Late-onset renal TMA and tubular injury in cobalamin C disease: a report of three cases and literature review

Abstract

Background

Mutation of MMACHC gene causes cobalamin C disease (cblC), an inherited metabolic disorder, which presents as combined methylmalonic aciduria (MMA-uria) and hyperhomocysteinaemia in clinical. Renal complications may also be present in patients with this inborn deficiency. The most common histological change is thrombotic microangiopathy (TMA). However, to our acknowledge, renal tubular injury in the late-onset presentation of cblC is rarely been reported. This study provides a detailed description of the characteristics of kidney disease in cblC deficiency, aiming to improve the early recognition of this treatable disease for clinical nephrologists.

Case presentation

Here we described three teenage patients who presented with hematuria, proteinuria, and hypertension in clinical presentation. They were diagnosed with renal involvement due to cblC deficiency after laboratory tests revealing elevated serum and urine homocysteine, renal biopsy showing TMA and tubular injury, along with genetic testing showing heterogeneous compound mutations in MMACHC. Hydroxocobalamin, betaine, and L-carnitine were administered to these patients. All of them got improved, with decreased homocysteine, controlled blood pressure, and kidney outcomes recovered.

Conclusions

The clinical diagnosis of cblC disease associated with kidney injury should be considered in patients with unclear TMA accompanied by a high concentration of serum homocysteine, even in teenagers or adults. Early diagnosis and timely intervention are vital to improving the prognosis of cobalamin C disease.

Clinical Trial Number

Not applicable.

Background

Combined methylmalonic aciduria (MMA-uria) and hyperhomocysteinaemia, cobalamin C (cblC) type, is the most common genetic type of functional cobalamin (a cofactor form of vitamin B12) deficiency, which is a kind of inherited metabolic disorder presented with diverse clinical manifestations combining multisystem damage [1, 2]. Patients with early-onset cblC may present in the first month of life with lethargy, hypotonia, poor oral intake, and failure to thrive [3]. While patients with late-onset cblC may present neuropsychiatric symptoms, like lower limb weakness, psychiatric disturbances, gait instability, or brain or spinal cord changes by imaging [4]. Kidney injury associated with cblC is uncommon [1]. Limited research works showed renal histopathology with glomerular lesions typical of thrombotic microangiopathy (TMA) [5]. Hemolytic uremic syndrome and thrombotic thrombocytopenic purpura are the 2 most frequent causes of TMA. Other causes of TMA include drugs, malignancies, pregnancy, autoimmune diseases, or malignant hypertension [5, 6]. Metabolic disease associated TMA are often ignored or hard to diagnose. Here we present three cases of kidney involvement in patients with cblC, presented with TMA and tubular injury in kidney biopsy. We also conducted a literature review of cblC disease with renal involvement in a summary of clinical and pathological impairment, aiming to enhance awareness and understanding of this rare, but treatable disease.

Case presentation

Patient 1

A 15-year-old young boy was admitted to our department due to proteinuria and hematuria with elevated blood pressure (170/120 mmHg) for 1 week. The laboratory examination showed macrocytic anemia (hemoglobin 9.3 g/dl, MCV 105.3 fl.), proteinuria (3+), and hematuria (20–30 cells/HPF). The 24-hour urinary protein was 2.12 g/day (total urine volume 500 ml) with decreased serum albumin level of 34.4 g/L. Serum creatinine (SCr) was 110.2 µmol/L (reference range: 56–102 µmol/L). Serum level of homocysteine (HCY) was over 200 µmol/L (reference range: 6–17 µmol/L), which exceeded the detection limit in routine test. He had a comparatively low level of complement component (C3 0.587 g/L, reference range: 0.6–1.5 g/L; C4 0.106 g/L, reference range: 0.12–0.36 g/L) and a low titer of positive antinuclear antibody (ANA) (1:100 ). However, no systemic immune or infection related disease was observed. He had impaired urine concentration function, with significantly low morning urinary osmotic pressure (310 mOsm/kg, reference range: 600–1000 mOsm/kg), and blood osmotic pressure was normal (293 mOsm/kg, reference range: 275–305 mOsm/kg). Urine glucose was negative and urine acidification examination was normal, without hypophosphatemia and hypouricemia. Schistocytes on the blood smear could be seen, accounting for 1.2%, with thrombocytopenia (platelet 118,000/mm³), and increased lactate dehydrogenase (LDH 573 IU/L), which indicated the microangiopathic hemolytic anemia (MAHA), without any history of thrombosis. However, von Willebrand factor protease (ADAMTS13) and the concentration of factor H were both normal, and the anti-factor H antibody was negative..

