Case Report

Association of a novel in-flame deletion mutation of the MYH9 gene with end-stage renal failure: case report and review of the literature

M. Ishida1*,Y. Mori1*, N. Ota1,K. Machida1,M. Nakayama1, K. Sonomura1,Y. Shiotsu1, E. Matsuoka1,K.Tamagaki1,T. Inaba2,S. Kunishima3, and H. Matsubara1

M. Ishida1*,Y. Mori1*, N. Ota1,K. Machida1, M. Nakayama1, K. Sonomura1,Y. Shiotsu1, E. Matsuoka1, R. Ishida1, H. Kado1, T. Adachi1, K. Tamagaki1, T. Inaba 2, S. Kunishima 3, and H. Matsubara1

1Department of Cardiology and Nephrology,

2Department of Infection Control and Laboratory Medicine,

Kyoto Prefectual University of Medicine, Kyoto, Japan

3Department of Advanced Diagnosis, Clinical research Center, National Hospital Organization Nagoya Medical Center,Nagoya, Japan

*These authors contributed equally to the report.

Address for correspondence:

Yasukiyo Mori, MD, PhD

Department of Cardiology and Nephrology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyou-ku, Kyoto, Japan

TEL: 81-75-251-5511; FAX: 81-75-251-5514; E-mail:

Charcter count of manuscript and abstract:16345 and 966

Running title: MYH9 disorders and ESRD

Abstract

MYH9 disorders are autosomal dominant diseases characterized by giant platelets, thrombocytopenia, and granulocyte inclusion bodies. These diseases are caused by mutations in the MYH9 gene that encodes non-muscle myosin heavy chain IIA. We describe a case of a 27-year-old male who presented with macrothrombocytopenia and leukocyte inclusion bodies. He had chronic kidney disease due to glomerulonephritis, and high-tone sensorineural deafness. Although deterioration of his renal function necessitated renal replacement therapy using peritoneal dialysis, , we reconsidered the etiology of the kidney disease with his clinical history. We diagnosed Fechtner syndrome, a member of MYH9 disorders, and identified an in-flame deletion mutation in exon 24 of MYH9 gene that resulted in the removal of 21 nucleotides. As this deletion is a novel abnormality of nucleotide arrangement, we report the case and compare it with previous cases and their associated phenotype.

Keywords: non-muscle myosin heavy chain IIA-MYH9 disorders-chronic kidney disease-peritoneal dialysis

Introduction

May-Hegglin anomaly (MHA) was originally reported as an autosomal dominant platelet disorder and is characterized by a triad of giant platelets, thrombocytopenia, and characteristic Dohle body-like cytoplasmic inclusions in granulocytes[1]. Several groups mapped the locus for myosin heavy chain (MHC) on chromosome 22q13.1 using genome-wide linkage analysis and found by positional cloning that MYH9, the gene for the nonmuscle MHC IIA (NMMHC IIA), is responsible for MHA[2,3,4, 5]. MHA, Sebastian syndrome (SBS), Fechtner syndrome (FTNS), and Epstein Syndrome were found to be allelic disorders of the NMMHC IIA gene. These four conditions are now called MYH9 disorders/MYH9-related diseases1. FTNS and Epstein Syndrome often show non-hematological complications such as glomerulonephritis, sensorineural deafness, and cataracts[5,6,7].

More than 200 families with MYH9 disorders have been investigated and more than 40 different MYH9 mutations have been identified across 12 of the 40 coding exons. A majority of MYH9 mutations (approximately 80%) are point mutations and cluster in a limited lesion[1, 7]. To our knowledge, cases of in-flame deletion are relatively rare,with only 7 having been reported in the English literature[8,9,10,11].Here we describe a case of FTNS with a novel in-flame deletion mutation of the MYH9 gene that developed end-stage renal failure. We alsoreview the existing literature on this condition.

