Roxanne Fischer

Autoimmune Lymphoproliferative Syndrome with Defective Fas: Genotype Influences Penetrance

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance. Most patients have a heterozygous mutation in the APT1 gene, which encodes Fas (CD95, APO-1), mediator of an apoptotic pathway crucial to lymphocyte homeostasis. Of 17 unique APT1 mutations in unrelated ALPS probands, 12 (71%) occurred in exons 7-9, which encode the intracellular portion of Fas. In vitro, activated lymphocytes from all 17 patients showed apoptotic defects when exposed to an anti-Fas agonist monoclonal antibody. Similar defects were found in a Fas-negative cell line transfected with cDNAs bearing each of the mutations. In cotransfection experiments, Fas constructs with either intra- or extracellular mutations caused dominant inhibition of apoptosis mediated by wild-type Fas. Two missense Fas variants, not restricted to patients with ALPS, were identified. Variant A(-1)T at the Fas signal-sequence cleavage site, which mediates apoptosis less well than wild-type Fas and is partially inhibitory, was present in 13% of African American alleles. Among the ALPS-associated Fas mutants, dominant inhibition of apoptosis was much more pronounced in mutants affecting the intracellular, versus extracellular, portion of the Fas receptor. Mutations causing disruption of the intracellular Fas death domain also showed a higher penetrance of ALPS phenotype features in mutation-bearing relatives. Significant ALPS-related morbidity occurred in 44% of relatives with intracellular mutations, versus 0% of relatives with extracellular mutations. Thus, the location of mutations within APT1 strongly influences the development and the severity of ALPS.

Characteristics of Congenital Hepatic Fibrosis in a Large Cohort of Patients With Autosomal Recessive Polycystic Kidney Disease

BACKGROUND & AIMS Autosomal recessive polycystic kidney disease (ARPKD), the most common ciliopathy of childhood, is characterized by congenital hepatic fibrosis and progressive cystic degeneration of kidneys. We aimed to describe congenital hepatic fibrosis in patients with ARPKD, confirmed by detection of mutations in PKHD1. METHODS Patients with ARPKD and congenital hepatic fibrosis were evaluated at the National Institutes of Health from 2003 to 2009. We analyzed clinical, molecular, and imaging data from 73 patients (age, 1-56 years; average, 12.7 ± 13.1 years) with kidney and liver involvement (based on clinical, imaging, or biopsy analyses) and mutations in PKHD1. RESULTS Initial symptoms were liver related in 26% of patients, and others presented with kidney disease. One patient underwent liver and kidney transplantation, and 10 others received kidney transplants. Four presented with cholangitis and one with variceal bleeding. Sixty-nine percent of patients had enlarged left lobes on magnetic resonance imaging, 92% had increased liver echogenicity on ultrasonography, and 65% had splenomegaly. Splenomegaly started early in life; 60% of children younger than 5 years had enlarged spleens. Spleen volume had an inverse correlation with platelet count and prothrombin time but not with serum albumin level. Platelet count was the best predictor of spleen volume (area under the curve of 0.88905), and spleen length corrected for patients height correlated inversely with platelet count (R(2) = 0.42, P < .0001). Spleen volume did not correlate with renal function or type of PKHD1 mutation. Twenty-two of 31 patients who underwent endoscopy were found to have varices. Five had variceal bleeding, and 2 had portosystemic shunts. Forty-percent had Caroli syndrome, and 30% had an isolated dilated common bile duct. CONCLUSIONS Platelet count is the best predictor of the severity of portal hypertension, which has early onset but is underdiagnosed in patients with ARPKD. Seventy percent of patients with ARPKD have biliary abnormalities. Kidney and liver disease are independent, and variability in severity is not explainable by type of PKHD1 mutation; ClinicalTrials.gov number, NCT00068224.

Correlation of Kidney Function, Volume and Imaging Findings, and PKHD1 Mutations in 73 Patients with Autosomal Recessive Polycystic Kidney Disease

