĞŶĐǇ 16q22 rs2303790 D442G Elevated HDL 16q23 cholesterol level

16q24 NLRC5

Figure 3. The region of chromosome 16 including SLC12A3 and CETP genes. Physical distance between the two mutations was calculated according to the NCBI database (Genbank accession no. NC000016). CETP: cholesteryl ester transfer protein

cused region [Fig. S2, generated by Haploview (17)]. Since variant of Bartter’s syndrome, inherited hypokalemic alkalosis, is the two polymorphisms are located in separate LD blocks, it caused by mutations in the thiazide-sensitive NaCl transporter. Nat is unlikely that the two are in complete LD. Considering the Genet 12: 24-30, 1996. parental consanguinity and the homozygosity for both muta- 6. Tago N, Kokubo Y, Inamoto N, et al. A high prevalence of Gitel- man’s syndrome mutations in Japanese. Hypertens Res 27: 327- tions, the patient could possess an autozygous chromosomal 331, 2004. region including the two adjacent mutations. In conclusion, 7. Monkawa T, Kurihara I, Kobayashi K, et al. Novel mutations in this report highlights the novel co-existence of Gitelman’s thiazide-sensitive Na-Cl cotransporter gene of patients with Gitel- syndrome with CETP deficiency that is incidentally discov- man’s syndrome. J Am Soc Nephrol 11: 65-70, 2000. ered in a patient with influenza. 8. Cruz DN, Shaer AJ, Bia MJ, et al. Gitelman’s syndrome revisited: an evaluation of symptoms and health-related quality of life. Kid- ney Int 59: 710-717, 2001. The authors state that they have no Conflict of Interest (COI). 9. Nagano M, Yamashita S, Hirano K, et al. Molecular mechanisms of cholesteryl ester transfer protein deficiency in Japanese. J Athe- roscler Thromb 11: 110-121, 2004. References 10. Song GJ, Han GH, Chae JJ, et al. The effects of the cholesterol ester transfer protein gene and environmental factors on the 1. Gitelman HJ, Grahan JB, Welt LG. A new familial disorder char- plasma high density lipoprotein cholesterol levels in the Korean acterized by hypokalemia and hypomagnesemia. Trans Assoc Am population. Mol Cells 7: 615-619, 1997. Physicians 79: 221-235, 1966. 11. Zhuang Y, Wang J, Qiang H, et al. Cholesteryl ester transfer pro- 2. Hirano K, Yamashita S, Nakajima N, et al. Genetic cholesteryl es- tein levels and gene deficiency in Chinese patients with caedio- ter transfer protein deficiency is extremely frequent in the Oma- cerebrovascular diseases. Chin Med J 115: 371-374, 2002. gari area of Japan. Marked hyperalphalipoproteinemia caused by 12. van der Steeg WA, Hovingh GK, Klerkx AH, et al. Cholesteryl es- CETP gene mutation is not associated with longevity. Arterioscler ter transfer protein levels and hyperalphalipoproteinemia in Cauca- Thromb Vasc Biol 17: 1053-1059, 1997. sians. J Lipid Res 48: 674-682, 2007. 3. Fukuyama S, Okudaira S, Yamazato S, et al. Analysis of renal tu- 13. Moriyama Y, Okamura T, Inazu A, et al. A low prevalence of bular electrolyte transporter genes in seven patients with hypoka- coronary heart disease among subjects with increased high-density lemic metabolic alkalosis. Kidney Int 64: 808-816, 2003. lipoprotein cholesterol levels, including those with plasma choles- 4. Nagano M, Yamashita S, Hirano K, et al. Two novel missense mu- teryl ester transfer protein deficiency. Prev Med 27: 659-667, tations in the CETP gene in Japanese hyperalphalipoproteinemic 1998. subjects: high-throughput assay by Invader assay. J Lipid Res 43: 14. Curb JD, Abbott RD, Rodriguez BL, et al. A prospective study of 1011-1018, 2002. HDL-C and cholesteryl ester transfer protein gene mutations and 5. Simon DB, Nelson-Williams C, Johson Bia M, et al. Gitelman’s the risk of coronary heart disease in the elderly. J Lipid Res 45:

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948-953, 2004. the human genome. Nature 437: 1299-1320, 2005. 15. Inazu A, Jiang XC, Haraki T, et al. Genetic cholesteryl ester trans- 17. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and fer protein deficiency caused by two prevalent mutations as a ma- visualization of LD and haplotype maps. Bioinformatics 21: 263- jor determinant of increased levels of high density lipoprotein cho- 265, 2005. lesterol. J Clin Invest 94: 1872-1882, 1994. 16. Altshuler D, Brooks LD, Chakravarti A, et al. A haplotype map of

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