Pathophysiology of cerebral small vessel disease: a journey through recent discoveries.

Cerebral small vessel disease (cSVD) encompasses a heterogeneous group of age-related small vessel pathologies that affect multiple regions. Disease manifestations range from lesions incidentally detected on neuroimaging (white matter hyperintensities, small deep infarcts, microbleeds, or enlarged perivascular spaces) to severe disability and cognitive impairment. cSVD accounts for approximately 25% of ischemic strokes and the vast majority of spontaneous intracerebral hemorrhage and is also the most important vascular contributor to dementia. Despite its high prevalence and potentially long therapeutic window, there are still no mechanism-based treatments. Here, we provide an overview of the recent advances in this field. We summarize recent data highlighting the remarkable continuum between monogenic and multifactorial cSVDs involving NOTCH3, HTRA1, and COL4A1/A2 genes. Taking a vessel-centric view, we discuss possible cause-and-effect relationships between risk factors, structural and functional vessel changes, and disease manifestations, underscoring some major knowledge gaps. Although endothelial dysfunction is rightly considered a central feature of cSVD, the contributions of smooth muscle cells, pericytes, and other perivascular cells warrant continued investigation.

TGFß Signaling Dysregulation May Contribute to COL4A1-Related Glaucomatous Optic Nerve Damage.

Purpose: Mutations in the genes encoding type IV collagen alpha 1 (COL4A1) and alpha 2 (COL4A2) cause a multisystem disorder that includes ocular anterior segment dysgenesis (ASD) and glaucoma. We previously showed that transforming growth factor beta (TGFß) signaling was elevated in developing anterior segments from Col4a1 mutant mice and that reducing TGFß signaling ameliorated ASD, supporting a role for the TGFß pathway in disease pathogenesis. Here, we tested whether altered TGFß signaling also contributes to glaucoma-related phenotypes in Col4a1 mutant mice. Methods: To test the role of TGFß signaling in glaucoma-relevant phenotypes, we genetically reduced TGFß signaling using mice with mutated Tgfbr2, which encodes the common receptor for all TGFß ligands in Col4a1+/G1344D mice. We performed slit-lamp biomicroscopy and optical coherence tomography for qualitative and quantitative analyses of anterior and posterior ocular segments, histological analyses of ocular tissues and optic nerves, and intraocular pressure assessments using rebound tonometry. Results: Col4a1+/G1344D mice showed defects of the ocular drainage structures, including iridocorneal adhesions, and phenotypes consistent with glaucomatous neurodegeneration, including thinning of the nerve fiber layer, retinal ganglion cell loss, optic nerve head excavation, and optic nerve degeneration. We found that reducing TGFß receptor 2 (TGFBR2) was protective for ASD, ameliorated ocular drainage structure defects, and protected against glaucomatous neurodegeneration in Col4a1+/G1344D mice. Conclusions: Our results suggest that elevated TGFß signaling contributes to glaucomatous neurodegeneration in Col4a1 mutant mice.

Coexisting presentation of two rare genetic variants of autosomal dominant polycystic kidney disease and Alport syndrome.

Alport syndrome and autosomal dominant polycystic kidney disease are monogenic causes of chronic kidney disease and end-stage kidney failure. We present a case of a man in his 60s with progressive chronic kidney disease, bilateral sensorineural hearing loss and multiple renal cysts. Genetic analysis revealed a heterozygous variant in COL4A3 (linked to Alport syndrome) and in the GANAB gene (associated with a milder form of autosomal dominant polycystic kidney disease). Although each variant confers a mild risk of developing end-stage kidney disease, the patient presented a pronounced and accelerated progression of chronic kidney disease, which goes beyond what would be predicted by adding up their individual effects. This suggests a potential synergic effect of both variants, which warrants further investigation.

[Precision diagnosis and therapeutic intervention of Alport syndrome].

