Focal segmental glomerulosclerosis and mild intellectual disability in a patient with a novel de novo truncating TRIM8 mutation.

Mutations in the TRIM8 gene have been described in patients with severe developmental delay, intellectual disability and epilepsy. Only six patients have been described to date. All the previous mutations were truncating variants clustered in the C-terminus of the protein. A previous patient with TRIM8-related epileptic encephalopathy was reported to have nephrotic syndrome. Here we describe the clinical, radiological and histological features of an 8-year-old male patient with a TRIM8 mutation who, in contrast to previous patients, had only mild intellectual disability and well-controlled epilepsy. The patient was found to have proteinuria at 2 years of age. Renal biopsy findings were suggestive of focal segmental glomerulosclerosis. His kidney function declined and peritoneal dialysis was started at 5 years of age. He underwent renal transplant at 7 years of age. Trio-based whole genome sequencing identified a novel de novo heterozygous frameshift mutation in TRIM8 (NM_030912.2) c.1198_1220del, p.(Tyr400ArgfsTer2). This patient is further evidence that TRIM8 mutations cause a syndrome with both neurological and renal features. Our findings suggest the spectrum of TRIM8-related disease may be wider than previously thought with the possibility of milder neurodevelopmental problems and/or a more severe, progressive renal phenotype. We highlight the need for proteinuria screening in patients with TRIM8 mutations.

International Porphyria Molecular Diagnostic Collaborative: an evidence-based database of verified pathogenic and benign variants for the porphyrias.

With the advent of precision and genomic medicine, a critical issue is whether a disease gene variant is pathogenic or benign. Such is the case for the three autosomal dominant acute hepatic porphyrias (AHPs), including acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria, each resulting from the half-normal enzymatic activities of hydroxymethylbilane synthase, coproporphyrinogen oxidase, and protoporphyrinogen oxidase, respectively. To date, there is no public database that documents the likely pathogenicity of variants causing the porphyrias, and more specifically, the AHPs with biochemically and clinically verified information. Therefore, an international collaborative with the European Porphyria Network and the National Institutes of Health/National Center for Advancing Translational Sciences/National Institute of Diabetes and Digestive and Kidney Diseases (NIH/NCATS/NIDDK)-sponsored Porphyrias Consortium of porphyria diagnostic experts is establishing an online database that will collate biochemical and clinical evidence verifying the pathogenicity of the published and newly identified variants in the AHP-causing genes. The overall goal of the International Porphyria Molecular Diagnostic Collaborative is to determine the pathogenic and benign variants for all eight porphyrias. Here we describe the overall objectives and the initial efforts to validate pathogenic and benign variants in the respective heme biosynthetic genes causing the AHPs.

Best practice guidelines on first-line laboratory testing for porphyria.

The porphyrias are disorders of haem biosynthesis which present with acute neurovisceral attacks or disorders of sun-exposed skin. Acute attacks occur mainly in adults and comprise severe abdominal pain, nausea, vomiting, autonomic disturbance, central nervous system involvement and peripheral motor neuropathy. Cutaneous porphyrias can be acute or chronic presenting at various ages. Timely diagnosis depends on clinical suspicion leading to referral of appropriate samples for screening by reliable biochemical methods. All samples should be protected from light. Investigation for an acute attack: • Porphobilinogen (PBG) quantitation in a random urine sample collected during symptoms. Urine concentration must be assessed by measuring creatinine, and a repeat requested if urine creatinine <2 mmol/L. • Urgent porphobilinogen testing should be available within 24 h of sample receipt at the local laboratory. Urine porphyrin excretion (TUP) should subsequently be measured on this urine. • Urine porphobilinogen should be measured using a validated quantitative ion-exchange resin-based method or LC-MS. • Increased urine porphobilinogen excretion requires confirmatory testing and clinical advice from the National Acute Porphyria Service. • Identification of individual acute porphyrias requires analysis of urine, plasma and faecal porphyrins. Investigation for cutaneous porphyria: • An EDTA blood sample for plasma porphyrin fluorescence emission spectroscopy and random urine sample for TUP. • Whole blood for porphyrin analysis is essential to identify protoporphyria. • Faeces need only be collected, if first-line tests are positive or if clinical symptoms persist. Investigation for latent porphyria or family history: • Contact a specialist porphyria laboratory for advice. Clinical, family details are usually required.