He was diagnosed with cblC 2 months after his birth, and treated with hydroxocobalamin, L-carnitine and betaine ever since. Comprehensive genetic analyses revealed compound heterozygous variants c.609 G > A (p.W203*) (The American College of Medical Genetics and Genomics, ACMG classification: pathogenic) and c.658_660del (p.K220del) (ACMG classification: likely pathogenic) mutation in the MMACHC gene (Fig. 1). He developed well in spite with stable disease. He had no signs of involvement of intelligence, sensibility or central nervous system symptoms, such as seizures, ataxia, or hypotonia, until he interrupted his therapy 4 months before admission.

Fig. 1

Sanger electropherogram figure of MMACHC mutation in patient 1. A to C and D to F shows the mutated locus of patient 1, his father, and his mother on c.609 G > A (p.W203*) and c.658_660del (p.K220del), separately

A renal biopsy was performed (Fig. 2). Light microscopic examination showed that 1/28 glomeruli was ischemic and sclerotic. The rest of the glomeruli showed mild mesangial expansion, accompanied by segmental endothelial cell proliferation. The glomerular basement membrane thickened diffusely, with the formation of “double contours”. The tubules displayed loss of brush border with epithelial simplification. Direct immunofluorescence revealed trace staining for segmental granular deposition of IgA (+) and IgM (+) on mesangial area, and negative for IgG, C3, and C1q. He was diagnosed with renal TMA and acute tubular injury as the renal complication of cblC. Electron microscopy confirmed the diagnosis, and no enlarged mitochondria was detected. The patient was treated with hydroxocobalamin (3 mg/day, i.m.), betaine (9 g/day, p.o.), L-carnitine (2 g/day, p.o.) as well as Vitamin AD and vitamin B compounds daily. Besides, angiotensin receptor blockers and calcium channel blockers combinations were used to treat hypertension. After 6 weeks of treatment, his blood pressure, hemoglobin, LDH and urine protein returned to normal. HCY decreased to 85.56µmol/L and SCr decreased to 65 µmol/L which indicated that an event of acute kidney disease occurred in this patient.

Fig. 2

Kidney biopsy findings. (A) The glomerulus exhibited segmental endothelial cell proliferation with diffusely thickened glomerular basement membrane and the formation of ‘double contours’, and a granuloma consisted of mainly foam cells at beside interstitium (PAS ×400). (B) The loss of brush border with epithelial simplification of tubular epithelia was presented, along with focal infiltration of lymphocyte and mononuclear cells in renal interstitium (HE×400). (C) Electron microscopy showed widening of subendothelial area of glomerular basement membrane with narrowed cavity of capillary loops (original magnification ×5,000). (D) Proliferation of mesangial cells and endothelial cells with occlusion of glomerular capillary lumen was observed (original magnification×6,000)

Patient 2

A 15-year-old boy was admitted to our department due to proteinuria and hematuria for 1 year. His blood pressure was 135/85 mmHg. Urinary protein excretion was 2.05 g/day (urine volume 1000 ml) and his SCr was 72 µmol/L (reference range 44–133 µmol/L). Urinary N-acetyl-beta-D-glucoaminidase (NAG) was elevated to 18.7 U/L. HCY was 149.01 µmol/L, which was also significantly increased. Urine glucose was negative, along with hyperuricemia but normal serum phosphorus. ANA was positive (1:320). Anti-dsDNA, anticardiolipin antibodies, and anti-β2-GP1 antibodies were all negative. Echocardiography showed an enlarged right heart with pulmonary arterial hypertension (86 mmHg). The level of hemoglobin and platelet count were both normal. There were no evidence of MAHA and no signs of thrombotic event in examinations. Renal biopsy showed TMA, combined with focal segmental glomerulosclerosis (FSGS) like lesions. Tubular epithelial cells presented with vacuolar and granular degeneration.