Alport syndrome is one of the most common inherited kidney diseases caused by mutations in the type Ⅳ collagen genes. It has a complex pattern of inheritance and diverse clinical manifestations, and severe cases will rapidly progress to end-stage kidney disease. With the rapid development of genetic testing technology, there is a deeper understanding of the genetic spectrum of Alport syndrome, the effectiveness of clinical therapies, and the prediction of disease prognosis. Therefore, the purpose of the article is to introduce the advances in the diagnosis and treatment of Alport syndrome, aiming to improve the early diagnosis and standardized treatment of this disease.

A chemiluminescence immunoassay for type IV collagen as a promising marker for liver fibrosis and cirrhosis.

Herein, a magnetic bead-based chemiluminescence assay is reported to detect type IV collagen (col-IV) in serum samples. Magnetic beads (MBs) exhibit biocompatibility. Taking advantage of this property, they were conjugated with the col-IV antibody. For the determination of col-IV, the interaction of the col-IV sample, anti-(col-IV)-alkaline phosphatase (anti-(col-IV)-ALP) and anti-col-IV-magnetic beads (anti-(col-IV)-MBs) was performed to generate chemiluminescence. Under the optimized conditions, the developed method displayed good linearity in the concentration range of 20-2000 ng mL-1 with the limit of 0.79 ng mL-1. The repeatability coefficient of variation (CV) for col-IV detection ranged from 3.16% to 7.50%. The col-IV level in samples collected from a hospital was assessed by the chemiluminescence assay. Satisfactory recoveries were obtained ranging from 93.30% to 100.14%. In conclusion, the magnetic bead-based chemiluminescence assay may be used as a routine and efficient tool to detect type IV collagen in clinical diagnosis.

Genetic reprogramming with stem cells regenerates glomerular epithelial podocytes in Alport syndrome.

Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFß1 and using allogenic bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.

A novel COL4A5 splicing mutation causes alport syndrome in a Chinese family.

BACKGROUND: Alport syndrome (AS) is characterised by haematuria, proteinuria, a gradual decline in kidney function, hearing loss, and eye abnormalities. The disease is caused by mutations in COL4An (n = 3, 4, 5) that encodes 3-5 chains of type IV collagen in the glomerular basement membrane. AS has three genetic models: X-linked, autosomal recessive, and autosomal dominant. The most common type of AS is X-linked AS, which is caused by COL4A5. METHODS: We enrolled children with renal insufficiency and a family history of kidney disorders. The proband was identified using whole-exome sequencing. Sanger sequencing was performed to verify the mutation site. Minigene technology was used to analyse the influence of mutant genes on pre-mRNA shearing, and the Iterative Threading ASSEmbly Refinement (I-TASSER) server was used to analyse the protein structure changes. RESULTS: The proband, together with her mother and younger brother, displayed microscopic haematuria and proteinuria, Pathological examination revealed mesangial hyperplasia and sclerosis. A novel mutation (NM_000495.5 c.4298-8G > A) in the intron of the COL4A5 gene in the proband was discovered, which was also present in the proband's mother, brother, and grandmother. In vitro minigene expression experiments verified that the c.4298-8G > A mutation caused abnormal splicing, leading to the retention of six base pairs at the end of intron 46. The I-TASSER software predicted that the mutation affected the hydrogen-bonding structure of COL4A5 and the electrostatic potential on the surface of the protein molecules. CONCLUSIONS: Based on the patient's clinical history and genetic traits, we conclude that the mutation at the splicing site c.4298-8G > A of the COL4A5 gene is highly probable to be the underlying cause within this particular family. This discovery expands the genetic spectrum and deepens our understanding of the molecular mechanisms underlying AS.

An AluYa5 Insertion in the 3'UTR of COL4A1 and Cerebral Small Vessel Disease.