BACKGROUND AND OBJECTIVES Renal function and imaging findings have not been comprehensively and prospectively characterized in a broad age range of patients with molecularly confirmed autosomal recessive polycystic kidney disease (ARPKD). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Ninety potential ARPKD patients were examined at the National Institutes of Health Clinical Center. Seventy-three fulfilled clinical diagnostic criteria, had at least one PKHD1 mutation, and were prospectively evaluated using magnetic resonance imaging (MRI), high-resolution ultrasonography (HR-USG), and measures of glomerular and tubular function. RESULTS Among 31 perinatally symptomatic patients, 25% required renal replacement therapy by age 11 years; among 42 patients who became symptomatic beyond 1 month (nonperinatal), 25% required kidney transplantation by age 32 years. Creatinine clearance (CrCl) for nonperinatal patients (103 +/- 54 ml/min/1.73 m(2)) was greater than for perinatal patients (62 +/- 33) (P = 0.002). Corticomedullary involvement on HR-USG was associated with a significantly worse mean CrCl (61 +/- 32) in comparison with medullary involvement only (131 +/- 46) (P < 0.0001). Among children with enlarged kidneys, volume correlated inversely with function, although with wide variability. Severity of PKHD1 mutations did not determine kidney size or function. In 35% of patients with medullary-only abnormalities, standard ultrasound was normal and the pathology was detectable with HR-USG. CONCLUSIONS In ARPKD, perinatal presentation and corticomedullary involvement are associated with faster progression of kidney disease. Mild ARPKD is best detected by HR-USG. Considerable variability occurs that is not explained by the type of PKHD1 mutation.

IL2RGbase: a database of γc-chain defects causing human X-SCID

Abstract X-linked severe combined immunodeficiency (X-SCID) is an immune disorder caused by mutations in the X-linked gene IL2RG, which encodes the common γ chain of the lymphocyte receptors for interleukin 2 (IL-2) and many other cytokines. A database of human X-SCID mutations (IL2RGbase) has been assembled, and this article summarizes the first 136 entries from unrelated patients.

DNA Variations in Oculocutaneous Albinism: An Updated Mutation List and Current Outstanding Issues in Molecular Diagnostics

Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. There are four types of OCA caused by mutations in TYR (OCA‐1), OCA2 (OCA‐2), TYRP1 (OCA‐3), or SLC45A2 (OCA‐4). Here we report 22 novel mutations in the OCA genes; 14 from a cohort of 61 patients seen as part of the NIH OCA Natural History Study and eight from a prior study at the University of Minnesota. We also include a comprehensive list of almost 600 previously reported OCA mutations along with ethnicity information, carrier frequencies, and in silico pathogenicity predictions as a supplement. In addition to discussing the clinical and molecular features of OCA, we address the cases of apparent missing heritability. In our cohort, 26% of patients did not have two mutations in a single OCA gene. We demonstrate the utility of multiple detection methods to reveal mutations missed by Sanger sequencing. Finally, we review the TYR p.R402Q temperature‐sensitive variant and confirm its association with cases of albinism with only one identifiable TYR mutation.

PKHD1 Sequence Variations in 78 Children and Adults with Autosomal Recessive Polycystic Kidney Disease and Congenital Hepatic Fibrosis

PKHD1, the gene mutated in autosomal recessive polycystic kidney disease (ARPKD)/congenital hepatic fibrosis (CHF), is an exceptionally large and complicated gene that consists of 86 exons and has a number of alternatively spliced transcripts. Its longest open reading frame contains 67 exons that encode a 4074 amino acid protein called fibrocystin or polyductin. The phenotypes caused by PKHD1 mutations are similarly complicated, ranging from perinatally-fatal PKD to CHF presenting in adulthood with mild kidney disease. To date, more than 300 mutations have been described throughout PKHD1. Most reported cohorts include a large proportion of perinatal-onset ARPKD patients; mutation detection rates vary between 42% and 87%. Here we report PKHD1 sequencing results on 78 ARPKD/CHF patients from 68 families. Differing from previous investigations, our study required survival beyond 6 months and included many adults with a CHF-predominant phenotype. We identified 77 PKHD1 variants (41 novel) including 19 truncating, 55 missense, 2 splice, and 1 small in-frame deletion. Using computer-based prediction tools (GVGD, PolyPhen, SNAP), we achieved a mutation detection rate of 79%, ranging from 63% in the CHF-predominant group to 82% in the remaining families. Prediction of the pathogenicity of missense variants will remain challenging until a functional assay is available. In the meantime, use of PKHD1 sequencing data for clinical decisions requires caution, especially when only novel or rare missense variants are identified.

Mutation spectrum of homogentisic acid oxidase (HGD) in alkaptonuria

Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder, characterized by accumulation of homogentisic acid, leading to darkened urine, pigmentation of connective tissue (ochronosis), joint and spine arthritis, and destruction of cardiac valves. AKU is due to mutations in the homogentisate dioxygenase gene (HGD) that converts homogentisic acid to maleylacetoacetic acid in the tyrosine catabolic pathway. Here we report a comprehensive mutation analysis of 93 patients enrolled in our study, as well as an extensive update of all previously published HGD mutations associated with AKU. Within our patient cohort, we identified 52 HGD variants, of which 22 were novel. This yields a total of 91 identified HGD variations associated with AKU to date, including 62 missense, 13 splice site, 10 frameshift, 5 nonsense, and 1 no‐stop mutation. Most HGD variants reside in exons 3, 6, 8, and 13. We assessed the potential effect of all missense variations on protein function, using five bioinformatic tools specifically designed for interpretation of missense variants (SIFT, POLYPHEN, PANTHER, PMUT, and SNAP). We also analyzed the potential effect of splice‐site variants using two different tools (BDGP and NetGene2). This study provides valuable resources for molecular analysis of alkaptonuria and expands our knowledge of the molecular basis of this disease. Hum Mutat 30:1–9, 2009. Published 2009 Wiley‐Liss, Inc.

