Heterozygous COL4A3/COL4A4 mutations: the hidden part of the iceberg?

BACKGROUND: Single mutations in COL4A3/COL4A4 genes have been described in patients with autosomal dominant Alport syndrome and thin basement membrane nephropathy, without a shared definition of these patients within the medical community. We aimed to better categorize this clinical entity by examining clinical manifestations, family history, pathological features and genetics. METHODS: We retrospectively analyzed patients with causative heterozygous COL4A3/COL4A4 mutations referred to us between 1990 and 2019. Index cases were defined as children who were the first to be diagnosed in their families. RESULTS: The study included 24 index cases and 29 affected relatives, belonging to 25 families with a heterozygous mutation in the COL4A3/COL4A4 genes. During the follow-up, nine patients developed proteinuria [median age 15.7 years (range 5.6-33)], six at clinical diagnosis and four with progression toward chronic kidney disease (CKD) (three required kidney replacement therapy at 25, 45 and 53 years and one had CKD Stage 2 at 46 years). Extrarenal involvement was observed in 24.5% of patients. Hematuria was transmitted in consecutive generations, while CKD was reported in nonconsecutive generations of 11 families [median age 53 years (range 16-80)]. Seventeen patients (32%) underwent kidney biopsy: findings were consistent with Alport syndrome in 12 cases and with thin basement membrane nephropathy in 5 cases. CONCLUSIONS: Despite the benign course for these patients described in the literature, a significant percentage is at risk for disease progression. Consequently, we suggest that the assessment of these patients must take into account family history, genetic analysis and pathologic findings. After comparison with the literature, our data suggest that a different definition for Alport syndrome must be considered.

Vestibular and audiological findings in the Alport syndrome.

Alport syndrome (AS) is caused by mutations in collagen IV, which is widespread in the basement membranes of many organs, including the kidneys, eyes, and ears. Whereas the effects of collagen IV changes in the cochlea are well known, no changes have been described in the posterior labyrinth. The aim of this study was to investigate both the auditory and the vestibular function of a group of individuals with AS. Seventeen patients, aged 9-52, underwent audiological tests including pure-tone and speech audiometry, immittance test and otoacoustic emissions and vestibular tests including video head impulse test, rotatory test, and vestibular evoked myogenic potentials. Hearing loss affected 25% of the males and 27.3% of the females with X-linked AS. It was sensorineural with a cochlear localization and a variable severity. 50% of the males and 45.4% of the females had a hearing impairment in the high-frequency range. Otoacoustic emissions were absent in about one-third of the individuals. A peripheral vestibular dysfunction was present in 75% of the males and 45.4% of the females, with no complaints of vertigo or dizziness. The vestibular impairment was compensated and the vestibulo-ocular reflex asymmetry was more evident in rotatory tests carried out at lower than higher speeds; a vestibular hypofunction was present in all hearing impaired ears although it was also found in subjects with normal hearing. A posterior labyrinth injury should be hypothesized in AS even when the patient does not manifest hearing disorders or evident signs of renal failure.

Alport syndrome and pregnancy: a case series and literature review.

PURPOSE: To assess pregnancy outcome in women with Alport syndrome and the impact of pregnancy on the disease progression. METHODS: We describe one of the largest series of pregnancies in Alport syndrome. Seven pregnancies of six women were monitored by a multidisciplinary team of nephrologists and gynecologists. After delivery, patients were followed for at least 3 years. We compare our results with those in the literature. RESULTS: Pregnancy course was uneventful in the patient with isolated microscopic hematuria. In the other cases, all presenting mild proteinuria at conception, some complications occurred. Proteinuria worsened during the last trimester, reaching nephrotic ranges in five out of six pregnancies and was associated with fluid overload leading to hospitalizations and early delivery. The majority of the newborns had a low birth weight. The two patients with arterial hypertension at conception and twin pregnancy developed pre-eclampsia and renal function deterioration persisted after delivery. The one with pre-pregnancy renal dysfunction reached end-stage renal disease. In the other patients, in which renal function and blood pressure were and remained normal, proteinuria improved after delivery and no signs of disease progression were recorded at last observation. CONCLUSIONS: Our observations suggest that Alport syndrome should be considered a potential risk factor for pregnancy in proteinuric patients due to the development of pre-eclampsia, renal function deterioration, and/or full-blown nephrotic syndrome that results in anasarca, slowing of fetal growth and pre-term delivery. Thus, all women with Alport syndrome should receive pre-conceptional counseling and be kept in close follow-up during pregnancy.

