Received: 12 January 2017
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Revised: 3 April 2017
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Accepted: 4 April 2017
DOI: 10.1002/jca.21549
CASE REPORT
An extremely rare splice site mutation in the gene encoding complement factor I in a patient with atypical hemolytic uremic syndrome Tina S. Ipe1
| Jooeun Lim2 | Meredith Anne Reyes3 | Mike Ero2 |
Christopher Leveque1 | Bradley Lewis2 | Jamey Kain2 1
Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, Texas 2
Machaon Diagnostics, Oakland, California 3 Department of Pathology and Laboratory Medicine, Baylor College of Medicine, Houston, Texas
Correspondence Tina S. Ipe, Department of Pathology and Genomic Medicine, Houston Methodist Hospital, 6565 Fannin St., MS205, Houston, TX 77030, USA. Email:
[email protected]
Abstract Background: Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney failure. The disease is difficult to diagnose due to its similarity with other hematologic disorders, such as thrombotic thrombocytopenic purpura (TTP). However, genetic mutations are found in 50–70% of patients with aHUS and can be useful in its diagnosis. Study design and methods: A 40-year-old male presented to our hospital with acute kidney injury, evidenced by high creatinine levels (8.3 mg/dL) and kidney biopsy results. The patient was preliminarily diagnosed with TTP and therapeutic plasma exchange (TPE) was initiated. After four treatments, TPE was discontinued due to lack of ADAMTS13 activity and inhibitor assay results that were not consistent with TTP, improved hematologic laboratory results, and aHUS genetic testing results. Results: Next-generation sequencing showed a rare mutation at a splice site in the gene encoding complement factor I (CFI). Implication of this mutation in aHUS has not been previously described. Treatment with eculizumab reduced creatinine levels below 4.0 mg/dL, and the patient remained on maintenance dosage of eculizumab (1200 mg/14 days) to prevent aHUS recurrence. Conclusion: An extremely rare, heterozygous mutation in the gene encoding CFI likely affecting splicing was associated for the first time with aHUS. Sequencing was critical for rapid diagnosis and subsequent timely treatment with eculizumab, which resulted in improved renal function. KEYWORDS aHUS, CFI, eculizumab, novel mutation, thrombotic microangiopathy
1 | CASE PRESENTATION Atypical hemolytic uremic syndrome (aHUS), also known as complement-mediated thrombotic microangiopathy (TMA), is a rare, life-threatening, chronic disease with an incidence of 2 cases per million in adults.1,2 A triggering event (eg, J Clin Apher. 2017;1–5.
infection, transplant, pregnancy) in a susceptible person leads to inappropriate activation of complement. Patients frequently progress to end-stage renal failure, in addition to many other extra-renal symptoms, and/or death. Genetic mutations can be identified in aHUS patients in 50–70% of cases and can be helpful in confirming the diagnosis.3,4 Prior
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to rapid mutational analysis, aHUS was diagnosed by excluding the following as causes: (1) thrombotic thrombocytopenic purpura (TTP), (2) Shiga-like toxin-producing E. coli (STEC-HUS), and (3) secondary thrombotic microangiopathies due to an underlying disease (eg, malignant hypertension).5,6 Knowing the affected gene can also have some prognostic value.3 Mutations associated with aHUS—usually single nucleotide variants or small insertion/deletions (indels) —have been identified in complement factor H (CFH), membrane cofactor protein (MCP)/CD46, CFI, CFB, complement component 3 (C3), complement factor H related 5 (CFHR5), thrombomodulin (THBD), and diacylglycerol kinase-epsilon (DGKE); large deletions have been found in CFHR1, CFHR3, and CFHR4.3,7,8 Additional genes have been recently implicated, such as the gene encoding plasminogen (PLG), and more are likely to be discovered in the near future.4 A 40-year-old male presented to our hospital with acute kidney injury. Approximately 5 months prior to hospitalization, his primary care physician performed routine laboratory tests that showed elevated creatinine levels (1.7 mg/dL). The patient was advised to discontinue the use of protein supplements for muscle building. Despite discontinuing the supplements, his creatinine levels remained elevated and a kidney biopsy was performed. The biopsy revealed acute and chronic interstitial nephritis, global sclerosis, mild-tomoderate interstitial fibrosis, and moderate arteriosclerosis. The patient was treated with oral and intravenous steroids but his creatinine levels continued to rise. The patient also developed bilateral lower extremity edema, which improved with diuretics. On admission, the laboratory findings were indicative