He went to the other medical center due to dyspnea 1 year ago. During hospitalization, examination showed pulmonary arterial hypertension and megaloblastic anemia. Electromyography (EMG) and nerve conduction velocity (NCV) showed peripheral nerve injury in the upper and lower limbs. Genetic analyses showed compound heterozygous variants of the c.80 A > G (p.Q27R) (ACMG classification: pathogenic) and c.609G > A (p.W203X) (ACMG classification: pathogenic) in MMACHC gene, which confirmed the diagnosis was MMA-uria and hyperhomocysteinaemia (cblC type). The patient was 195 cm in height and weighed 70 kg but his physical strength was worse than his peers, and he had not attended physical education classes since the fifth grade of primary school. Intelligence Quotient (IQ) test showed he has a normal IQ score of over 90.

Treatment with mecobalamin (3 mg/day, p.o.), cobamamide (3 mg/day, i.m.), folic acid (5 mg/day, p.o.), betaine (9 g/day, p.o.), L-carnitine (2 g/day, p.o.), as well as Vitamin AD compounds and vitamin B supplements. Lotensin (10 mg/day, p.o.) was used to anti-hypertension and ambrisentan (5 mg/day, p.o.) was also used to reduce pulmonary artery pressure. Within the next 10 days, the serum HCY was decreased to 136.22 µmol/L and 24 h-urinary protein excretion was reduced to 1.18 g/day (total urine volume 650 ml). At the last follow-up, two months after the discharge, the patient’s serum HCY was decreased to a satisfactory level of 50.13 µmol/L, and blood pressure was well controlled under antihypertensive agents.

Patient 3

A 16-year-old boy without significant medical history presented with severe hypertension (up to 180/116 mmHg) with the chief complaint of a decrease in activity tolerance, mucosal hemorrhage, and oliguria. Further examination showed anemia (Hb 6.3 g/dl) with schistocytes, thrombocytopenia (90,000/mm³), kidney injury (SCr 197 µmol/L), proteinuria, elevated lactate dehydrogenase (LDH 779 U/L) and abnormal NT-proBNP (6152 pg/ml). Ultrasonography revealed enlargement of left atrial and the thickening of left ventricular wall and splenomegaly. Renal biopsy showed typical signs of TMA in glomerulus, which presented as duplication of the glomerular basement membrane (GBM), endothelial swelling, mesangiolysis and intraglomerular thrombi, along with swelling of the tubular epithelium, and a patchy infiltration of lymphocytes and monocytes was present within the interstitial area, with interstitial fibrosis. ANA, serum complement level, H factor and ADAMTS 13 activity were normal. There was no evidence of any bacterial and viral infection, including HIV, as well as the effect of drugs or toxic exposure. Glucocorticoid, plasmapheresis and antihypertensive drugs (including RAASi) were used for TMA therapy. However, although hemolysis was rapidly controlled with normalization of the LDH and platelet count, his kidney injury was still progressive to end-stage kidney disease. In addition, he also presented with renal tubular acidosis and persistent hyperkalemia. This phenomenon forced us to continue searching for the cause of TMA.

The intellectually disabled sister in family history and a marked rise in serum level of HCY (247.8 µmol/L) put the focus on cobalamin C metabolism. A higher level of urine HCY (65.71 µmol/L) supported our suspicion. Nutritional B12 deficiency was excluded by a normal serum level. However, chromatographic evaluation of the urine showed methylmalonic aciduria. A cobalamin C (cblC) deficiency presented with MMA-uria and hyperhomocysteinaemia was subsequently confirmed by genetic testing of the two heterozygous likely pathogenic mutations of c.1 A > G (p.Met1?) (ACMG classification: likely pathogenic) and c.80 A > G (p.Q27R) (ACMG classification: likely pathogenic) in the MMACHC gene. The same genetic mutation was also found in the blood sample of his mother and father, respectively.

After a definite diagnosis of cblC deficiency, the patient was treated with hydroxocobalamin, betaine, calcium folinate and L-carnitine. After one month of therapy, serum HCY decreased to 83 µmol/l, and urine HCY decreased to 28 µmol/l, while the patient was still hemodialysis-dependent. It was reassuring that after 10 months of drug therapy, the level of hemoglobin, platelet and LDH recovered. Furthermore, this patient was finally dialysis-free with the level of SCr maintained at185 µmol/L.