Alport syndrome: the effects of spironolactone on proteinuria and urinary TGF-ß1.

BACKGROUND: Alport syndrome (AS) is a progressive hereditary glomerular disease. Recent data indicate that aldosterone promotes fibrosis mediated by the transforming growth factor-ß1 (TGF-ß1) pathway, which may worsen proteinuria. Spironolactone (SP) antagonizes aldosterone and this study aimed to evaluate the efficacy of SP in reducing proteinuria and urinary TGF-ß1 excretion in proteinuric AS patients. METHODS: The study involved ten children with AS, normal renal function, and persistent proteinuria (>6 months; uPr/uCr ratio >1). SP 25 mg once a day for 6 months was added to existing ACE inhibitor treatment with or without angiotensin-II receptor blockade. Urine and blood samples were examined monthly. Urinary TGF-ß1 levels were measured twice before and three times during SP treatment. Plasma renin activity (PRA) and serum aldosterone levels were also measured. In eight patients, uProt/uCreat was also assessed after 9 months and 12 months of SP treatment. RESULTS: After beginning SP therapy, all patients showed significant decrease in mean uProt/uCreat ratio (1.77 ± 0.8 to 0.86 ± 0.6; p < 0.001) and mean urinary TGF-ß1 levels (104 ± 54 to 41 ± 20 pg/mgCreatinine; p < 0.01), beginning after 30 days of treatment and remaining stable throughout SP administration. PRA remain unchanged, and mean serum aldosterone increased from 105 ± 72 pg/ml to 303 ± 156 pg/ml (p < 0.001). The only side effect was gynecomastia in an obese boy. After 1 year of therapy, mean uProt/uCreat remains low (0.82 ± 0.48). CONCLUSIONS: Addition of SP to ACE-I treatment with or without angiotensin II receptor blokers (ARB) significantly reduced proteinuria. This was mediated by decreased urinary TGF-ß1 levels and not associated with major side effects.

Single, Double and Triple Blockade of RAAS in Alport Syndrome: Different Tools to Freeze the Evolution of the Disease.

BACKGROUND: The goal of the treatment of Alport syndrome (AS) is to delay the progression of kidney damage. The current standard of care is the use of Renin Angiotensin Aldosterone System (RAAS) blockers: angiotensin-converting enzyme inhibition (ACEi), angiotensin receptor blockade, and, recently, spironolactone (SP). AIM OF THE STUDY: the purpose of this retrospective study is to evaluate the efficacy (reduction of proteinuria and changes of glomerular function) and safety of a sequential introduction of RAAS blockers up to a triple RAAS blockade in pediatric proteinuric patients with AS. METHODS: in this retrospective study (1995 to 2019), we evaluated proteinuria values in AS patients, during the 12 months following the beginning of a new RAAS blocker, up to a triple blockade. ACEi was always the first line of treatment; then ARB and SP were sequentially added if uPCR increased by 50% from the basal level in 2 consecutive samples during a 3-months observation period, or when uPCR ratio was >2 mg/mg. RESULTS: 26 patients (mean age at treatment onset was 10.55 ± 5.02 years) were enrolled. All patients were on ACEi, 14/26 were started on a second drug (6/14 ARB, 8/14 SP) after a mean time of 2.2 ± 1.7 years, 7/26 were on triple RAAS blockade after a further period of 5.5 ± 2.3 years from the introduction of a second drug. Repeated Measure Anova analysis of log-transformed data shows that the reduction of uPCR values after Time 0 from the introduction of the first, second and third drug is highly significant in all three cases (p values = 0.0016, 0.003, and 0.014, respectively). No significant changes in eGFR were recorded in any group, apart from a 15-year-old boy with X-linked AS, who developed kidney failure. One patient developed mild hyperkaliemia, and one gynecomastia and symptomatic hypotension. No life-threatening events were recorded. CONCLUSIONS: double and triple RAAS blockade is an effective and safe strategy to reduce proteinuria in children with AS. Nevertheless, we suggest monitoring eGFR and Kaliemia during follow-up.

