Pathological and sonographic review of early isolated severe lower urinary tract obstruction and implications for prenatal treatment.

OBJECTIVE: To identify favorable renal histology in fetuses with early severe lower urinary tract obstruction (LUTO) and determine the best timing and selection criteria for prenatal surgery. METHODS: This multicenter, retrospective study included male fetuses with severe LUTO which died before 24 weeks of gestation during the period January 2000 to December 2018. Age-matched controls were used as reference standard for renal histology. Prenatal ultrasound features and fetal serum and/or urine ß2microglobulin level were retrieved and kidney histology slides (hematein-eosin-safran and α-smooth-muscle-actin (αSMA) immunostaining) were prepared and reviewed. αSMA-positive staining of the blastema is due to its aberrant differentiation into myofibroblastic cells. Cases were sorted into histopathologic groups (favorable or unfavorable) according to the blastema's morphology and αSMA labeling and the data of these groups were compared. RESULTS: Included in the study were 74 fetuses with a median gestational age at outcome of 17 + 6 (range, 13 + 0 to 23 + 5) weeks. Parenchymal organization was preserved in 48% of the kidneys. A blastema was present in 90% of the kidneys, but it was morphologically normal in only 9% and αSMA-negative in only 1% of them. Most (82%) fetuses had an unfavorable prognosis, and 36% of fetuses died ≤ 18 weeks and had severe renal lesions detected on histology (early unfavorable prognosis). A favorable renal prognosis was associated with an earlier gestational age (P = 0.001). Fetuses with LUTO had a significantly lower number of mature glomeruli (P < 0.001) compared with controls. However, there was no significant difference in the number of glomeruli generations between the early-unfavorable-prognosis group (≤ 18 weeks) and the group with a favorable prognosis (P = 0.19). A comparison of prenatal ultrasound features and biochemical markers between groups could not identify any prenatal selection criteria. CONCLUSIONS: Before 18 weeks, around 30% of fetuses with severe LUTO still have potential for kidney development. Identification of these cases would enable them to be targeted for prenatal therapy. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Sequential fetal serum ß2-microglobulin to predict postnatal renal function in bilateral or low urinary tract obstruction.

OBJECTIVE: Fetal serum ß2-microglobulin has been shown to predict postnatal renal outcome in cases of fetal obstructive uropathy. We assessed the value of serial measurements of fetal serum ß2-microglobulin in the prediction of postnatal renal outcome. METHODS: We retrospectively studied renal outcome in 42 fetuses with bilateral or low urinary tract obstruction that had fetal blood sampling on at least two occasions to assay serum levels of ß2-microglobulin. Amniotic fluid volume at the time of each sampling was recorded. We classified renal outcome as either favorable (when postnatal renal function was normal) or adverse (when postnatal chronic renal failure occurred or when renal dysplasia at autopsy was noted). A ß2-microglobulin cut-off of 5 mg/L and amniotic fluid index of 5 cm were used to predict postnatal renal outcome. RESULTS: Renal outcome was adverse in 28 cases and favorable in 14. In 12 (28.6%) cases, fetal serum ß2-microglobulin concentration differed between the first and last measurement. Prediction of postnatal renal outcome was correct in 11 of these cases based on the last ß2-microglobulin measurement. The sensitivity of ß2-microglobulin in predicting renal outcome was significantly higher (P = 0.005) when using the last rather than the first measurement (96.4% vs 64.3%), with similar specificity for both measurements (85.7% vs 78.6%, non-significant). The sensitivity of amniotic fluid volume was also significantly higher (P = 0.005) when using the last rather than the first measurement (75.0% vs 35.7%), with similar specificity for both measurements (64.3% vs 71.4%, non-significant). CONCLUSION: Sequential measurement of serum ß2-microglobulin, performed for adverse ultrasound findings, such as renal parenchymal abnormality or decreasing amniotic fluid volume, predicts postnatal renal outcome more accurately than does a single assay. This may be due to possible worsening of renal injury with increasing duration of urinary tract obstruction. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Benign familial hematuria associated with a novel COL4A4 mutation.

We describe a father and three offspring with hematuria. The father and one girl also complained of flank pain. Renal function tests and ophthalmological examinations were normal in all. The father had very mild neural deafness. The renal biopsy samples of two affected siblings showed changes compatible with thin basement membrane disease. Genetic analysis revealed a novel missense mutation in exon 32 of COL4A4 to be responsible for the phenotype in this family. We suggest that thin basement membrane disease may have overlapping clinical features with other causes of hematuria; genetic analysis may help in the differential diagnosis and help us further understand the disease processes.

