The importance of clinician, patient and researcher collaborations in Alport syndrome.

Alport syndrome is caused by mutations in the genes COL4A3, COL4A4 or COL4A5 and is characterised by progressive glomerular disease, sensorineural hearing loss and ocular defects. Occurring in less than 1:5000, Alport syndrome is a rare genetic disorder but still accounts for > 1% of the prevalent population receiving renal replacement therapy. There is also increasing awareness about the risk of chronic kidney disease in individuals with heterozygous mutations in Alport syndrome genes. The mainstay of current therapy is the use of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, yet potential new therapies are now entering clinical trials. The 2017 International Workshop on Alport Syndrome in Glasgow was a pre-conference workshop ahead of the 50th anniversary meeting of the European Society for Pediatric Nephrology. It focussed on updates in clinical practice, genetics and basic science and also incorporated patient perspectives. More than 80 international experts including clinicians, geneticists, researchers from academia and industry, and patient representatives took part in panel discussions and breakout groups. This report summarises the workshop proceedings and the relevant contemporary literature. It highlights the unique clinician, patient and researcher collaborations achieved by regular engagement between the groups.

Coinheritance of COL4A5 and MYO1E mutations accentuate the severity of kidney disease.

BACKGROUND: Mutations in podocyte and basement membrane genes are associated with a growing spectrum of glomerular disease affecting adults and children. Investigation of familial cases has helped to build understanding of both normal physiology and disease. METHODS: We investigated a consanguineous family with a wide clinical phenotype of glomerular disease using clinical, histological, and new genetic studies. RESULTS: We report striking variability in severity of nephropathy within an X-linked Alport syndrome (XLAS) family. Four siblings each carried a mutant COL4A5 allele, p.(Gly953Val) and p.(Gly1033Arg). Two boys had signs limited to hematuria and mild/moderate proteinuria. In striking contrast, a sister presented with end-stage renal disease (ESRD) at 8 years of age and an infant brother presented with nephrotic syndrome, progressing to ESRD by 3 years of age. Both were subsequently found to have homozygous variants in MYO1E, p.(Lys118Glu) and p.(Thr876Arg). MYO1E is a gene implicated in focal segmental glomerulosclerosis and it encodes a podocyte-expressed non-muscle myosin. Bioinformatic modeling demonstrated that the collagen IV-alpha3,4,5 extracellular network connected via known protein-protein interactions to intracellular myosin 1E. CONCLUSIONS: COL4A5 and MYO1E mutations may summate to perturb common signaling pathways, resulting in more severe disease than anticipated independently. We suggest screening for MYO1E and other non-COL4 'podocyte gene' mutations in XLAS when clinical nephropathy is more severe than expected for an individual's age and sex.

COL4A3/COL4A4 mutations and features in individuals with autosomal recessive Alport syndrome.

Alport syndrome is an inherited disease characterized by hematuria, progressive renal failure, hearing loss, and ocular abnormalities. Autosomal recessive Alport syndrome is suspected in consanguineous families and when female patients develop renal failure. Fifteen percent of patients with Alport syndrome have autosomal recessive inheritance caused by two pathogenic mutations in either COL4A3 or COL4A4. Here, we describe the mutations and clinical features in 40 individuals including 9 children and 21 female individuals (53%) with autosomal recessive inheritance indicated by the detection of two mutations. The median age was 31 years (range, 6-54 years). The median age at end stage renal failure was 22.5 years (range, 10-38 years), but renal function was normal in nine adults (29%). Hearing loss and ocular abnormalities were common (23 of 35 patients [66%] and 10 of 18 patients [56%], respectively). Twenty mutation pairs (50%) affected COL4A3 and 20 pairs affected COL4A4. Of the 68 variants identified, 39 were novel, 12 were homozygous changes, and 9 were present in multiple individuals, including c.2906C>G (p.(Ser969*)) in COL4A4, which was found in 23% of the patients. Thirty-six variants (53%) resulted directly or indirectly in a stop codon, and all 17 individuals with early onset renal failure had at least one such mutation, whereas these mutations were less common in patients with normal renal function or late-onset renal failure. In conclusion, patient phenotypes may vary depending on the underlying mutations, and genetic testing should be considered for the routine diagnosis of autosomal recessive Alport syndrome.

Atypical Neurogenesis in Induced Pluripotent Stem Cells From Autistic Individuals.