Successful medical treatment of EBV smooth muscle tumor in a renal transplant recipient.

EBV is associated with various malignancies in patients with acquired or induced immune impairment. EBV-SMT is very uncommon in immunocompromised patients, and a kidney localization has been described only anecdotally. We report the case of a 17-yr-old kidney transplant recipient diagnosed as having an EBV-SMT inside the renal graft, which was successfully managed by minimizing isolated immunosuppression.

Clinical and Pathophysiological Insights Into Immunological Mediated Glomerular Diseases in Childhood.

The kidney is often the target of immune system dysregulation in the context of primary or systemic disease. In particular, the glomerulus represents the anatomical entity most frequently involved, generally as the expression of inflammatory cell invasion or circulant or in situ immune-complex deposition. Glomerulonephritis is the most common clinical and pathological manifestation of this involvement. There are no universally accepted classifications for glomerulonephritis. However, recent advances in our understanding of the pathophysiological mechanisms suggest the assessment of immunological features, biomarkers, and genetic analysis. At the same time, more accurate and targeted therapies have been developed. Data on pediatric glomerulonephritis are scarce and often derived from adult studies. In this review, we update the current understanding of the etiologic events and genetic factors involved in the pathogenesis of pediatric immunologically mediated primitive forms of glomerulonephritis, together with the clinical spectrum and prognosis. Possible new therapeutic targets are also briefly discussed.

X-Linked Alport Syndrome in Women: Genotype and Clinical Course in 24 Cases.

Objectives: X-linked Alport syndrome (XLAS) females are at risk of developing proteinuria and chronic kidney damage (CKD). The aim of this study is to evaluate the genotype-phenotype correlation in this rare population. Materials and Methods: This is a prospective, observational study of XLAS females, confirmed by a pathogenic mutation in COL4A5 and renal ultrastructural evaluation. Proteinuria, renal function and extrarenal involvement were monitored during follow-up. Patients were divided in 2 groups, according to mutations in COL4A5: missense (Group 1) and non-missense variants (Group 2). Results: Twenty-four XLAS females, aged 10.6 ± 10.4 years at clinical onset (mean follow-up: 13.1 ± 12.6 years) were recruited between 2000 and 2017 at a single center. In group 1 there were 10 patients and in group 2, 14 (mean age at the end of follow-up: 24.9 ± 13.6 and 23.2 ± 13.8 years, respectively). One patient in Group 1 and 9 in Group 2 (p = 0.013) developed proteinuria during follow-up. Mean eGFR at last follow-up was lower in Group 2 (p = 0.027), where two patients developed CKD. No differences in hearing loss were documented among the two groups. Two patients in Group 2 carried one mutation in both COL4A5 and COL4A3 (digenic inheritance) and were proteinuric. In one family, the mother presented only hematuria while the daughter was proteinuric and presented a greater inactivation of the X chromosome carrying the wild-type allele. Conclusions: The appearance of proteinuria and CKD is more frequent in patients with severe variants. Carrying digenic inheritance and skewed XCI seem to be additional risk factors for proteinuria in XLAS females.

Alport syndrome cold cases: Missing mutations identified by exome sequencing and functional analysis.