Diffuse esophageal leiomyomatosis with perirectal involvement mimicking Hirschsprung disease.

We describe a 25-year-old woman with diffuse esophageal leiomyomatosis. During childhood, achalasia was mistakenly diagnosed in this patient. Subsequently, she underwent cardiomyotomy and developed symptoms of Hirschsprung disease. These symptoms were caused by infiltration of the esophageal and rectal walls by benign muscular hypertrophy. The pseudo-Hirschsprung disorder was manifested by chronic severe constipation, with consistent manometric findings. Clitoral hypertrophy and vulvar and periurethral leiomyoma were also present. Genetic analysis demonstrating deletion of the COL4A5/COL4A6 locus and the discovery of microscopic hematuria implied that the patient could transmit both diffuse leiomyomatosis and the Alport syndrome.

Autosomal dominant Alport syndrome caused by a COL4A3 splice site mutation.

BACKGROUND: Alport syndrome (AS) is a clinically and genetically heterogeneous renal disorder, predominantly affecting the type IV collagen alpha 3/alpha 4/alpha 5 network of the glomerular basement membrane (GBM). AS can be caused by mutations in any of the three genes encoding these type IV collagen chains. The majority of AS families (85%) are X-linked (XL-AS) involving mutations in the COL4A5 gene. Mutations in the COL4A3 and COL4A4 genes cause autosomal recessive AS (AR-AS), accounting for approximately 14% of the cases. Recently, autosomal dominant AS (AD-AS) was linked to the COL4A3/COL4A4 locus in a large family. METHODS: COL4A3 and COL4A4 cDNAs were generated by nested reverse transcription-polymerase chain reaction and were analyzed by DNA sequence analysis. Denaturating high-performance liquid chromatography (DHPLC) was used for mutation and segregation analysis at the genomic DNA level. RESULTS: In the AD-AS family, a splice site mutation resulting in skipping of exon 21 of the COL4A3 gene was detected. The mutation does not alter the reading frame and is predicted to result in a COL4A3 chain with an internal deletion. CONCLUSION: As the NC domain is intact, this chain may be incorporated and distort the collagen triple helix, thereby causing the dominant effect of the mutation. The finding of a specific COL4A3 mutation in AD-AS completes the spectrum of type IV collagen mutations in all genetic forms of AS.

Glomerular expression of type IV collagen chains in normal and X-linked Alport syndrome kidneys.

Alport syndrome is an inherited nephropathy characterized by alterations of the glomerular basement membrane because of mutations in type IV collagen genes. COL4A5 mutations, causing X-linked Alport syndrome, frequently result in the loss of the alpha5 chains of type IV collagen in basement membranes. This is associated with the absence of the alpha3(IV) and alpha4(IV) chains and increased amounts of alpha1(IV) and alpha2(IV) in glomerular basement membranes. The mechanisms resulting in such a configuration are still controversial and are of fundamental importance for understanding the pathology of the disease and for considering gene therapy. In this article we studied, for the first time, type IV collagen expression in kidneys from X-linked Alport syndrome patients, using in situ hybridization and immunohistochemistry. We show that, independent of the type of mutation and of the level of COL4A5 transcription, both COL4A3 and COL4A4 genes are actively transcribed in podocytes. Moreover, using immunofluorescence amplification, we were able to demonstrate that the alpha3 chain of type IV collagen was present in the podocytes of all patients. Finally, the alpha1(IV) chain, which accumulates within glomerular basement membranes, was found to be synthesized by mesangial/endothelial cells. These results strongly suggest that, contrary to what has been found in dogs affected with X-linked Alport syndrome, there is no transcriptional co-regulation of COL4A3, COL4A4, and COL4A5 genes in humans, and that the absence of alpha3(IV) to alpha5(IV) in glomerular basement membranes in the patients results from events downstream of transcription, RNA processing, and protein synthesis.

Alport syndrome and diffuse leiomyomatosis. Clinical aspects, pathology, molecular biology and extracellular matrix studies. A synthesis.

