Real-life use of tolvaptan in ADPKD: a retrospective analysis of a large Canadian cohort.

Tolvaptan is the first disease-modifying drug proven to slow eGFR decline in high-risk patients with ADPKD. However, barriers from the patient perspective to its use in real-life settings have not been systemically examined in a large cohort. This was a single-center, retrospective study of 523 existing or new patients with ADPKD followed at the Center for Innovative Management of PKD in Toronto, Ontario, between January 1, 2016 to December 31, 2018. All patients underwent clinical assessment including total kidney volume measurements and Mayo Clinic Imaging Class (MCIC). Those who were deemed to be at high risk were offered tolvaptan with their preference (yes or no) and reasons for their choices recorded. Overall, 315/523 (60%) patients had MCIC 1C-1E; however, only 96 (30%) of them were treated with tolvaptan at their last follow-up. Among these high-risk patients, those not treated versus treated with tolvaptan were more likely to have a higher eGFR (82 ± 26 vs. 61 ± 27 ml/min/1.73 m2), CKD stages 1-2 (79% vs. 41%), and MCIC 1C (63% vs. 31%). The most common reasons provided for not taking tolvaptan were lifestyle preference related to the aquaretic effect (51%), older age ≥ 60 (12%), and pregnancy/family planning (6%). In this real-world experience, at least 60% of patients with ADPKD considered to be at high risk for progression to ESKD by imaging were not treated with tolvaptan; most of them had early stages of CKD with well-preserved eGFR and as such, were prime targets for tolvaptan therapy to slow disease progression. Given that the most common reason for tolvaptan refusal was the concern for intolerability of the aquaretic side-effect, strategies to mitigate this may help to reduce this barrier to tolvaptan therapy.

Nemaline myopathy: reclassification of previously reported variants according to ACMG guidelines, and report of novel genetic variants.

Nemaline myopathy (NM) is a heterogeneous genetic neuromuscular disorder characterized by rod bodies in muscle fibers resulting in multiple complications due to muscle weakness. NM patients and their families could benefit from genetic analysis for early diagnosis, carrier and prenatal testing; however, clinical classification of variants is subject to change as further information becomes available. Reclassification can significantly alter the clinical management of patients and their families. We used the newly published data and ACMG/AMP guidelines to reassess NM-associated variants previously reported by clinical laboratories (ClinVar). Our analyses on rare variants that were not canonical loss-of-function (LOF) resulted in the downgrading of ~29% (28/97) of variants from pathogenic or likely-pathogenic (P/LP) to variants of uncertain significance (VUS). In addition, we analyzed the splicing effect of variants identified in NM patients by clinical laboratories or research, using an accurate in silico prediction tool that applies a deep-learning network. We identified 55 rare variants that may impact splicing (cryptic splicing). We also analyzed six new NM families and identified eight variants in NEB and ACTA1, including three novel variants: homozygous pathogenic c.164A > G (p.Tyr55Cys), and homozygous likely pathogenic c.980T > C (p.Met327Thr) in ACTA1, and heterozygous VUS c.18694-3T > G in NEB. This study demonstrates the importance of reclassifying variants to facilitate more definitive "calls" on causality or no causality in clinical genetic testing of patients with NM. Reclassification of ~150 variants is now available for improved clinical management, risk counseling and screening of NM patients.

Atypical Polycystic Kidney Disease as defined by Imaging.