Alport syndrome (AS) is an inherited progressive renal disease caused by mutations in COL4A3, COL4A4, and COL4A5 genes. Despite simultaneous screening of these genes being widely available, mutation detection still remains incomplete in a non-marginal portion of patients. Here, we applied whole-exome sequencing (WES) in 3 Italian families negative after candidate-gene analyses. In Family 1, we identified a novel heterozygous intronic variant (c.2245-40A>G) -outside the conventionally screened candidate region for diagnosis- potentially disrupting COL4A5 exon29 splicing. Using a minigene-based approach in HEK293 cells we demonstrated that this variant abolishes exon29 branch site, causing exon skipping. Moreover, skewed X-inactivation of the c.2245-40A>G allele correlated with disease severity in heterozygous females. In Family 2, WES highlighted a novel COL4A5 hemizygous missense mutation (p.Gly491Asp), which segregates with the phenotype and impacts on a highly-conserved residue. Finally, in Family 3, we detected a homozygous 24-bp in-frame deletion in COL4A3 exon1 (NM_000091.4:c.30_53del:p.Val11_Leu18del or c.40_63del24:p.Leu14_Leu21del), which is ambiguously annotated in databases, although it corresponds to a recurrent AS mutation. Functional analyses showed that this deletion disrupts COL4A3 signal peptide, possibly altering protein secretion. In conclusion, WES -together with functional studies- was fundamental for molecular diagnosis in 3 AS families, highlighting pathogenic variants that escaped previous screenings.

Heavy chain myosin 9-related disease (MYH9 -RD): neutrophil inclusions of myosin-9 as a pathognomonic sign of the disorder.

MYH9-related disease ( MYH9-RD) is an autosomal dominant thrombocytopenia with giant platelets variably associated with young-adult onset of progressive sensorineural hearing loss, presenile cataract, and renal damage. MYH9-RD is caused by mutations of MYH9 , the gene encoding for non-muscle heavy-chain myosin-9. Wild-type and mutant myosin-9 aggregate as cytoplasmic inclusions in patients' leukocytes, the identification of which by immunofluorescence has been proposed as a suitable tool for the diagnosis of MYH9-RD. Since the predictive value of this assay, in terms of sensitivity and specificity, is unknown, we investigated 118 consecutive unrelated patients with a clinical presentation strongly consistent with MYH9-RD. All patients prospectively underwent both the immunofluorescence assay for myosin-9 aggregate detection and molecular genetic analysis of the MYH9 gene. Myosin-9 aggregates were identified in 82 patients, 80 of which (98%) had also a MYH9 mutation. In the remaining 36 patients neither myosin-9 aggregates nor MYH9 mutations were found. Sensitivity and specificity of the immunofluorescence assay was evaluated to be 100% and 95%, respectively. Except for the presence of aggregates, we did not find any other significant difference between patients with or without aggregates, demonstrating that the myosin-9 inclusions in neutrophils are a pathognomonic sign of the disease. However, the identification of the specific MYH9 mutation is still of importance for prognostic aspects of MYH9-RD.

Urine erythrocyte morphology in patients with microscopic haematuria caused by a glomerulopathy.

The evaluation of urinary erythrocyte morphology (UEM) has been proposed for patients with isolated microscopic haematuria (IMH) to early orientate the diagnosis towards a glomerular or a nonglomerular disease. However, to date, the role of this test in patients with IMH has very rarely been investigated. Sixteen patients (ten children, six adults) with persistent IMH classified as glomerular on the basis of repeated UEM evaluations (55 urine samples, two to eight per patient) were submitted to renal biopsy. This showed a glomerular disease in 14/16 patients (87.5%) (nine thin basement membrane disease; three Alport syndrome; two other), whereas in two patients, no abnormalities were found. Of four microscopic criteria investigated to define a IMH as glomerular, >80% dysmorphic erythrocytes were not found in any sample, >or=40% dysmorphic erythrocytes alone were seen in seven samples (12.7%), >or=5% acanthocytes alone in 15 samples (27.3%) and erythrocytic casts in six samples (10.9%). There was >or=40% dysmorphic erythrocytes associated with >or=5% acanthocytes in 25 samples (45.5%). Sensitivity and positive predictive values in diagnosing a glomerular haematuria were 59.2% and 90.6%, respectively, for >or=40% dysmorphic erythrocytes, 69.4% and 85% for >or=5% acanthocytes/G1 cells and 12.2% and 100% for erythrocytic casts. Our findings demonstrate that the evaluation of UEM is useful to identify patients with an IMH of glomerular origin.