Case Report

A 27-year-old Japanese male was hospitalized for undergoing peritoneal dialysis at our hospital. At his first visit to our clinic, the patient was 22 years old and already had chronic kidney disease. At this visit, urinalysis showed proteinuria (2.18 g/day) without hematuria. Blood chemistry findings were blood urea nitrogen (BUN), (BUN), 22.1 mg/dl; serum creatinine (SCr), 1.55 mg/dl; uric acid, 8.0 mg/dl; total protein, 6.5 g/dl; and albumin, 3.1 g/dl. Creatinine clearance (CCr) was 46.9 ml/min/1.73m2. Hematologicalexamination showed thrombocytopenia of55.0×104/μl and large platelets. In addition, ultrasound showed bilateral atrophic changes in the kidneys. This results in us being unable toperform subcutaneous renal biopsy. From the above-mentioned clinical information, we diagnosed chronic kidney disease induced by a chronic nephritic syndrome. Because of the patient’s past history,he had been referred to a pediatrician for evaluation of thrombocytopenia, when he underwent an operation for inguinal hernia at 2 years of age. However, the etiology of the thrombocytopenia was not clarified. He had no history of a bleeding tendency until his current admission. Although proteinuria was identified at his school work-up at age 11 years, no further examination had been performed.

After the patient’s first visit to our clinic when he was 22 years old, we prescribed diet therapy (restriction of protein and salt) combined with administration of an angiotensin II receptor blocker (Candesartan, 6 mg/day). Despite this treatment, his renal dysfunction deteriorated gradually. We finally recommended the patient receive renal replacement therapy when he was aged 27 years old. At the current admission, his blood pressure was 154/114 mmHg, pulse 80 beats/min, and body temperature 36.9°C. Physical examination confirmed edema in the legs, whereas lung, heart, abdomen, and the neurological system were normal on examination. We demonstrated the patient had high-tone sensorineural deafness. Urinalysis revealed nephrotic range proteinuria (9.436 g/day) without hematuria.The blood chemistry findings were BUN, 65.9 mg/dl; SCr 14.85 mg/dl; uric acid, 9.4 mg/dl; albumin, 2.9 g/dl; Na, 140 mEq/l; K, 6.3mEq/l; Cl, 100mEq/l; Ca, 5.7 mg/dl; IP, 7.1 mg/dl; and CCr, 2.3 ml/min/1.73 m2.Hematological examination showed erythrocytes, 296×104/µl; hemoglobin, 8.7g/dl; leukocytes, 5600/µl; and platelets, 29.0104/μl. Conspicuous granulocyte inclusion bodies were identified in peripheral blood smears. The inclusion bodies were basophilic and had an oval- to spindle-shape morphology (Figure 1A). Aftercatheterization, the patient was started on peritoneal dialysis.

The existence of chronic kidney disease associated with hematological symptoms (thrombocytopenia with large platelets and granulocyte inclusion bodies) and sensorineural deafness is compatible with FTNS. Accordingly, we examined neutrophil myosin IIA localization using immunofluorescence analysis, and confirmed type I cytoplasmic myosin IIA-positive granules (Figure 1B). MYH9 gene analysis showed a novel in-flame deletion mutation, which resulted in the removal of 21 nucleotides, c.3202_3222 del, in exon 24 (Figure 1C, upper panel). This gene mutationcaused a p.Q1068_L1074 del of NMMHC IIA protein. Interestingly, the amino acid sequence of p.Q1068_L1074 del resulted in the same derangement of the amino acid sequence (p.Q1066_L1072 del) as the in-flame gene deletion (C.3195_3215 del) found in two previous cases (Figure 1C, lower panel)[8,11].

Discussion

Peterson et al.[12] first described FTNS in 1985 in a four-generation family who had macrothrombocytopenia, inclusion bodies in granulocytes, sensorineural deafness, nephritis, and cataracts. However, the pathogenesis and phenotype-genotyperelationship of MYH9 disorders-related nephritis, including cases with FTNS remains unclear[7]. MYH9 spans 110 kb of the genomic sequence with 41 exons. The mRNA transcript is 7.5 kb in size and encodes a protein of 960 amino acid sequences. Exons 2–17 encode the head domain, while exons 18–41 encode the rod domain[13].More than 40 different MYH9 mutations have been identified in patients with MYH9 disorders. The majority of MYH9 mutations (approximately 80%) are point mutations and cluster in a limited exon[1]. Arundel et al. described wide expression of MYH9 in mature kidneys and showed that NMMHC IIA was located mainly in podocytes[6]. They also demonstrated variable penetrance and different extra-hematological phenotypes such as nephritis, cataract, and deafness (so called Alport-like syndrome) within a given family and between families that carried the same missense mutation (D1424N or R1165L substitution). Further analysis of an Italian family carrying the D1424N substitution in the coiled-coil domain of MYH9 revealed that the renal phenotype was observed with atypical distribution of NMMHC IIA in tubular epithelia, the focal and segmental effacement of podocytes, and the co-segregation of a specific haplotype of podocin[13]. It has also been confirmed that NMMHC IIA is a major component of the contractile apparatus in the podocyte foot process, with changes in NMMHC IIA structure caused by MYH9R702 mutations altering podocyte structure and function[14, 15]. Taken together, these results indicate it is likely, at least in cases of a missense mutation, that the additional predisposing conditions and/or environmental factors cause nephropathy in MYH9 disorders including FTNS.