Using age- and height-adjusted total kidney volume, the Mayo Clinic Imaging Classification provides a validated approach to assess the risk of chronic kidney disease (CKD) progression in autosomal dominant polycystic kidney disease (ADPKD), but requires excluding patients with atypical imaging patterns, whose clinical characteristics have been poorly defined. We report an analysis of the prevalence, clinical and genetic characteristics of patients with atypical polycystic kidney disease by imaging. Patients from the extended Toronto Genetic Epidemiology Study of Polycystic Kidney Disease recruited between 2016 and 2018 completed a standardized clinical questionnaire, kidney function assessment, genetic testing, and kidney imaging by magnetic resonance or computed tomography. We compared the prevalence, clinical features, genetics, and renal prognosis of atypical versus typical polycystic kidney disease by imaging. Forty-six of the 523 (8.8%) patients displayed atypical polycystic kidney disease by imaging; they were older (55 vs. 43 years; P < 0.001), and less likely to have a family history of ADPKD (26.1% vs. 74.6%; P < 0.001), a detectable PKD1 or PKD2 mutation (9.2% vs. 80.4%; P < 0.001), or progression to CKD stage 3 or stage 5 (P < 0.001). Patients with atypical polycystic kidney disease by imaging represent a distinct prognostic group with a low likelihood of progression to CKD.

Urinary epidermal growth factor/monocyte chemotactic peptide 1 ratio as non-invasive predictor of Mayo clinic imaging classes in autosomal dominant polycystic kidney disease.

BACKGROUND: Age- and height-adjusted total kidney volume is currently considered the best prognosticator in patients with autosomal dominant polycystic kidney disease. We tested the ratio of urinary epidermal growth factor and monocyte chemotactic peptide 1 for the prediction of the Mayo Clinic Imaging Classes. METHODS: Urinary epidermal growth factor and monocyte chemotactic peptide 1 levels were measured in two independent cohorts (discovery, n = 74 and validation set, n = 177) and healthy controls (n = 59) by immunological assay. Magnetic resonance imaging parameters were used for total kidney volume calculation and the Mayo Clinic Imaging Classification defined slow (1A-1B) and fast progressors (1C-1E). Microarray and quantitative gene expression analysis were used to test epidermal growth factor and monocyte chemotactic peptide 1 gene expression. RESULTS: Baseline ratio of urinary epidermal growth factor and monocyte chemotactic peptide 1 correlated with total kidney volume adjusted for height (r = - 0.6, p < 0.001), estimated glomerular filtration rate (r = 0.69 p < 0.001), discriminated between Mayo Clinic Imaging Classes (p < 0.001), and predicted the variation of estimated glomerular filtration rate at 10 years (r = - 0.51, p < 0.001). Conditional Inference Trees identified cut-off levels of the ratio of urinary epidermal growth factor and monocyte chemotactic peptide 1 for slow and fast progressors at > 132 (100% slow) and < 25.76 (89% and 86% fast, according to age), with 94% sensitivity and 66% specificity (p = 6.51E-16). Further, the ratio of urinary epidermal growth factor and monocyte chemotactic peptide 1 at baseline showed a positive correlation (p = 0.006, r = 0.36) with renal outcome (delta-estimated glomerular filtration rate per year, over a mean follow-up of 4.2 ± 1.2 years). Changes in the urinary epidermal growth factor and monocyte chemotactic peptide 1 were mirrored by gene expression levels in both human kidney cysts (epidermal growth factor: - 5.6-fold, fdr = 0.001; monocyte chemotactic peptide 1: 3.1-fold, fdr = 0.03) and Pkd1 knock-out mouse kidney (Egf: - 14.8-fold, fdr = 2.37E-20, Mcp1: 2.8-fold, fdr = 6.82E-15). CONCLUSION: The ratio of urinary epidermal growth factor and monocyte chemotactic peptide 1 is a non-invasive pathophysiological biomarker that can be used for clinical risk stratification in autosomal dominant polycystic kidney disease.