Diagnostic strategies for autosomal dominant acute porphyrias: retrospective analysis of 467 unrelated patients referred for mutational analysis of the HMBS, CPOX, or PPOX gene.

BACKGROUND: Clinically indistinguishable attacks of acute porphyria occur in acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP). There are few evidence-based diagnostic strategies for these disorders. METHODS: The diagnostic sensitivity of mutation detection was determined by sequencing and gene-dosage analysis to search for mutations in 467 sequentially referred, unrelated patients. The diagnostic accuracy of plasma fluorescence scanning, fecal porphyrin analysis, and porphobilinogen deaminase (PBGD) assay was assessed in mutation-positive patients (AIP, 260 patients; VP, 152 patients; HCP, 31 patients). RESULTS: Sensitivities (95% CI) for mutation detection were as follows: AIP, 98.1% (95.6%-99.2%); HCP, 96.9% (84.3%-99.5%); VP, 100% (95.7%-100%). We identified 5 large deletions in the HMBS gene (hydroxymethylbilane synthase) and one in the CPOX gene (coproporphyrinogen oxidase). The plasma fluorescence scan was positive more often in VP (99% of patients) than in AIP (68%) or HCP (29%). The wavelength of the fluorescence emission peak and the fecal coproporphyrin isomer ratio had high diagnostic specificity and sensitivity for differentiating between AIP, HCP, and VP. DNA analysis followed by PBGD assay in mutation-negative patients had greater diagnostic accuracy for AIP than either test alone. CONCLUSIONS: When PBG excretion is increased, 2 investigations (plasma fluorescence scanning, the coproporphyrin isomer ratio) are sufficient, with rare exceptions, to identify the type of acute porphyria. When the results of PBG, 5-aminolevulinate, and porphyrin analyses are within reference intervals and clinical suspicion that a past illness was caused by an acute porphyria remains high, mutation analysis of the HMBS gene followed by PBGD assay is an effective strategy for diagnosis or exclusion of AIP.

Seasonal palmar keratoderma in erythropoietic protoporphyria indicates autosomal recessive inheritance.

Erythropoietic protoporphyria (EPP) is an inherited disorder that results from partial deficiency of ferrochelatase (FECH). It is characterized clinically by acute photosensitivity and, in 2% of patients, liver disease. Inheritance is usually autosomal dominant with low penetrance but is recessive in about 4% of families. A cross-sectional study of 223 patients with EPP in the United Kingdom identified six individuals with palmar keratoderma. We now show that these and three additional patients, from six families, have an inherited subtype of EPP which is characterized by seasonal palmar keratoderma, relatively low erythrocyte protoporphyrin concentrations, and recessive inheritance. No patient had evidence of liver dysfunction; four patients had neurological abnormalities. Patients were hetero- or homoallelic for nine different FECH mutations; four of which were previously unreported. Prokaryotic expression predicted that FECH activities were 2.7-25% (mean 10.6%) of normal. Neither mutation type nor FECH activity provided an explanation for the unusual phenotype. Our findings show that palmar keratoderma is a clinical indicator of recessive EPP, identify a phenotype that occurs in 38% of reported families with recessive EPP that to our knowledge is previously unreported, and suggest that patients with this phenotype may carry a lower risk of liver disease than other patients with recessive EPP.

C-terminal deletions in the ALAS2 gene lead to gain of function and cause X-linked dominant protoporphyria without anemia or iron overload.

All reported mutations in ALAS2, which encodes the rate-regulating enzyme of erythroid heme biosynthesis, cause X-linked sideroblastic anemia. We describe eight families with ALAS2 deletions, either c.1706-1709 delAGTG (p.E569GfsX24) or c.1699-1700 delAT (p.M567EfsX2), resulting in frameshifts that lead to replacement or deletion of the 19-20 C-terminal residues of the enzyme. Prokaryotic expression studies show that both mutations markedly increase ALAS2 activity. These gain-of-function mutations cause a previously unrecognized form of porphyria, X-linked dominant protoporphyria, characterized biochemically by a high proportion of zinc-protoporphyrin in erythrocytes, in which a mismatch between protoporphyrin production and the heme requirement of differentiating erythroid cells leads to overproduction of protoporphyrin in amounts sufficient to cause photosensitivity and liver disease.