Importance: Cerebral small vessel diseases (CSVDs) account for one-fifth of stroke cases. Numerous familial cases remain unresolved after routine screening of known CSVD genes. Objective: To identify novel genes and mechanisms associated with familial CSVD. Design, Setting, and Participants: This 2-stage study involved linkage analysis and a case-control study; linkage analysis and whole exome and genome sequencing were used to identify candidate gene variants in 2 large families with CSVD (9 patients with CSVD). Then, a case-control analysis was conducted on 246 unrelated probands, including probands from these 2 families and 244 additional probands. All probands (clinical onset

A brain-specific angiogenic mechanism enabled by tip cell specialization.

Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals3-5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-ß-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.

Integrating single-cell and spatial transcriptomes reveals COL4A1/2 facilitates the spatial organisation of stromal cells differentiation in breast phyllodes tumours.

BACKGROUND: Breast phyllodes tumours (PTs) are a unique type of fibroepithelial neoplasms with metastatic potential and recurrence tendency. However, the precise nature of heterogeneity in breast PTs remains poorly understood. This study aimed to elucidate the cell subpopulations composition and spatial structure and investigate diagnostic markers in the pathogenesis of PTs. METHODS: We applied single-cell RNA sequencing and spatial transcriptomes on tumours and adjacent normal tissues for integration analysis. Immunofluorescence experiments were conducted to verify the tissue distribution of cells. Tumour cells from patients with PTs were cultured to validate the function of genes. To validate the heterogeneity, the epithelial and stromal components of tumour tissues were separated using laser capture microdissection, and microproteomics data were obtained using data-independent acquisition mass spectrometry. The diagnostic value of genes was assessed using immunohistochemistry staining. RESULTS: Tumour stromal cells harboured seven subpopulations. Among them, a population of widely distributed cancer-associated fibroblast-like stroma cells exhibited strong communications with epithelial progenitors which underwent a mesenchymal transition. We identified two stromal subpopulations sharing epithelial progenitors and mesenchymal markers. They were inferred to further differentiate into transcriptionally active stromal subpopulations continuously expressing COL4A1/2. The binding of COL4A1/2 with ITGA1/B1 facilitated a growth pattern from the stroma towards the surrounding glands. Furthermore, we found consistent transcriptional changes between intratumoural heterogeneity and inter-patient heterogeneity by performing microproteomics studies on 30 samples from 11 PTs. The immunohistochemical assessment of 97 independent cohorts identified that COL4A1/2 and CSRP1 could aid in accurate diagnosis and grading. CONCLUSIONS: Our study demonstrates that COL4A1/2 shapes the spatial structure of stromal cell differentiation and has important clinical implications for accurate diagnosis of breast PTs.

Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues.

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin ß1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/ß1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor ß1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.

Alport syndrome and Alport kidney diseases - elucidating the disease spectrum.

PURPOSE OF REVIEW: With the latest classification, variants in three collagen IV genes, COL4A3 , COL4A4 , and COL4A5 , represent the most prevalent genetic kidney disease in humans, exhibiting diverse, complex, and inconsistent clinical manifestations. This review breaks down the disease spectrum and genotype-phenotype correlations of kidney diseases linked to genetic variants in these genes and distinguishes "classic" Alport syndrome (AS) from the less severe nonsyndromic genetically related nephropathies that we suggest be called "Alport kidney diseases". RECENT FINDINGS: Several research studies have focused on the genotype-phenotype correlation under the latest classification scheme of AS. The historic diagnoses of "benign familial hematuria" and "thin basement membrane nephropathy" linked to heterozygous variants in COL4A3 or COL4A4 are suggested to be obsolete, but instead classified as autosomal AS by recent expert consensus due to a significant risk of disease progression. SUMMARY: The concept of Alport kidney disease extends beyond classic AS. Patients carrying pathogenic variants in any one of the COL4A3/A4/A5 genes can have variable phenotypes ranging from completely normal/clinically unrecognizable, hematuria without or with proteinuria, or progression to chronic kidney disease and kidney failure, depending on sex, genotype, and interplays of other genetic as well as environmental factors.

Acquired curved hair is caused by fusion of multiple hair matrix cells.