Shared pathobiology identifies AMPK as a therapeutic target for obesity and autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common Mendelian kidney disease, affecting approximately one in 1,000 births and accounting for 5% of end-stage kidney disease in developed countries. The pathophysiology of ADPKD is strongly linked to metabolic dysregulation, which may be secondary to defective polycystin function. Overweight and obesity are highly prevalent in patients with ADPKD and constitute an independent risk factor for progression. Recent studies have highlighted reduced AMP-activated protein kinase (AMPK) activity, increased mammalian target of rapamycin (mTOR) signaling, and mitochondrial dysfunction as shared pathobiology between ADPKD and overweight/obesity. Notably, mTOR and AMPK are two diametrically opposed sensors of energy metabolism that regulate cell growth and proliferation. However, treatment with the current generation of mTOR inhibitors is poorly tolerated due to their toxicity, making clinical translation difficult. By contrast, multiple preclinical and clinical studies have shown that pharmacological activation of AMPK provides a promising approach to treat ADPKD. In this narrative review, we summarize the pleiotropic functions of AMPK as a regulator of cellular proliferation, macromolecule metabolism, and mitochondrial biogenesis, and discuss the potential for pharmacological activation of AMPK to treat ADPKD and obesity-related kidney disease.

Patients with Protein-Truncating PKD1 Mutations and Mild ADPKD.

BACKGROUND AND OBJECTIVES: Progression of autosomal dominant polycystic kidney disease (ADPKD) is highly variable. On average, protein-truncating PKD1 mutations are associated with the most severe kidney disease among all mutation classes. Here, we report that patients with protein-truncating PKD1 mutations may also have mild kidney disease, a finding not previously well recognized. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: From the extended Toronto Genetic Epidemiologic Study of Polycystic Kidney Disease, 487 patients had PKD1 and PKD2 sequencing and typical ADPKD imaging patterns by magnetic resonance imaging or computed tomography. Mayo Clinic Imaging Classification on the basis of age- and height-adjusted total kidney volume was used to assess their cystic disease severity; classes 1A or 1B were used as a proxy to define mild disease. Multivariable linear regression was performed to test the effects of age, sex, and mutation classes on log-transformed height-adjusted total kidney volume and eGFR. RESULTS: Among 174 study patients with typical imaging patterns and protein-truncating PKD1 mutations, 32 (18%) were found to have mild disease on the basis of imaging results (i.e., Mayo Clinic Imaging class 1A-1B), with their mutations spanning the entire gene. By multivariable analyses of age, sex, and mutation class, they displayed mild disease similar to patients with PKD2 mutations and Mayo Clinic Imaging class 1A-1B. Most of these mildly affected patients with protein-truncating PKD1 mutations reported a positive family history of ADPKD in preceding generations and displayed significant intrafamilial disease variability. CONCLUSIONS: Despite having the most severe mutation class, 18% of patients with protein-truncating PKD1 mutations had mild disease on the basis of clinical and imaging assessment. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2021_02_18_CJN11100720_final.mp3.

Insights into Autosomal Dominant Polycystic Kidney Disease from Genetic Studies.

Autosomal dominant polycystic kidney disease is the most common monogenic cause of ESKD. Genetic studies from patients and animal models have informed disease pathobiology and strongly support a "threshold model" in which cyst formation is triggered by reduced functional polycystin dosage below a critical threshold within individual tubular epithelial cells due to (1) germline and somatic PKD1 and/or PKD2 mutations, (2) mutations of genes (e.g., SEC63, SEC61B, GANAB, PRKCSH, DNAJB11, ALG8, and ALG9) in the endoplasmic reticulum protein biosynthetic pathway, or (3) somatic mosaicism. Genetic testing has the potential to provide diagnostic and prognostic information in cystic kidney disease. However, mutation screening of PKD1 is challenging due to its large size and complexity, making it both costly and labor intensive. Moreover, conventional Sanger sequencing-based genetic testing is currently limited in elucidating the causes of atypical polycystic kidney disease, such as within-family disease discordance, atypical kidney imaging patterns, and discordant disease severity between total kidney volume and rate of eGFR decline. In addition, environmental factors, genetic modifiers, and somatic mosaicism also contribute to disease variability, further limiting prognostication by mutation class in individual patients. Recent innovations in next-generation sequencing are poised to transform and extend molecular diagnostics at reasonable costs. By comprehensive screening of multiple cystic disease and modifier genes, targeted gene panel, whole-exome, or whole-genome sequencing is expected to improve both diagnostic and prognostic accuracy to advance personalized medicine in autosomal dominant polycystic kidney disease.