Discussion and conclusions

Combined MMA-uria and hyperhomocysteinaemia, cobalamin C (cblC) type, is the most common genetic type of functional cobalamin deficiency. This metabolic disorder is characterized by marked heterogeneity of the neurocognitive disease and variable extra-central nervous system involvement, including renal, cardiovascular and ocular [1]. Neonates typically show lethargy, seizures and muscular hypotonia. Visual impairment such as nystagmus on the background of retinopathy or optic atrophy exhibit in up to 50% of infants. Nevertheless, older children, adolescents and adults may present with acute or chronic behavioral or psychiatric, cognitive impairment, signs of peripheral neuropathy and ataxia or rarely venous thromboembolism [2]. The pathophysiology of cblC deficiency is not comprehensively understood and it is likely that the synergistic effect of different mechanisms [7]. Previous studies illustrated that cobalamin C defect causes the impaired conversion of dietary cobalamin into its two metabolically active forms, methylcobalamin and adenosylcobalamin. Methylcobalamin is the cofactor for methionine synthase, which catalyzes the remethylation of homocysteine into methionine in the cytoplasm and is also responsible for the regeneration of methionine from homocysteine. Adenosylcobalamin is the cofactor for the methylmalonyl-CoA mutase, which converts methylmalonyl-CoA into succinyl-CoA in mitochondria. When these two reactions are impaired, accumulation of homocysteine and MMA could appear, as well as the reduction synthesis of methionine [7,8,9]. Toxicity of methylmalonate and homocysteine may play a crucial role in mitochondrial energy metabolism and vascular endothelial injury, which is responsible for the multisystem organ involvement [5, 7].

We have herein described three patients, who were presented with consistent clinical features of proteinuria and hematuria, and renal function ranged from normal to kidney dysfunction in their teenage. Renal biopsies of all 3 cases were presented as glomerular lesions of TMA and acute tubular injury at the same time (Table 1). Even though there was no evidence of MAHA in patient 2, we could still find TMA lesions in his kidney. Hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are the 2 most frequent causes of TMA. Other causes of TMA include malignant hypertension, infection, autoimmune diseases, or drugs that also should be considered for differentiation of TMA. However, certain laboratory investigations, such as infectious, endocrinological, and immunological factors were performed to exclude the above potential causes. In a previous study, Lemoine M et al. revealed arteriolar and glomerular TMA in cblC patients and showed that GBM vacuoles and IgM deposition were significantly more frequent, compared to a control population with cblC-independent TMA [5]. In the present study, we showed typical glomerular TMA lesions with thickening of capillary wall in all patients and intraglomerular thrombi in one patient, and all of them presented typical tubular necrosis. Obviously, in all these cases, the homocysteine was elevated significantly, which could be a clue for the differential diagnosis of cblC. The diagnosis of cblC disease is mainly based on biochemical hallmarks of elevated plasma and urine methylmalonic acid, hyperhomocysteinaemia, low blood methionine and genetic testing. c.80 A > G and c.609G > A were two common mutations in Chinese cblC type patients and were reported to be associated with late-onset cblC disease [10]. This deficiency is a treatable disease and timely recognition of cblC deficiency is of utmost importance for treatment. Patients with elevation of biochemical hallmarks that are associated with severe disease should be treated immediately and the acute management of patients even cannot wait for the results of genetic analyses. The supplements of hydroxocobalamin, betaine, and L-carnitine in our three patients resulted in a significant decrease in serum HCY and urinary protein, as well as significant kidney function improvement. One patient reached complete kidney function recovery at the last follow-up and the other patient still remains CKD but not dependent on dialysis.