Gene dosage analysis identifies large deletions of the FECH gene in 10% of families with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is an inherited cutaneous porphyria characterized by partial deficiency of ferrochelatase (FECH), accumulation of protoporphyrin IX in erythrocytes, skin, and liver, and acute photosensitivity. Genetic counseling in EPP requires identification of FECH mutations, but current sequencing-based procedures fail to detect mutations in about one in six families. We have used gene dosage analysis by quantitative PCR to identify large deletions of the FECH gene in 19 (58%) of 33 unrelated UK patients with EPP in whom mutations could not be detected by sequencing. Seven deletions were identified, six of which were previously unreported. Breakpoints were identified for six deletions (c.1-7887-IVS1+2425insTTCA; c.1-9629-IVS1+2437; IVS2-1987-IVS4+352del; c.768-IVS7+244del; IVS7+2784-IVS9+108del; IVS6+2350-TGA+95del). Five breakpoints were in intronic repeat sequences (AluSc, AluSq, AluSx, L1MC4). The remaining deletion (Del Ex3-4) is likely to be a large insertion-deletion. Combining quantitative PCR with routine sequencing increased the sensitivity of mutation detection in 189 unrelated UK patients with EPP from 83% (95% CI: 76-87%) to 93% (CI: 88-96%) (P=0.003). Our findings show that large deletions of the FECH gene are an important cause of EPP. Gene dosage analysis should be incorporated into routine procedures for mutation detection in EPP.

Exonic deletions as a cause of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is an inherited disorder that results from partial deficiency of ferrochelatase (FECH), the terminal enzyme of haem biosynthesis. Current methods that examine the exons and their flanking regions of the FECH gene fail to identify mutations in about one in seven of families with EPP. The presence in some families of intragenic deletions that are not identifiable by current methods for sequencing the FECH gene may partly explain the low sensitivity of mutation detection in EPP. Here we describe the identification by gene dosage analysis of a deletion of exons 3 and 4 in a family with EPP in whom a mutation had not been found by sequencing-based methods.

NTNG1 mutations are a rare cause of Rett syndrome.

A translocation that disrupted the netrin G1 gene (NTNG1) was recently reported in a patient with the early seizure variant of Rett syndrome (RTT). The netrin G1 protein (NTNG1) has an important role in the developing central nervous system, particularly in axonal guidance, signalling and NMDA receptor function and was a good candidate gene for RTT. We recruited 115 patients with RTT (females: 25 classic and 84 atypical; 6 males) but no mutation in the MECP2 gene. For those 52 patients with epileptic seizure onset in the first 6 months of life, CDKL5 mutations were also excluded. We aimed to determine whether mutations in NTNG1 accounted for a significant subset of patients with RTT, particularly those with the early onset seizure variant and other atypical presentations. We sequenced the nine coding exons of NTNG1 and identified four sequence variants, none of which were likely to be pathogenic. Mutations in the NTNG1 gene appear to be a rare cause of RTT but NTNG1 function demands further investigation in relation to the central nervous system pathophysiology of the disorder.

Photosensitivity and acute liver injury in myeloproliferative disorder secondary to late-onset protoporphyria caused by deletion of a ferrochelatase gene in hematopoietic cells.

Late-onset erythropoietic protoporphyria (EPP) is a rare complication of myelodysplastic syndrome (MDS) but has not been described in association with a myeloproliferative disorder (MPD). EPP is normally an inherited disorder characterized by photosensitivity that starts in early childhood and results from overproduction of protoporphyrin secondary to ferrochelatase (FECH) deficiency. Severe liver disease occurs in 1% to 2% of patients. Here we report that severe photosensitivity and cholestatic liver disease in a patient with a myeloproliferative disorder was caused by excess protoporphyrin production from a clone of hematopoietic cells in which one FECH allele had been deleted. Our observations suggest that the usual explanation for the association of late-onset EPP with MPD and MDS is acquired somatic mutation of one FECH allele in bone marrow and show for the first time that the consequent overproduction of protoporphyrin may be severe enough to cause acute liver damage.

Dimensional phenotypic analysis and functional categorisation of mutations reveal novel genotype-phenotype associations in Rett syndrome.