Global microRNA profiling in human urinary exosomes reveals novel disease biomarkers and cellular pathways for autosomal dominant polycystic kidney disease.

MicroRNAs (miRNAs) play an important role in regulating gene expression in health and disease but their role in modifying disease expression in Autosomal Dominant Polycystic Kidney Disease (ADPKD) remains uncertain. Here, we profiled human urinary exosome miRNA by global small RNA-sequencing in an initial discovery cohort of seven patients with ADPKD with early disease (eGFR over 60ml/min/1.73m2), nine with late disease (eGFR under 60ml/min/1.73m2), and compared their differential expression with six age and sex matched healthy controls. Two kidney-enriched candidate miRNA families were identified (miR-192/miR-194-2 and miR-30) and selected for confirmatory testing in a 60 patient validation cohort by quantitative polymerase chain reaction. We confirmed that miR-192-5p, miR-194-5p, miR-30a-5p, miR-30d-5p and miR-30e-5p were significantly downregulated in patient urine exosomes, in murine Pkd1 cystic kidneys and in human PKD1 cystic kidney tissue. All five miRNAs showed significant correlations with baseline eGFR and ultrasound-determined mean kidney length and improved the diagnostic performance (area under the curve) of mean kidney length for the rate of disease progression. Finally, inverse correlations of these two miRNA families with increased expression in their predicted target genes in patient PKD1 cystic tissue identified dysregulated pathways and transcriptional networks including novel interactions between miR-194-5p and two potentially relevant candidate genes, PIK3R1 and ANO1. Thus, our results identify a subset of urinary exosomal miRNAs that could serve as novel biomarkers of disease progression and suggest new therapeutic targets in ADPKD.

Intrafamilial Variability of ADPKD.

INTRODUCTION: Discordance in kidney disease severity between affected relatives is a recognized feature of autosomal dominant polycystic kidney disease (ADPKD). Here, we report a systematic study of a large cohort of families to define the prevalence and clinical features of intrafamilial discordance in ADPKD. METHODS: The extended Toronto Genetic Epidemiology Study of Polycystic Kidney Disease (eTGESP) cohort includes 1390 patients from 612 unrelated families with ADPKD ascertained in a regional polycystic kidney disease center. All probands underwent comprehensive PKD1 and PKD2 mutation screening. Total kidney volume by magnetic resonance imaging (MRI) was available in 500 study patients. RESULTS: Based on (i) rate of estimated glomerular filtration rate (eGFR) decline, (ii) age at onset of end-stage renal disease (ESRD), and (iii) Mayo Clinic Imaging Classification (MCIC), 20% of patients were classified as having mild disease, and 33% as having severe disease. Intrafamilial ADPKD discordance with at least 1 mild and 1 severe case was observed in 43 of 371 (12%) families, at a similar frequency regardless of the responsible gene (PKD1/PKD2/no mutation detected) or mutation type (protein-truncating versus nontruncating). Intrafamilial discordance was more common in larger families and was present in 30% of families with more than 5 affected members. The heritability of age at onset of ESRD was similar between different mutation types. CONCLUSION: Extreme kidney disease discordance is present in at least 12% of families with ADPKD, regardless of the underlying mutated gene or mutation class. Delineating genetic and environmental modifiers underlying the observed intrafamilial ADPKD variability will provide novel insights into the mechanisms of progression in ADPKD.

Integration of Genetic Testing and Pathology for the Diagnosis of Adults with FSGS.