BACKGROUND: "Curved hair" caused by acquired factors is considered to have adverse cosmetic effects, but the detailed mechanism behind curved hair remains obscure. OBJECTIVE: We attempted to clarify the causes of curved hair that appeared to have occurred via acquired factors. METHODS: Outer root sheath cells (ORSC) isolated from plucked human hair follicles were used to evaluate the expression of type IV collagen. Straight and curved hairs with hair follicle tissue attached were also collected from the same individuals and subjected to morphological, immunohistochemical, and gene expression analyses. RESULTS: The amount of type IV collagen increased upon inducing endoplasmic reticulum stress in ORSC. Meanwhile, in curved hair follicle tissue, the gene expression of type IV collagen decreased. In addition, the curved hair follicle tissue obtained from participants in their 30 s to 50 s had distorted shapes compared with that of straight hair from the same individuals. It was also observed that hair matrix cells based on multiple hair germs fused to eventually form a single hair follicle and hair shaft. In curved hair follicle tissue, KRT71 protein, a marker of inner root sheath differentiation, was unevenly distributed and there was elevated expression of Dickkopf-1 (DKK1) protein, an inhibitor of the Wnt signaling pathway. CONCLUSION: Our study revealed the fusion of hair matrix cells during hair follicle regeneration as a cause of acquired curved hair. We consider that such fusion causes hair follicle tissue to abnormally differentiate, resulting in asymmetric hair follicle shapes and curved hair.

Molecular Imaging of Diffuse Cardiac Fibrosis with a Radiotracer That Targets Proteolyzed Collagen IV.

Purpose To develop an approach for in vivo detection of interstitial cardiac fibrosis using PET with a peptide tracer targeting proteolyzed collagen IV (T-peptide). Materials and Methods T-peptide was conjugated to the copper chelator MeCOSar (chemical name, 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid) and radiolabeled with copper 64 (64Cu). PET/CT scans were acquired following intravenous delivery of 64Cu-T-peptide-MeCOSar (0.25 mg/kg; 18 MBq ± 2.7 [SD]) to male transgenic mice overexpressing ß2-adrenergic receptors with intermediate (7 months of age; n = 4 per group) to severe (10 months of age; n = 11 per group) cardiac fibrosis and their wild-type controls. PET scans were also performed following coadministration of the radiolabeled probe with nonlabeled T-peptide in excess to confirm binding specificity. PET data were analyzed by t tests for static scans and analysis of variance tests (one- or two-way) for dynamic scans. Results PET/CT scans revealed significantly elevated (2.24-4.26-fold; P < .05) 64Cu-T-peptide-MeCOSar binding in the fibrotic hearts of aged transgenic ß2-adrenergic receptor mice across the entire 45-minute acquisition period compared with healthy controls. The cardiac tracer accumulation and presence of diffuse cardiac fibrosis in older animals were confirmed by gamma counting (P < .05) and histologic evaluation, respectively. Coadministration of a nonradiolabeled probe in excess abolished the elevated radiotracer binding in the aged transgenic hearts. Importantly, PET tracer accumulation was also detected in younger (7 months of age) transgenic mice with intermediate cardiac fibrosis, although this was only apparent from 20 minutes following injection (1.6-2.2-fold binding increase; P < .05). Conclusion The T-peptide PET tracer targeting proteolyzed collagen IV provided a sensitive and specific approach of detecting diffuse cardiac fibrosis at varying degrees of severity in a transgenic mouse model. Keywords: Diffuse Cardiac Fibrosis, Molecular Peptide Probe, Molecular Imaging, PET/CT © RSNA, 2024.

Genetic diagnosis of Alport syndrome in 16 Chinese families.