BACKGROUND: Autism is a heterogeneous collection of disorders with a complex molecular underpinning. Evidence from postmortem brain studies have indicated that early prenatal development may be altered in autism. Induced pluripotent stem cells (iPSCs) generated from individuals with autism with macrocephaly also indicate prenatal development as a critical period for this condition. But little is known about early altered cellular events during prenatal stages in autism. METHODS: iPSCs were generated from 9 unrelated individuals with autism without macrocephaly and with heterogeneous genetic backgrounds, and 6 typically developing control individuals. iPSCs were differentiated toward either cortical or midbrain fates. Gene expression and high throughput cellular phenotyping was used to characterize iPSCs at different stages of differentiation. RESULTS: A subset of autism-iPSC cortical neurons were RNA-sequenced to reveal autism-specific signatures similar to postmortem brain studies, indicating a potential common biological mechanism. Autism-iPSCs differentiated toward a cortical fate displayed impairments in the ability to self-form into neural rosettes. In addition, autism-iPSCs demonstrated significant differences in rate of cell type assignment of cortical precursors and dorsal and ventral forebrain precursors. These cellular phenotypes occurred in the absence of alterations in cell proliferation during cortical differentiation, differing from previous studies. Acquisition of cell fate during midbrain differentiation was not different between control- and autism-iPSCs. CONCLUSIONS: Taken together, our data indicate that autism-iPSCs diverge from control-iPSCs at a cellular level during early stage of neurodevelopment. This suggests that unique developmental differences associated with autism may be established at early prenatal stages.

Germline selection shapes human mitochondrial DNA diversity.

Approximately 2.4% of the human mitochondrial DNA (mtDNA) genome exhibits common homoplasmic genetic variation. We analyzed 12,975 whole-genome sequences to show that 45.1% of individuals from 1526 mother-offspring pairs harbor a mixed population of mtDNA (heteroplasmy), but the propensity for maternal transmission differs across the mitochondrial genome. Over one generation, we observed selection both for and against variants in specific genomic regions; known variants were more likely to be transmitted than previously unknown variants. However, new heteroplasmies were more likely to match the nuclear genetic ancestry as opposed to the ancestry of the mitochondrial genome on which the mutations occurred, validating our findings in 40,325 individuals. Thus, human mtDNA at the population level is shaped by selective forces within the female germ line under nuclear genetic control, which ensures consistency between the two independent genetic lineages.

A two-tier approach to mutation detection in the COL4A5 gene for Alport syndrome.

About 85% of Alport syndrome is an X-linked semi-dominant condition caused by mutations in the collagen gene, COL4A5. The large size and high GC content of this gene have presented diagnostic laboratories with problems in identifying mutations with greater than about a 50% success rate since the gene was first cloned 16 years ago. An RNA based approach is adopted here for a first pass mutation scanning coupled with more traditional exon-by-exon screening to increase the rate of mutation identification. Twenty-one mutations were identified in twenty-five patients with clear Alport syndrome including four gross deletions, two deep intronic mutations, three frameshifts, three splice site mutations, eight missense mutations and one inframe deletion.

Benefits and drawbacks of preimplantation genetic diagnosis (PGD) for reciprocal translocations: lessons from a prospective cohort study.

Preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridisation probes was carried out for 59 couples carrying reciprocal translocations. Before treatment, 85% of pregnancies had resulted in spontaneous miscarriage and five couples had achieved a healthy live-birth delivery. Following treatment, 33% of pregnancies failed and 21 of 59 couples had a healthy live-born child. The accuracy of diagnosis was 92% (8% false abnormal and 0% false normal results). The overall incidence of 2:2 alternate segregation products was 44%; however, products consistent with 2:2 adjacent segregation were ~twice as likely from male heterozygotes, and those with 3:1 disjunction were three times more likely from female heterozygotes. Our results indicate that up to three stimulation cycles per couple would give an ~50% chance of a successful live birth, with the risk of miscarriage reduced to the level found in the general population. In our study, 87% of all normal/balanced embryos available were identified as being suitable for transfer. We conclude that PGD provides benefit for couples with high-risk translocations by reducing the risk of miscarriage and avoiding a pregnancy with an unbalanced form of the translocation; however, for fertile carriers of translocations with a low risk of conceiving a chromosomally unbalanced offspring, natural conception may be a more viable option.

Unusual deep intronic mutations in the COL4A5 gene cause X linked Alport syndrome.

The X-linked form of Alport syndrome is caused by mutations in the COL4A5 gene in Xq22. This large multiexonic gene has, in the past, been difficult to screen, with several studies detecting only about 50% of mutations. We report three novel intronic mutations that may, in part, explain this poor success rate and demonstrate that single base changes deep within introns can, and do, cause disease: one mutation creates a new donor splice site within an intron resulting in the inclusion of a novel in-frame cryptic exon; a second mutation results in a new exon splice enhancer sequence (ESE) that promotes splicing of a cryptic exon containing a stop codon; a third patient exhibits exon skipping as a result of a base substitution within the polypyrimidine tract that precedes the acceptor splice site. All three cases would have been missed using an exon-by-exon DNA screening approach.