BACKGROUND AND OBJECTIVES: FSGS and nephrotic syndrome studies have shown that single gene causes are more likely to be found in pediatric cases than adults. Consequently, many studies have examined limited gene panels in largely pediatric cohorts. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Whole-exome sequencing was performed in adults with FSGS diagnosed between 1976 and 2017 in the Toronto GN Registry. An expanded panel of 109 genes linked to FSGS, glomerular basement membrane abnormalities, as well as causes of pediatric ESKD including congenital abnormalities of the kidney and urinary tract (CAKUT) and nephronophthisis, were examined. RESULTS: The cohort was composed of 193 individuals from 179 families. Nearly half (49%) developed ESKD at a mean age of 47±17 years. The genetic diagnostic rate was 11%. Of definitely pathogenic variants, 55% were in COL4A (A3/A4/A5), 40% were in podocyte genes, and 5% were in CAKUT genes. Many, but not all individuals with COL4A definitely pathogenic variants had some evidence of glomerular basement membrane abnormalities. The estimated mean survival/age of kidney failure for individuals with COL4A definitely pathogenic variants was 58 years (95% confidence interval, 49 to 69), far later than what has been reported in the literature. Likely pathogenic variants were identified in an additional 9% of the cohort, with most in COL4A. Correlation with glomerular basement membrane morphology suggested a causal role for at least some of these likely pathogenic variants. CONCLUSIONS: Even with an expanded gene panel, we find that COL4A disorders are the leading monogenic cause in adults diagnosed with FSGS. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2019_01_15_CJASNPodcast_19_02_.mp3.

Evolving role of genetic testing for the clinical management of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused primarily by mutations of two genes, PKD1 and PKD2. In the presence of a positive family history of ADPKD, genetic testing is currently seldom indicated as the diagnosis is mostly based on imaging studies using well-established criteria. Moreover, PKD1 mutation screening is technically challenging due to its large size, complexity (i.e. presence of six pseudogenes with high levels of DNA sequence similarity) and extensive allelic heterogeneity. Despite these limitations, recent studies have delineated a strong genotype-phenotype correlation in ADPKD and begun to unravel the role of genetics underlying cases with atypical phenotypes. Furthermore, adaptation of next-generation sequencing (NGS) to clinical PKD genetic testing will provide a high-throughput, accurate and comprehensive screen of multiple cystic disease and modifier genes at a reduced cost. In this review, we discuss the evolving indications of genetic testing in ADPKD and how NGS-based screening promises to yield clinically important prognostic information for both typical as well as unusual genetic (e.g. allelic or genic interactions, somatic mosaicism, cystic kidney disease modifiers) cases to advance personalized medicine in the era of novel therapeutics for ADPKD.

Prevalence Estimates of Polycystic Kidney and Liver Disease by Population Sequencing.

BACKGROUND: Estimating the prevalence of autosomal dominant polycystic kidney disease (ADPKD) is challenging because of age-dependent penetrance and incomplete clinical ascertainment. Early studies estimated the lifetime risk of ADPKD to be about one per 1000 in the general population, whereas recent epidemiologic studies report a point prevalence of three to five cases per 10,000 in the general population. METHODS: To measure the frequency of high-confidence mutations presumed to be causative in ADPKD and autosomal dominant polycystic liver disease (ADPLD) and estimate lifetime ADPKD prevalence, we used two large, population sequencing databases, gnomAD (15,496 whole-genome sequences; 123,136 exome sequences) and BRAVO (62,784 whole-genome sequences). We used stringent criteria for defining rare variants in genes involved in ADPKD (PKD1, PKD2), ADPLD (PRKCSH, SEC63, GANAB, ALG8, SEC61B, LRP5), and potential cystic disease modifiers; evaluated variants for quality and annotation; compared variants with data from an ADPKD mutation database; and used bioinformatic tools to predict pathogenicity. RESULTS: Identification of high-confidence pathogenic mutations in whole-genome sequencing provided a lower boundary for lifetime ADPKD prevalence of 9.3 cases per 10,000 sequenced. Estimates from whole-genome and exome data were similar. Truncating mutations in ADPLD genes and genes of potential relevance as cyst modifiers were found in 20.2 cases and 103.9 cases per 10,000 sequenced, respectively. CONCLUSIONS: Population whole-genome sequencing suggests a higher than expected prevalence of ADPKD-associated mutations. Loss-of-function mutations in ADPLD genes are also more common than expected, suggesting the possibility of unrecognized cases and incomplete penetrance. Substantial rare variation exists in genes with potential for phenotype modification in ADPKD.