BACKGROUND: Alport syndrome (AS) is a genetically heterogeneous disorder resulting from mutations in the collagen IV genes COL4A3, COL4A4, and COL4A5. The genetic diagnosis of AS is very important to make precise diagnosis and achieve optimal outcomes. METHODS: In this study, 16 Chinese families with suspected AS were recruited after pedigree analysis, and the clinical presentations were analyzed by a nephrologist. The genetic diagnosis was performed by whole-exome sequencing (WES) and the disease-causing variants were confirmed by Sanger sequencing. RESULTS: The cohort of probands included seven men and nine women, with a mean age of 19.9 years. Pathological analysis showed slight-to-moderate mesangial proliferation, and thin basement membrane was the main findings. Pathogenic variants were revealed by WES in each family, and the co-segregation with renal presentation was confirmed by PCR. In addition, RT-PCR analysis showed that the intronic variant led to aberrant splicing. CONCLUSION: Our findings expand the spectrum of AS gene variation, which will inform genetic diagnosis and add to the theoretical basis for the prevention of AS.

Quantitative assessment of glomerular basement membrane collagen IV α chains in paraffin sections from patients with focal segmental glomerulosclerosis and Alport gene variants.

Focal segmental glomerulosclerosis (FSGS) lesions have been linked to variants in COL4A3/A4/A5 genes, which are also mutated in Alport syndrome. Although it could be useful for diagnosis, quantitative evaluation of glomerular basement membrane (GBM) type IV collagen (colIV) networks is not widely used to assess these patients. To do so, we developed immunofluorescence imaging for collagen α5(IV) and α1/2(IV) on kidney paraffin sections with Airyscan confocal microscopy that clearly distinguishes GBM collagen α3α4α5(IV) and α1α1α2(IV) as two distinct layers, allowing quantitative assessment of both colIV networks. The ratios of collagen α5(IV):α1/2(IV) mean fluorescence intensities (α5:α1/2 intensity ratios) and thicknesses (α5:α1/2 thickness ratios) were calculated to represent the levels of collagen α3α4α5(IV) relative to α1α1α2(IV). The α5:α1/2 intensity and thickness ratios were comparable across all 11 control samples, while both ratios were significantly and markedly decreased in all patients with pathogenic or likely pathogenic Alport COL4A variants, supporting validity of this approach. Thus, with further validation of this technique, quantitative measurement of GBM colIV subtype abundance by immunofluorescence, may potentially serve to identify the subgroup of patients with FSGS lesions likely to harbor pathogenic COL4A variants who could benefit from genetic testing.

PDGF-D Is Dispensable for the Development and Progression of Murine Alport Syndrome.

Alport syndrome is an inherited kidney disease, which can lead to glomerulosclerosis and fibrosis, as well as end-stage kidney disease in children and adults. Platelet-derived growth factor-D (PDGF-D) mediates glomerulosclerosis and interstitial fibrosis in various models of kidney disease, prompting investigation of its role in a murine model of Alport syndrome. In vitro, PDGF-D induced proliferation and profibrotic activation of conditionally immortalized human parietal epithelial cells. In Col4a3-/- mice, a model of Alport syndrome, PDGF-D mRNA and protein were significantly up-regulated compared with non-diseased wild-type mice. To analyze the therapeutic potential of PDGF-D inhibition, Col4a3-/- mice were treated with a PDGF-D neutralizing antibody. Surprisingly, PDGF-D antibody treatment had no effect on renal function, glomerulosclerosis, fibrosis, or other indices of kidney injury compared with control treatment with unspecific IgG. To characterize the role of PDGF-D in disease development, Col4a3-/- mice with a constitutive genetic deletion of Pdgfd were generated and analyzed. No difference in pathologic features or kidney function was observed in Col4a3-/-Pdgfd-/- mice compared with Col4a3-/-Pdgfd+/+ littermates, confirming the antibody treatment data. Mechanistically, lack of proteolytic PDGF-D activation in Col4a3-/- mice might explain the lack of effects in vivo. In conclusion, despite its established role in kidney fibrosis, PDGF-D, without further activation, does not mediate the development and progression of Alport syndrome in mice.

Three exonic variants in the COL4A5 gene alter RNA splicing in a minigene assay.