Autosomal recessive Alport syndrome: an in-depth clinical and molecular analysis of five families.

BACKGROUND: Alport syndrome (ATS) is a progressive inherited nephropathy characterized by irregular thinning, thickening and splitting of the glomerular basement membrane (GBM) often associated with hearing loss and ocular symptoms. ATS has been shown to be caused by COL4A5 mutations in its X-linked form and by COL4A3 and COL4A4 mutations in its autosomal forms. METHODS: Five families with a suspicion of ATS were investigated both from a clinical and molecular point of view. COL4A3 and COL4A4 genes were analysed by DHPLC. Automated sequencing was performed to identify the underlying mutation. RESULTS: Molecular analysis indicated that in all 5 cases the correct diagnosis was autosomal recessive ATS. In three families in which parental consanguinity clearly pinpointed to autosomal recessive ATS, we found COL4A4 homozygous mutations in two of them and COL4A3 homozygous mutation in the other one. In the remaining two families a differential diagnosis including X-linked ATS, autosomal recessive ATS and thin basement membrane nephropathy was considered. The molecular analysis demonstrated that the probands were genetic compounds for two different mutations in the COL4A4 gene pinpointing to the correct diagnosis of autosomal recessive ATS. CONCLUSIONS: A clinical evaluation of probands and their relatives of the five families carrying mutations in either the COL4A3 or the COL4A4 gene was carried out to underline the natural history of the autosomal recessive ATS. In addition, this paper stresses the complexity of the clinics and genetics of ATS and how a correct diagnosis is based on a combination of: (i) an in-depth clinical investigation; (ii) a detailed formal genetic analysis; (iii) a correct technical choice of the gene to be investigated; (iv) a correct technical choice of the family member to be included in the mutational screening. A correct diagnosis is the basis for an appropriate genetic counselling dealing with both the correct prognosis and the accurate recurrence risk for the patients and family members.

Simultaneous mutations in the CLCNKB and SLC12A3 genes in two siblings with phenotypic heterogeneity in classic Bartter syndrome.

Two siblings (brother and sister) with renal tubular hypokalemic alkalosis underwent clinical, biochemical and molecular investigations. Although the biochemical findings were similar (including hypokalemia, metabolic alkalosis, hyperreninemia, hyperaldosteronism and normal blood pressure), the clinical findings were different: the boy, who also presented syndromic signs, developed glomerular proteinuria and renal biopsy revealed focal segmental glomerular sclerosis; the girl showed the typical signs of classic Bartter syndrome. As described in a previous paper, a heterozygous mutation (frameshift 2534delT) was demonstrated in the gene encoding the thiazide-sensitive NaCl co-transporter (SLC12A3) of the distal convoluted tubule; the second molecular analysis revealed a compound heterozygous mutation (A61D/V149E) in the CLCNKB chloride channel gene in both subjects, inherited in trans from the parents. The children were finally diagnosed as having classic Bartter syndrome. These cases represent the first report of the simultaneous presence of heterozygous and compound heterozygous mutations in the SLC12A3 and CLCNKB genes, both of which are involved in renal salt losing tubulopathies, and confirm previous observations regarding classic Bartter syndrome phenotype variability in the same kindred.