Pharmacogenomics in diabetes: outcomes of thiamine therapy in TRMA syndrome.

AIMS/HYPOTHESIS: Diabetes is one of the cardinal features of thiamine-responsive megaloblastic anaemia (TRMA) syndrome. Current knowledge of this rare monogenic diabetes subtype is limited. We investigated the genotype, phenotype and response to thiamine (vitamin B1) in a cohort of individuals with TRMA-related diabetes. METHODS: We studied 32 individuals with biallelic SLC19A2 mutations identified by Sanger or next generation sequencing. Clinical details were collected through a follow-up questionnaire. RESULTS: We identified 24 different mutations, of which nine are novel. The onset of the first TRMA symptom ranged from birth to 4 years (median 6 months [interquartile range, IQR 3-24]) and median age at diabetes onset was 10 months (IQR 5-27). At presentation, three individuals had isolated diabetes and 12 had asymptomatic hyperglycaemia. Follow-up data was available for 15 individuals treated with thiamine for a median 4.7 years (IQR 3-10). Four patients were able to stop insulin and seven achieved better glycaemic control on lower insulin doses. These 11 patients were significantly younger at diabetes diagnosis (p = 0.042), at genetic testing (p = 0.01) and when starting thiamine (p = 0.007) compared with the rest of the cohort. All patients treated with thiamine became transfusion-independent and adolescents achieved normal puberty. There were no additional benefits of thiamine doses >150 mg/day and no reported side effects up to 300 mg/day. CONCLUSIONS/INTERPRETATION: In TRMA syndrome, diabetes can be asymptomatic and present before the appearance of other features. Prompt recognition is essential as early treatment with thiamine can result in improved glycaemic control, with some individuals becoming insulin-independent. DATA AVAILABILITY: SLC19A2 mutation details have been deposited in the Decipher database ( https://decipher.sanger.ac.uk/ ).

Molecular diagnosis of autosomal dominant polycystic kidney disease.

INTRODUCTION: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease that accounts for 5-10% of end-stage renal disease in developed countries. Mutations in PKD1 and PKD2 account for a majority of cases. Mutation screening of PKD1 is technically challenging largely due to the complexity resulting from duplication of its first 33 exons in six highly homologous pseudogenes (i.e. PKD1P1-P6). Protocol using locus-specific long-range and nested PCR has enabled comprehensive PKD1 mutation screening but is labor-intensive and costly. Here, the authors review how recent advances in Next Generation Sequencing are poised to transform and extend molecular diagnosis of ADPKD. Areas covered: Key original research articles and reviews of the topic published in English identified through PubMed from 1957-2017. Expert commentary: The authors review current and evolving approaches using targeted resequencing or whole genome sequencing for screening typical as well as challenging cases (e.g. cases with no detectable PKD1 and PKD2 mutations which may be due to somatic mosaicism or other cystic disease; and complex genetics such as bilineal disease).

Genetics of GNE myopathy in the non-Jewish Persian population.