We aimed to improve the understanding of genotype-phenotype correlations in Rett syndrome (RS) by adopting a novel approach to categorising phenotypic dimensions - separating typicality of presentation, outcome severity and age of onset - and by classifying MECP2 mutations strictly by predicted functional attributes. MECP2 mutation screening results were available on 190 patients with a clinical diagnosis of RS (140 cases with classic RS, 50 with atypical RS). 135 cases had identified mutations. Of the 140 patients, 116 with classic RS (82.9%) had an identified mutation compared with 19 of 50 patients (38%) with an atypical presentation. Cases with early onset of regression and seizures, and those with clinical features that might indicate alternative aetiologies, were less likely to have mutations. Individuals with late truncating mutations had a less typical presentation than cases with missense and early truncating mutations, presumably reflecting greater residual function of MECP2 protein. Individuals with early truncating mutations had a more severe outcome than cases with missense and late truncating mutations. These findings held when restricting the analysis to cases over 15 years of age and classic cases only. Previous findings of variation in severity among the common mutations were confirmed. The approach to phenotypic and genotypic classification adopted here allowed us to identify genotype-phenotype associations in RS that may aid our understanding of pathogenesis and also contribute to clinical knowledge on the impact of different types of mutations.

Early onset seizures and Rett-like features associated with mutations in CDKL5.

Mutations in the CDKL5 gene (also known as STK9) have recently been shown to cause early onset epilepsy and severe mental retardation (ISSX or West syndrome). Patients with CDKL5 mutations sometimes also show features similar to those seen in Rett Syndrome (RTT). We have screened the CDKL5 gene in 94 patients with RTT or a RTT-like phenotype who had tested negative for MECP2 mutations (13 classical RTT female subjects, 25 atypical RTT female subjects, 40 RTT-like female and 16 RTT-like male subjects; 33 of the patients had early onset seizures). Novel pathogenic CDKL5 mutations were identified in three girls, two of whom had initially been diagnosed with the early onset seizure variant of RTT and the other with early onset seizures and some features of RTT. In addition, the 33 patients with early seizures were screened for the most common mutations in the ARX gene but none were found. Combining our three new cases with the previously published cases, 13/14 patients with CDKL5 mutations presented with seizures before the age of 3 months.

Variation in exon 1 coding region and promoter of MECP2 in Rett syndrome and controls.

Mutations in MECP2 are a cause of Rett syndrome. Recently, a new isoform of MeCP2 was described, which has an alternative N-terminus, transcribed from exon 1. We screened exon 1 and the promoter region of MECP2 in 97 mutation-negative Rett syndrome cases. We found two sequence variants, but there was no evidence that they are pathogenic. Mutations in exon 1 and the promoter of MECP2 are not a common cause of Rett syndrome.

Large deletions of the MECP2 gene detected by gene dosage analysis in patients with Rett syndrome.

MECP2 mutations are responsible for Rett syndrome (RTT). Approximately a quarter of classic RTT cases, however, do not have an identifiable mutation of the MECP2 gene. We hypothesized that larger deletions arising from a deletion prone region (DPR) occur commonly and are not being routinely detected by the current PCR-mediated screening strategies. We developed and applied a quantitative PCR strategy (qPCR) to samples referred for diagnostic assessment from 140 patients among whom RTT was strongly suspected and from a second selected group of 31 girls with classical RTT. Earlier MECP2 mutation screening in both groups of patients had yielded a wild-type result. We identified 10 large deletions (7.1%) within the first group and five deletions in the second group (16.1%). Sequencing of the breakpoints in 11 cases revealed that eight cases had one breakpoint within the DPR. Among seven cases, the breakpoint distant to the DPR involved one of several Alu repeats. Sequence analysis of the junction sequences revealed that eight cases had complex rearrangements. Examination of the MECP2 genomic sequence reveals that it is highly enriched for repeat elements, with the content of Alu repeats rising to 27.8% in intron 2, in which there was an abundance of breakpoints among our patients. Furthermore, a perfect chi sequence, known to be recombinogenic in E. coli, is located in the DPR. We propose that the chi sequence and Alu repeats are potent factors contributing to genomic rearrangement. We suggest that routine mutation screening in MECP2 should include quantitative analysis of the genomic sequences flanking the DPR.