Although deletion mutations are very rare, and only 7 in-flame deletion mutations in 7 patents have been reported to date (5 small deletion mutations; p.N76_S81 del, p.E1066_A1072 del, p.E1084 del, p.G1055_Q1068 del, and p.L1205_Q1207 del; and 1 large gene defect; p.V1092_R1162 del; Table 1), it is of interest comparatively, in that in-flame deletions in exons 24 and 25 positioned around the mid-portion of NMMHC IIA, the hinge of subfragment-2 and light meromyosin, often cause non-hematological complications[8,10,11]. Therefore, in-flame deletion mutations in exon 24 of MYH9 seem to cause unique effects on NMMHC IIA function that may hinder both motor activity of the head and normal assembly of the coiled-coil tail[11].Notably, p.Q1068_L1074 del in our case resulted in the same amino acid mutation of p.E1066_A1072 del as that reported in two earlier sporadic cases[8,11]. As shown in Figure 1C, the motif of AELKMQ with the surrounding amino acid sequences in p.Q1068_L1074 del and p.E1066_A1072 del may be essential for normal structure and function of NMMHC IIA in platelets and leucocytes as well as podocytes. Further experiments using an in vitro expression system are necessary to clarify this possibility.

The restricted location of in-frame deletion and insertion mutations in exon 24 indicates that this exon contains latent hot spots for small rearrangements. Gene rearrangements such as deletions and duplications/insertions may occur by unequal homologous recombination between non-allelic regions, such as two repetitive elements. In fact, identification of the first duplication was observed in the same nucleotides (p.E1066_A1072 dup)[16]. This suggests unequal crossing in the repetitive sequence found as a short unit of 16 nucleotides (C CAG ATC GCG GAG CTC), which is repeated six nucleotides downstream. It is interesting that this sequence is included in the deletion mutation in our case (Figure 1C).

In summary, we describe a case of a 27-year-old male with ESRD on peritoneal dialysis due to MYH9 disorders. New evidence suggests that MYH9 gene alterations are also associated with greater risk of focal segmental glomerulosclerosis in African Americans[7,13]. More precise investigations regarding the role of the MYH9 gene on renal injury in MYH9 disorders will be useful for a greater understanding of general glomerular pathology.

Acknowledgements

Support: None.

Financial Disclosure: The authors declare that they have no relevant financial interests.

References

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[3]Kelley MJ, Jawien W, Lin A, Hoffmeister K, Pugh EW, Doheny KF, Korczac, JF. Autosomal dominant macrothrombocytopenia with leukocyte inculusions (May-Hegglin anomaly) is linked to chromosome 22q12-13. Hum Genet 2000; 106: 557-564

[4]Martignetti JA, Heath KE, Harris J, Bizzaro N, Savoia A, Balduini CL, Desnick RJ. The gene for May-Hegglin anomaly localizes to a <1-Mb region on chromosome 22q12.3-13.1. Am J Hum Genet 2000; 66: 1449-1454

[5]Toren A, Rozenfeld-Granot G, Rocca B, Epstein CJ, Amariglio N, Laghi F, Landolfi R, Brok-Simoni F, Carlsson LE, Rechavi G, Greinacher A. Autosomal dominant giant platelet syndrome: a hint of the same genetic defect as in Fechtner syndrome owing to a similar linkage to chromosome 22q11-13. Blood 2000; 96: 3447-3451

[6]Arrondel C, Vodovar N, Knebelmann B, Grunfeld JP, Gubler MC, Antignac C, Heidet L. Expression of the nonmuscle myosin heavy chain IIA in the human kidney and screening for MYH9 mutations in Epstein and Fechtner syndromes. J Am Soc Nephrol 2001;13: 65-74

[7]Singh N, Nainani N, Arora P, Venuto RC. CKD in MYH9-Related Disorders. Am J Kidney Dis2009; 54: 732-740