GNE myopathy is an autosomal recessive adult-onset disorder characterized by progressive muscle atrophy and weakness, initially involving the distal muscles, while often sparing the quadriceps. It is caused by variants in the GNE gene that encodes a key bifunctional enzyme in the sialic acid biosynthetic pathway. We investigated the clinical and molecular characteristics of 18 non-Jewish Persian patients from 11 unrelated GNE myopathy families. In addition, we reviewed the previously reported cases and suggest genotype-phenotype correlations for the identified variants. Comprehensive clinical and laboratory evaluations were carried out. Sequencing of the GNE gene was performed using genomic DNA from the patients. Screening of the identified variants was performed in all relevant family members. Molecular analyses identified three causative homozygous GNE variants in 11 families: c.2228T>C (p. M743T) in 7, c.830G>A (p.R277Q) in 2, and one novel variation (c.804G>A) in 2 families that results in a synonymous codon change (p.L268=) and likely creates a novel splice site affecting the protein function. This study confirms that c.2228T>C (p.M743T) is the most prevalent disease-causing variant in the non-Jewish Persian population, but other GNE variants can cause GNE myopathy in this population. The patients with all three different variants had similar ages of onset. The youngest patient was an 18-year-old girl in whom the c.830G>A (p.R277Q) variant was identified, whereas the oldest onset age (31 years) was seen in a male patient with c.804G>A (p.L268=). The results of this investigation expand our knowledge about the genotype-phenotype correlations in GNE myopathy and aid in clinical management and therapeutic interventions.

Homozygosity mapping and whole exome sequencing reveal a novel homozygous COL18A1 mutation causing Knobloch syndrome.

The aim of this study was to identify the genetic basis of a chorioretinal dystrophy with high myopia of unknown origin in a child of a consanguineous marriage. The proband and ten family members of Iranian ancestry participated in this study. Linkage analysis was carried out with DNA samples of the proband and her parents by using the Human SNP Array 6.0. Whole exome sequencing (WES) was performed with the patients' DNA. Specific sequence alterations within the homozygous regions identified by whole exome sequencing were verified by Sanger sequencing. Upon genetic analysis, a novel homozygous frameshift mutation was found in exon 42 of the COL18A1 gene in the patient. Both parents were heterozygous for this sequence variation. Mutations in COL18A1 are known to cause Knobloch syndrome (KS). Retrospective analysis of clinical records of the patient revealed surgical removal of a meningocele present at birth. The clinical features shown by our patient were typical of KS with the exception of chorioretinal degeneration which is a rare manifestation. This is the first case of KS reported in a family of Iranian ancestry. We identified a novel disease-causing (deletion) mutation in the COL18A1 gene leading to a frameshift and premature stop codon in the last exon. The mutation was not present in SNP databases and was also not found in 192 control individuals. Its localization within the endostatin domain implicates a functional relevance of endostatin in KS. A combined approach of linkage analysis and WES led to a rapid identification of the disease-causing mutation even though the clinical description was not completely clear at the beginning.

Identification of a SLC19A2 nonsense mutation in Persian families with thiamine-responsive megaloblastic anemia.

Thiamine-responsive megaloblastic anemia (TRMA) is an autosomal recessive syndrome characterized by early-onset anemia, diabetes, and hearing loss caused by mutations in the SLC19A2 gene. We studied the genetic cause and clinical features of this condition in patients from the Persian population. A clinical and molecular investigation was performed in four patients from three families and their healthy family members. All had the typical diagnostic criteria. The onset of hearing loss in three patients was at birth and one patient also had a stroke and seizure disorder. Thiamine treatment effectively corrected the anemia in all of our patients but did not prevent hearing loss. Diabetes was improved in one patient who presented at the age of 8months with anemia and diabetes after 2months of starting thiamine. The coding regions of SLC19A2 were sequenced in all patients. The identified mutation was tested in all members of the families. Molecular analyses identified a homozygous nonsense mutation c.697C>T (p.Gln233*) as the cause of the disease in all families. This mutation was previously reported in a Turkish patient with TRMA and is likely to be a founder mutation in the Persian population.

Prenatal exposure to maternal cigarette smoking, amygdala volume, and fat intake in adolescence.