The Alport syndrome-diffuse leiomyomatosis association can be defined as a hereditary disease of type IV collagen combining features of Alport syndrome (hematuric nephropathy, deafness and ocular abnormalities: anterior lenticonus, maculopathy) and leiomyomatosis involving oesophagus (diffuse type), tracheobronchial tree, and genitals (only in women). This entity is transmitted as an X-linked dominant trait. Mutations of both the COL4A5 and COL4A6 genes, located head to head in Xq22 encoding the alpha 5 and alpha 6(IV) chains are responsible for the abnormalities. Molecular studies have shown deletions of the 5' end of both COL4A5 and COL4A6 including the intergenic region. The breakpoint in COL4A6 is always located within intron 2. Immunohistochemistry has shown significant alterations of basement membranes in the kidney and esophageal leiomyomas. Leiomyomas lack alpha 5 and alpha 6(IV) chains, fibronectin and laminin beta 1 chains in the muscle basement membranes where they are normally expressed. The tumors also show myocyte anomalies: irregular expression of the alpha 5 integrin subunits, and disorganization of actin and desmin filaments. It is hypothesized that a third as yet unknown gene, situated within the large intron 2 in a critical 90 kb region, is responsible for the smooth muscle proliferation. Abnormalities of the basement membranes could destabilize interactions between muscular cells and the extracellular matrix.

Diffuse leiomyomatosis of the esophagus: disorder of cell-matrix interaction?

Diffuse leiomyomatosis (DL) is rare condition characterized by proliferation of smooth muscle in the upper gastrointestinal tract. Most cases are associated with X-linked Alport syndrome and have partial deletions in the genes encoding both the alpha5 and alpha6 chains of collagen type IV. We studied aspects of cell-matrix interaction of myocytes in an esophagogastrectomy specimen from a 12-year-old patient with DL. Myocytes had central areas of cytoplasmic rarefaction, which were actin positive and desmin poor, with the reverse pattern of staining at the cell periphery. Electron microscopy (EM) showed that the areas of rarefaction consisted of disorganized aggregates of filaments. The basement membranes ranged from thickened to thinned or absent. Immunohistochemical staining for the alpha1-alpha4 chains of collagen type IV, the alpha1, alpha2, beta2, and gamma1 chains of laminin, nidogen, type VI collagen, and fibronectin was normal. There was loss of the alpha5 and alpha6 chains of collagen type IV and the beta1 chain of laminin. Normal staining for alpha1, alpha2, alpha3, alpha4, alpha6, alpha8, and beta1 integrins was noted. Staining for alpha5 integrin varied from normal to reduced or negative in different cells. In DL, a primary abnormality of basement membrane may be associated with disorganization of the contractile apparatus and alterations of certain integrins. This may reflect a disturbance of cell-matrix interactions that play a role in cell differentiation and internal organization.

Determination of the genomic structure of the COL4A4 gene and of novel mutations causing autosomal recessive Alport syndrome.

Autosomal recessive Alport syndrome is a progressive hematuric glomerulonephritis characterized by glomerular basement membrane abnormalities and associated with mutations in either the COL4A3 or the COL4A4 gene, which encode the alpha3 and alpha4 type IV collagen chains, respectively. To date, mutation screening in the two genes has been hampered by the lack of genomic structure information. We report here the complete characterization of the 48 exons of the COL4A4 gene, a comprehensive gene screen, and the subsequent detection of 10 novel mutations in eight patients diagnosed with autosomal recessive Alport syndrome. Furthermore, we identified a glycine to alanine substitution in the collagenous domain that is apparently silent in the heterozygous carriers, in 11.5% of all control individuals, and in one control individual homozygous for this glycine substitution. There has been no previous finding of a glycine substitution that is not associated with any obvious phenotype in homozygous individuals.

[Diffuse leiomyomatosis with genital involvement and Alport syndrome. Report of two cases]. / Léiomyomatose diffuse avec atteinte génitale et syndrome d'Alport. A propos de deux cas.

We observed the cases of two young women who both developed esophageal and perineal tumors successively. The esophageal component usually is the first manifestation. Esophagectomy, with or without gastrectomy is generally required. The genital affection involves the periclitoridian region, the minora and majora labia. Tracheobronchial localization is less common, but it may be lethal due to bronchospasm. An association between diffuse leiomyomatosis and Alport syndrome is not fortuitous. Recently, molecular biology has enabled to understand the combination of the two pathologies by showing the presence of a deletion on adjacent X chromosome genes, COL4A5 and COL4A6, which are involved in the synthesis of type IV collagen fibres. Leiomyomatosis and Alport syndrome are transmitted as X-linked dominant traits. Women with diffuse leiomyomatosis transmit Alport syndrome. An antenatal diagnosis can be proposed for such patients.