[8]Seri M, Pecci A, Bari FD, Cusano R, Savino M, Panza E, Nigro A, Noris P, Gangarossa S, Rocca B, Gresele P, Bizzaro N, Malatesta P, Koivisto P, Longo I, Musso R, Pecoraro C, Iolascon A, Magrini U, Soriano JR, Renieri A, Ghiggeri GM, Ravazzolo R, Balduini CL, Savoia A. MYH-Related Disease May-Hegglin Anomaly, Sebastiansyndrome, Fechtner syndrome, and Epstein syndrome are not disntinct entities but represent a variable expression of a single illness. Medicine 2003; 82: 203-215

[9] Kunishima S, Matsushita T, Shiratsuchi M, Ikuta T, Nishimura J, Hamaguchi M, Naoe T, Saito H. Detection of unique neutrophil non-muscle myosin heavy chain-A localization by immunofluorescence analysis in MYH9 disorder presented with macrothrombocytopenia without leukocyte inclusions and deafness. Eur J Haematol 2005; 74: 1-5

[10] Kunishima S, Matsushita T, Hamaguchi M, Sato H. Identification and characterization of the first large delerion of the MYH9 gene associated with MYH9 disorders. Eur J Haematol 2008; 80: 540-544

[11]Miyazaki K, Kunishima S, Fujii W, Higashihara M.Identification of three in-frame deletion mutations in MYH9 disorders suggesting an important hot spot for small rearrangements in MYH9 exon 24. European Journal of Haematology 2009; 83: 230-234

[12]Peterson LC, Rao KV, Crosson JT, White JG. Fechtner syndrome-a variant of Alport’s syndrome with leukocyte inclusion and macrothrombocytopnia. Blood 1985; 65: 397-406

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[14] Ghiggeri GM, Caridi G, Magrini U, Sessa A, Savoia A, Seri M, Pecci A, Romagnoli R, Gangarossa S, Noris P, Sartore S, Necchi V, Ravazzolo R, Balduini CL. Genetics, clinical and pathological features of glomerulonephritis associated with mutations of nonmuscle myosin IIA (Fechtner syndrome). Am J Kidney Dis 2003; 41: 95-104

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Table 1. Case Reports of deletion mutations in MYH9 gene

Case / Author / Age / Inheritance / Deleted
nucleotides / Deleted
amino acids / Exon / Nonhematological complications / Ref
Kidney impairment / Hearing
impairment / Cataract
1 / Seri et al.
[2003] / 1.5 / Sporadic / c.3195_3215del / p.E1066_A1072del / Exon24 / No / Bilateral / Yes / 8
2 / Seri et al.
[2003] / unknown / Sporadic / unknown / p.L1205_Q1207del / Exon26 / No / No / No / 8
3 / Kunishima et al
[2005] / 45 / Familial / c.228_245del / p.N76_S81del / Exon1 / No / Bilateral / No / 9
4 / Kunishimaet al.
[2008] / 37 / Sporadic / nt64823_nt63604 / p.V1092_R1162del / Exon25-Intron / nephritis / No / No / 10
5 / Miyazaki et al.
[2009] / 37 / Familial / c.3164_3205del / p.G1055_Q1068del / Exon24 / ESRD / Bilateral / No / 11
6 / Miyazaki et al.
[2009] / 47 / Sporadic / c.3195_3215del / p.E1066_A1072del / Exon24 / nephritis / Bilateral / Yes
Bilateral / 11
7 / Miyazaki et al.
[2009] / 21 / Familial / c.3250_3252del / p.E1084del / Exon24 / No / No / No / 11
8 / ours / 27 / Sporadic / c.3202_3222del / p.Q1068_L1074 del / Exon24 / ESRD / Bilateral / No

1

FIGURE LEGENDS

Figure 1.The light microscopic findings, immunofluorescence assay of granulocytes and platelets, and schematic presentation of the C.3202_3222 del mutation in the MYH9 gene in the patient. Conspicuous granulocyte inclusion bodies and giant platelets were identified in peripheral blood smears. The inclusion bodies were basophilic and had an oval- to spindle-shape morphology (A). The immunofluorescence analysis confirmed the type I cytoplasmic NMMHC IIA-positive granules (B). The asterisk (*) indicates

the cases reported previously in references 8 and 11 (C).

1