CONTEXT: Prenatal exposure to maternal cigarette smoking is a well-established risk factor for obesity, but the underlying mechanisms are not known. Preference for fatty foods, regulated in part by the brain reward system, may contribute to the development of obesity. OBJECTIVE: To examine whether prenatal exposure to maternal cigarette smoking is associated with enhanced fat intake and risk for obesity, and whether these associations may be related to subtle structural variations in brain regions involved in reward processing. DESIGN: Cross-sectional study of a population-based cohort. SETTING: The Saguenay Youth Study, Quebec, Canada. PARTICIPANTS: A total of 378 adolescents (aged 13 to 19 years; Tanner stage 4 and 5 of sexual maturation), half of whom were exposed prenatally to maternal cigarette smoking (mean [SD], 11.1 [6.8] cigarettes/d). MAIN OUTCOME MEASURES: Fat intake was assessed with a 24-hour food recall (percentage of energy intake consumed as fat). Body adiposity was measured with anthropometry and multifrequency bioimpedance. Volumes of key brain structures involved in reward processing, namely the amygdala, nucleus accumbens, and orbitofrontal cortex, were measured with magnetic resonance imaging. RESULTS: Exposed vs nonexposed subjects exhibited a higher total body fat (by approximately 1.7 kg; P = .009) and fat intake (by 2.7%; P = .001). They also exhibited a lower volume of the amygdala (by 95 mm3; P < .001) but not of the other 2 brain structures. Consistent with its possible role in limiting fat intake, amygdala volume correlated inversely with fat intake (r = -0.15; P = .006). CONCLUSIONS: Prenatal exposure to maternal cigarette smoking may promote obesity by enhancing dietary preference for fat, and this effect may be mediated in part through subtle structural variations in the amygdala.

Identification of homozygous WFS1 mutations (p.Asp211Asn, p.Gln486*) causing severe Wolfram syndrome and first report of male fertility.

Wolfram syndrome (WFS) is a neurodegenerative genetic condition characterized by juvenile-onset of diabetes mellitus and optic atrophy. We studied clinical features and the molecular basis of severe WFS (neurodegenerative complications) in two consanguineous families from Iran. A clinical and molecular genetic investigation was performed in the affected and healthy members of two families. The clinical diagnosis of WFS was confirmed by the existence of diabetes mellitus and optic atrophy in the affected patients, who in addition had severe neurodegenerative complications. Sequencing of WFS1 was undertaken in one affected member from each family. Targeted mutations were tested in all members of relevant families. Patients had most of the reported features of WFS. Two affected males in the first family had fathered unaffected children. We identified two homozygous mutations previously reported with apparently milder phenotypes: family 1: c.631G>A (p.Asp211Asn) in exon 5, and family 2: c.1456C>T (p.Gln486*) in exon 8. Heterozygous carriers were unaffected. This is the first report of male Wolfram patients who have successfully fathered children. Surprisingly, they also had almost all the complications associated with WFS. Our report has implications for genetic counseling and family planning advice for other affected families.

Identification of a novel nonsense mutation and a missense substitution in the AGPAT2 gene causing congenital generalized lipodystrophy type 1.

Congenital generalized lipodystrophy (CGL) is an autosomal recessive disease characterized by the generalized scant of adipose tissue. CGL type 1 is caused by mutations in gene encoding 1-acylglycerol-3-phosphate O-acyltransferase-2 (AGPAT2). A clinical and molecular genetic investigation was performed in affected and unaffected members of two families with CGL type 1. The AGPAT2 coding region was sequenced in index cases of the two families. The presence of the identified mutations in relevant parents was tested. We identified a novel nonsense mutation (c.685G>T, p.Glu229*) and a missense substitution (c.514G>A, p.Glu172Lys). The unaffected parents in both families were heterozygous carrier of the relevant mutation. The results expand genotype-phenotype spectrum in CGL1 and will have applications in prenatal and early diagnosis of the disease. This is the first report of Persian families identified with AGPAT2 mutations.