Somatic deletion of the 5' ends of both the COL4A5 and COL4A6 genes in a sporadic leiomyoma of the esophagus.

Leiomyomata of the esophagus are sporadic benign tumors of unknown etiology. We studied a collection of nine tumors for the expression of extracellular matrix components and found the same aberrant expression pattern as previously observed in inherited diffuse leiomyomatosis. We demonstrate here the occurrence of a somatic deletion at the COL4A5/COL4A6 locus at Xq22 in a frozen leiomyoma sample. These data confirm the hypothesis that the same underlying etiology is responsible for circumscribed smooth muscle proliferation in sporadic leiomyomata as for diffuse smooth muscle cell proliferation in inherited diffuse leiomyomatosis.

Sequence and structure of the human OXA1L gene and its upstream elements.

The genes encoding proteins involved in respiratory chain assembly represent candidate genes for nuclearly-encoded multiple respiratory chain deficiency. Using the long PCR amplification procedure, we have characterized the organization and complete sequence of OXA1L, a gene involved in the assembly of several complexes of the mitochondrial respiratory chain. The OXA1L gene (5 kb) is composed of 10 exons and 9 introns and contains a 24 N-terminal amino-acid stretch is characteristic of a mitochondrial presequence. The screening of OXA1L mutation in patients with multiple respiratory chain deficiency is now feasible.

Diffuse leiomyomatosis associated with X-linked Alport syndrome: extracellular matrix study using immunohistochemistry and in situ hybridization.

Inherited diffuse esophageal leiomyomatosis a benign tumor involving smooth muscle cells of the whole esophagus, is frequently associated with X-linked Alport syndrome, a hereditary disease of type IV collagen. Families with this condition are consistently found to have deletions encompassing the 5' ends of both the alpha 5 chain of type IV collagen (COL4A5) and the alpha 6 chain of type IV collagen (COL4A6) genes, always limited in COL4A6 to exons 1', 1, and 2. On the contrary, patients with COL4A5/COL4A6 deletions extending further into COL4A6 display no such tumors. Despite the deletion, a COL4A6 transcript including exon 4, but not exon 3, was found in a tumor sample, raising the possibility of the involvement of a truncated alpha 6(IV) chain in the tumorous process. Using immunohistochemistry and in situ hybridization methods, we analyzed the expression and distribution of the alpha 6 chain of type IV collagen in tumors in comparison with that of normal, fetal, and mature esophagus. We also studied associated changes in tumor basement membrane composition and in tumor-cell integrin subunit distribution. No labeling with alpha 6(IV) antibodies was detected in tumors, ruling out the hypothesis of a stably integrated truncated alpha 6(IV) chain in tumor basement membranes. In contrast, despite the deletions of the first two exons of the gene and its 5' end, a COL4A6 transcript is clearly expressed by tumor cells. This finding raises the question of a potential role for this RNA in the tumor process. The absence of the alpha 6(IV) chain is associated with the absence of the alpha 5(IV) chain, as was suggested by the COL4A5 deletion. An additional striking feature is the absence of the beta 1 chain of laminin in tumor basement membranes and the lack of or uneven expression of the alpha 5 integrin subunit. These findings show that dramatic changes in the composition of the matrix and the expression of integrin receptors also occur in this benign tumorous process.

Novel COL4A5/COL4A6 deletions and further characterization of the diffuse leiomyomatosis-Alport syndrome (DL-AS) locus define the DL critical region.

Diffuse leiomyomatosis (DL) with Alport syndrome (AS) has been shown to be associated with contiguous gene deletions of the COL4A5 and COL4A6 genes, with the COL4A6 breakpoint of the deletions invariably located in the large intron 2 of the gene. We describe four YAC clones covering the locus and a refined restriction map of the entire COL4A6 gene. These resources have allowed us to make a precise estimate of the size of COL4A6 introns 2 and 3, as well as the size of the gene itself. We also describe five novel deletions which, in conjunction with previous reports, allow the definition of a 90-kb critical region in which to search for a gene or other entity involved in the pathogenesis of DL.