Table 1 Clinical characteristics of the 3 cases at initial presentation

Two distinct phenotypes have been defined in terms of age of onset. Early-onset patients present in infants with multisystem disorders, including neurological, hematological abnormalities, ocular, renal, gastrointestinal, cardiac, and pulmonary manifestations and the survivors may have severe neurological impairment [11, 12]. Late-onset cblC disease was recognized in the 1980s (Shinnar and Singer 1984) and the term has been historically applied to patients that have overt symptoms after 4 years of age [4]. Patients usually present with a milder clinical phenotype characterized by acute or slowly progressive neurological symptoms, behavioral disturbances, and chronic kidney disease [7, 12]. HUS and pulmonary hypertension are often the main presenting symptoms in preschool children with late onset cblC. About 90% of patients which have been reported had an early onset of disease, but the late onset form is much rarer. Kalantari et al. presented an emblematic case of adult-onset cblC deficiency with the age of 45 at the onset of disease, who is the oldest patient reported up to now [13]. Despite therapeutic improvements in the last decades, chronic organ manifestations occur in many patients [12], especially in interrupted or irregular therapy. Patients with renal disease mostly had clinical features of hematuria and proteinuria (also up to the nephrotic range), hypertension (also malignant hypertension), chronic renal failure (from mild to severe), even some with intravascular hemolysis. Kalantari and his colleagues also performed a systemic review including 45 adult onset patients, to find glomerular nephropathy, kidney failure, proteinuria and HUS still common in renal manifestation [13]. Renal biopsies predominantly demonstrate thrombotic microangiopathy [1, 12], including glomerular fibrin thrombi, endothelial swelling, and duplication of the glomerular basement membrane, but also membranous nephropathy associated to cblC disease has been reported recently [14]. We present a summary of clinical and pathology findings of cblC disease with renal involvement in recent reviews and case reports (Table 2). The etiology of TMA in patients with cobalamin C defects remains unknown. It has been demonstrated that hyperhomocysteinaemia could induce damage to endothelial cells [1, 19]. Impairment of the nitric oxide-dependent inhibition of platelet aggregation or the procoagulant state of the endothelium leads to the formation of microthrombi [20].

Table 2 Clinical and pathological impairment of cblC disease with renal involvement

In patients with isolated MMA, chronic tubulointerstitial nephritis with progressive impairment of renal function could be observed [12]. Some patients with isolated MMA have proximal renal tubular acidosis [21]. However, cblC is hardly reported with renal tubular injury, like tubulointerstitial nephritis and interstitial fibrosis. Our three patients all had clinical evidence of renal tubular injury, which may lead to dysfunction of urine concentration and/or acidification. Kidney biopsy findings also confirmed tubulointerstitial nephritis with inflammation and patchy interstitial fibrosis, which is presumably associated with toxic metabolites interfering with intrarenal mitochondrial metabolism [22]. Since the urinary excretion of MMA and other putatively toxic metabolites of alternative propionate oxidation (e.g. 2-methylcitrate) diminishes with decreasing kidney function and thus accumulating toxic compounds could foster the naturally occurring disease progression despite hydroxocobalamin supplementation [23]. However, we assumed that these tubulointerstitial changes were too mild to account for the severity of the renal disease.

Treatment strategies usually consist of a combined approach including utilizing hydroxycobalamin to increase intracellular cobalamin and maximize deficient enzyme activities, betaine to provide a substrate for the conversion of homocysteine into methionine through betaine-homocysteine methyltransferase, and folic acid to enhance remethylation pathways [12]. However, there is no proven efficacy for carnitine and methionine supplementation, and dietary protein restriction in these patients [7]. Urinary methylmalonic acid excretion was often used to assess cobalamin responsiveness before and after administration of cobalamin [24]. The reported outcomes of MMA cases vary in the literature and range from insignificant to initiation of renal replacement therapy (RRT), which depends mainly on the type of disease, the onset, and clinical compliance [25]. Mortality was higher in infants (57%) compared with later-onset disease (35%), resulting in an overall mortality rate of 44% [1]. Some patients experienced complete recovery of kidney function, while the others had residual kidney dysfunction with persistent proteinuria and hematuria [25, 26] or some remaining dialysis-dependent [1]. However, when recognized and treated at an early stage, complete clinical recovery is possible.

The clinical diagnosis of cblC disease associated with kidney injury should be considered in patients with unclear TMA accompanied with a high concentration of serum homocysteine, even in teenagers or adults. Dosage of MMA and methionine should be tested to complete diagnostic work-up and to discriminate among the different forms of remethylation disorders. MMA in (fresh or optimally stored) urine or blood is the most sensitive intracellular marker. However, limitation should be noticed that urinary MMA is unreliable in individuals with renal functional impairment [2]. Additionally, it is significant to remind that microangiopathic nephropathy can be present in the complete absence of the laboratory alterations typical of TMA [13]. From a diagnostic standpoint, it would be important to perform genetic analysis in patients with a suspected intracellular cobalamin disorder, once homocysteine, methionine and MMA levels have been measured. Awareness should be taken for this rare, but treatable disease.