of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury (Table 1). A peripheral blood smear revealed fragmented red cells consistent with a thrombotic microangiopathy. Given the presumptive diagnosis of thrombotic thrombocytopenic purpura (TTP), therapeutic plasma exchange (TPE) with thawed plasma was performed empirically in case the patient had TTP. Daily TPE was planned until a platelet count of 150 3 103/mL was achieved for three consecutive days. Before initiating TPE, a blood sample was drawn to measure ADAMTS13 activity and an inhibitor assay was performed. In addition, a blood sample was sent for aHUS genetic panel testing. A 1.0 plasma volume exchange was performed daily with a Spectra Optia, version 11.0 (Terumo BCT) for 4 consecutive days using thawed plasma and citrate as the anticoagulant. The ratio of inlet volume to anticoagulant was 12:1. The ADAMTS13 activity was 32% with no neutralizing inhibitor. The aHUS testing using next-generation sequencing was positive for a mutation in the CFI gene (Machaon Diagnostics, Oakland, CA). In light of the ADAMTS13 activity and inhibitor assay
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Patient’s laboratory values upon hospital admission
Laboratory parameter
Patient’s results
Normal ranges
Hemoglobin
8.5 g/L
14.0–18.0 g/dL
Platelet count
71 3 103/mL
150–400 k/lL
Lactate dehydrogenase (LDH)
900 U/L
87–225 U/L
Haptoglobin
65
ADAMTS13 inhibitor
Negative
Positive/negative
results and the positive aHUS genetic testing, the stabilization of the platelet count (>120 3 103/mL), decreasing LDH (900–286 U/L), and the rising creatinine levels (from 8.3 to 11.0 mg/dL), TPE was discontinued after the fourth procedure (Figure 1A). At this point, aHUS was suspected and eculizumab treatment was initiated. Sequencing showed a rare, heterozygous splice site variant (NM_000204.3: c.1429 1 1 G > C) 1 bp downstream from the 30 end of exon 11 of the CFI gene. As the first two base pairs after the end of an exon are strictly conserved, this variant should alter splicing.9 This variant has no reported minor allele frequency in the 1000 Genomes database and a frequency of 1 3 1024 in the 5000 Exomes database; thus, this variant is extremely rare in the general population. This mutation is not listed in the FH-HUS.org database of aHUSassociated variants. We confirmed this mutation by Sanger sequencing (Figure 1B). The patient also has a heterozygous missense variant (c.3148 A > T, p.Asn1050Tyr) in exon 20 of CFH. This variant has a minor allele frequency of 0.012 (1000 Genomes database) and is classified as a non-disease-causing polymorphism in the FH-HUS.org database. This frequency is probably not rare enough to be causative given how rare aHUS is in the population. The patient is also heterozygous for two polymorphisms in CFH and five polymorphisms in MCP/ CD46 that are common in the normal population but statistically enriched in the disease population.10,11 The patient is heterozygous for the large deletion of CFHR1–CFHR3, but only the homozygous deletion has been associated with disease.11 After starting eculizumab treatment, the patient’s creatinine levels declined over a period of weeks before eventually
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F I G U R E 1 A, Patient’s creatinine levels (mg/dL) are shown. Arrows indicate TPE treatments; arrowhead indicates initiation of eculizumab treatment. B, Chromatogram data showing Sanger DNA sequencing confirmation of the CFI splice site mutation. Note the presence of a single peak in the control (top panel) versus two peaks in the patient sample (bottom panel, blue shading). The amino acid sequence of CFI is shown along the bottom, the red line indicates the conserved splice site (GT), and the blue line indicates the intronic sequence
stabilizing below 4.0 mg/dL. Owing to the suspicious nature of the CFI splice site mutation, the patient has remained on maintenance dosage of eculizumab (1200 mg every 14 days) to prevent the recurrence of aHUS. A biopsy done approximately 4 months after initiation of treatment showed the presence of ongoing tubular injury and repair, consistent with chronic thrombotic microangiopathy. His creatinine continues to be elevated at 4 mg/dL despite normalization of LDH and platelet counts.
2 | DISCUSSION In patients presenting with signs and symptoms of TMAs, specifically diagnosing aHUS can be challenging because its clinical presentation is similar to other systemic TMAs, such as TTP. However, it is important to identify the underlying disease etiology for timely and targeted treatment. Using lab-
oratory results such as ADAMTS13 can help in differentiating the underlying disease etiology mediating the TMA. The ADAMTS13 test was ordered in our patient prior to the initiation of plasma exchange and while the activity was decreased, it was not severely decreased (