BACKGROUND: X-linked Alport syndrome (XLAS) is an inherited renal disease caused by rare variants of COL4A5 on chromosome Xq22. Many studies have indicated that single nucleotide variants (SNVs) in exons can disrupt normal splicing process of the pre-mRNA by altering various splicing regulatory signals. The male patients with XLAS have a strong genotype-phenotype correlation. Confirming the effect of variants on splicing can help to predict kidney prognosis. This study aimed to investigate whether single nucleotide substitutions, located within three bases at the 5' end of the exons or internal position of the exons in COL4A5 gene, cause aberrant splicing process. METHODS: We analyzed 401 SNVs previously presumed missense and nonsense variants in COL4A5 gene by bioinformatics programs and identified candidate variants that may affect the splicing of pre-mRNA via minigene assays. RESULTS: Our study indicated three of eight candidate variants induced complete or partial exon skipping. Variants c.2678G>C and c.2918G>A probably disturb classic splice sites leading to corresponding exon skipping. Variant c.3700C>T may disrupt splicing enhancer motifs accompanying with generation of splicing silencer sequences resulting in the skipping of exon 41. CONCLUSION: Our study revealed that two missense variants positioned the first nucleotides of the 5' end of COL4A5 exons and one internal exonic nonsense variant caused aberrant splicing. Importantly, this study emphasized the necessity of assessing the effects of SNVs at the mRNA level.

Collagen type IV alpha 1 chain (COL4A1) expression in the developing human lung.

BACKGROUND: Collagen type IV alpha 1 chain (COL4A1) in the basement membrane is an important component during lung development, as suggested from animal models where COL4A1 has been shown to regulate alveolarization and angiogenesis. Less is known about its role in human lung development. Our aim was to study COL4A1 expression in preterm infants with different lung maturational and clinical features. METHODS: COL4A1 expression in 115 lung samples from newborn infants (21-41 weeks' gestational age; 0-228 days' postnatal age [PNA]) was studied by immunohistochemistry combined with digital image analysis. Cluster analysis was performed to find subgroups according to immunohistologic and clinical data. RESULTS: Patients were automatically categorized into 4 Groups depending on their COL4A1 expression. Expression of COL4A1 was mainly extracellular in Group 1, low in Group 2, intracellular in Group 3, and both extra- and intracellular in Group 4. Intracellular/extracellular ratio of COL4A1 expression related to PNA showed a distinctive postnatal maturational pattern on days 1-7, where intracellular expression of COL4A1 was overrepresented in extremely preterm infants. CONCLUSIONS: COL4A1 expression seems to be highly dynamic during the postnatal life due to a possible rapid remodeling of the basement membrane. Intracellular accumulation of COL4A1 in the lungs of extremely premature infants occurs more frequently between 1 and 7 postnatal days than during the first 24 hours. In view of the lung arrest described in extremely preterm infants, the pathological and/or developmental role of postnatally increased intracellular COL4A1 as marker for basement membrane turnover, needs to be further investigated.

Infantile hemiparesis and porencephaly due to a COL4A1 mutation: Gould syndrome.

Gould syndrome is an autosomal dominant syndrome due to a COL4A1 or COL4A2 mutation that is commonly characterised by familial porencephaly, seizures, intracranial haemorrhages, cataracts, nephropathies and more. There have been up to 137 identified patients based on a review of the literature. In this case, we describe a male infant that presents with hemiparesis, developmental delay and gait abnormalities at his well-child check. Referral to neurology and a subsequent MRI demonstrated porencephaly and ocular lens abnormalities. Genetic sequencing uncovered a mutation to the COL4A1 gene, suggesting Gould syndrome. There are no family members with similar phenotypes. Mutations to the COL4A1 and COL4A2 genes result in disruption of collagen found in most basement membranes, resulting in a variety of phenotypes that can make diagnosis difficult. Genetic identification of these patients is critical as these patients require a multidisciplinary approach to care and specific counselling on risk reduction techniques.