Large homozygous deletions of the 2q13 region are a major cause of juvenile nephronophthisis.

Juvenile nephronophthisis (NPH) is a genetically heterogeneous disorder representing the most frequent inherited cause of chronic renal failure in children. We recently assigned a gene (NPH1) to the 2q13 region which is responsible for approximately 85% of cases. Cloning this region in a yeast artificial chromosome contig revealed the presence of low copy repeats. Large-scale rearrangements were detected in 80% of the patients belonging to inbred or multiplex NPH1 families and in 65% of the sporadic cases. Surprisingly, these rearrangements seem to be, in most cases, large homozygous deletions of approximately 250 kb involving an 100 kb inverted duplication. This suggests a common genetic disease-causing mechanism, which could be responsible for the highest frequency of large rearrangements reported in an autosomal recessive trait. Our findings are also of major clinical interest, as they permit the diagnosis in the majority of sporadic cases without the need for kidney biopsy.

Smooth muscle tumors associated with X-linked Alport syndrome: carrier detection in females.

X-linked Alport syndrome (AS) associated with diffuse esophageal leiomyomatosis (DL) has been reported to be due to deletions removing the 5' ends of both the COL4A5 and COL4A6 genes, encoding the alpha 5 and alpha 6 chains of type IV collagen, respectively, whereas a variety of mutations in COL4A5 has been identified in patients with AS alone. Here we report three additional DL-AS patients who also display deletions removing the 5' ends of both COL4A5 and COL4A6 genes. Furthermore, we tracked the mutation in 15 females belonging to six DL-AS families by gene copy number determination. We found that, like AS, DL is transmitted as an X-linked dominant trait but, contrary to AS, DL is fully penetrant and completely expressed in females. These results are in agreement with our previous work suggesting that DL could be due to a dominant effect of an abnormal alpha 6 (IV) collagen chain. Finally, we have detected a similar deletion of the COL4A5 and COl4A6 genes in a DL affected female who showed no sign of nephropathy, demonstrating the AS carrier status of this DL patient. These results emphasize the importance of molecular analysis of female DL patients for genetic counseling.

Deletions of both alpha 5(IV) and alpha 6(IV) collagen genes in Alport syndrome and in Alport syndrome associated with smooth muscle tumours.

Diffuse oesophageal leiomyomatosis (DL), an inherited smooth muscle proliferation process, has been reported to be associated with Alport syndrome (AS), a familial nephropathy, mainly dominant X-linked inherited, and characterized by ultrastructural changes of the glomerular basement membrane. The COL4A5 gene, encoding the alpha 5 chain of type IV collagen, has been identified as the site of mutations in families with X-linked AS. Recently, a novel alpha 6(IV) collagen chain encoding gene has been mapped closely upstream of COL4A5, and disruption of the 5' end of both genes has been reported in four patients with DL and AS (DL-AS). Here, we report a long-range restriction map around the COL4A6 locus, and show that the COL4A5/COL4A6 deletion observed in seven patients with DL-AS encompasses only the two first exons of COL4A6, with a breakpoint located in the second intron of COL4A6, whose size exceeds 65 kb. Furthermore, we demonstrate that three patients with AS without DL, known to have a deletion of the 5' part of the COL4A5 gene, display a larger deletion in COL4A6. Moreover, a COL4A6 mRNA product was detected by reverse-transcription-polymerase chain reaction in an oesophageal tumour sample of a patient with DL-AS. These results suggest that DL-AS could be caused by an abnormal truncated alpha 6(IV) chain.

Recurrence of de novo membranous glomerulonephritis on renal grafts.

An updated study of the glomerular lesions found in renal grafts in children showed 3 features of interest. 1) In our experience, the incidence of the occurrence of de novo membranous glomerulonephritis (MGN) remains around 9% (48 of 530 grafts). 2) Of the 29 patients we reported in a previous study [Antignac et al. 1988], 18 lost their grafts and 7 received a second graft. Four of these 7 patients recurred de novo MGN on their second graft. Their clinical course is reported in detail. 3) In our population of transplanted children, of the 55 patients who received a second graft, the only recipients who developed a de novo MGN were the 4 patients who had already developed de novo MGN on their first graft. The various factors possibly involved in the pathogenesis of de novo MGN are reviewed. The high incidence of recurrence of de novo MGN indicates that host factors play a major role in the development of this nephropathy.