Genetic alterations in caspase-10 may be causative or protective in autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by lymphadenopathy, elevated numbers of T cells with αβ-T cell receptors but neither CD4 nor CD8 co-receptors, and impaired lymphocyte apoptosis in vitro. Defects in the Fas receptor are the most common cause of ALPS (ALPS Ia), but in rare cases other apoptosis proteins have been implicated, including caspase-10 (ALPS II). We investigated the role of variants of caspase-10 in ALPS. Of 32 unrelated probands with ALPS who did not have Fas defects, two were heterozygous for the caspase-10 missense mutation I406L. Like the previously reported ALPS II-associated mutation L285F, I406L impaired apoptosis when transfected alone and dominantly inhibited apoptosis mediated by wild type caspase-10 in a co-transfection assay. Other variants in caspase-10, V410I and Y446C, were found in 3.4 and 1.6% of chromosomes in Caucasians, and in 0.5 and <0.5% of African Americans, respectively. In contrast to L285F and I406L, these variants had no dominant negative effect in co-transfection assays into the H9 lymphocytic cell line. We found healthy individuals homozygous for V410I, challenging the earlier suggestion that homozygosity for V410I alone causes ALPS. Moreover, an association analysis suggested protection from severe disease by caspase-10 V410I in 63 families with ALPS Ia due to dominant Fas mutations (P<0.05). Thus, different genetic variations in caspase-10 can produce contrasting phenotypic effects.

York platelet syndrome is a CRAC channelopathy due to gain-of-function mutations in STIM1

Store-operated Ca(2+) entry is the major route of replenishment of intracellular Ca(2+) in animal cells in response to the depletion of Ca(2+) stores in the endoplasmic reticulum. It is primarily mediated by the Ca(2+)-selective release-activated Ca(2+) (CRAC) channel, which consists of the pore-forming subunits ORAI1-3 and the Ca(2+) sensors, STIM1 and STIM2. Recessive loss-of-function mutations in STIM1 or ORAI1 result in immune deficiency and nonprogressive myopathy. Heterozygous gain-of-function mutations in STIM1 cause non-syndromic myopathies as well as syndromic forms of miosis and myopathy with tubular aggregates and Stormorken syndrome; some of these syndromic forms are associated with thrombocytopenia. Increased concentration of Ca(2+) as a result of store-operated Ca(2+) entry is essential for platelet activation. The York Platelet syndrome (YPS) is characterized by thrombocytopenia, striking ultrastructural platelet abnormalities including giant electron-opaque organelles and massive, multilayered target bodies and deficiency of platelet Ca(2+) storage in delta granules. We present clinical and molecular findings in 7 YPS patients from 4 families, demonstrating that YPS patients have a chronic myopathy associated with rimmed vacuoles and heterozygous gain-of-function STIM1 mutations. These findings expand the phenotypic spectrum of STIM1-related human disorders and define the molecular basis of YPS.

Analphoid marker chromosome in a patient with hyper-IgE syndrome, autism, and mild mental retardation

Abstract: Hyper-IgE syndrome with recurrent infections (HIES) is a primary immunodeficiency disease characterized by recurrent skin and lung abscesses and extreme elevations of serum IgE, but also involving dentition, bones, and connective tissue. Although the etiology of HIES is unknown, autosomal dominant inheritance has been observed in multiple kindreds. A 17 year old male with sporadic HIES, autism, and mild mental retardation was found to have a supernumerary marker chromosome in peripheral blood lymphocytes and skin fibroblasts. Microdissection and FISH analysis of the marker chromosome showed that it was derived from a small interstitial deletion of one homologue of chromosome 4q21. Lack of hybridization of probes specific for telomeres and alphoid centromeres, including a centromere 4 specific probe, established that the marker was an analphoid ring chromosome. Comparative genotyping of transformed B-cell subclones with (M+) and without (M–) the marker chromosome showed loss of the maternal alleles in M– cells between markers D4S1569 and D4S3010. FISH using YAC clones from 4q21 confirmed the size and location of the interstitial deletion. Thus our patients phenotypes were associated with de novo formation of a marker chromosome containing 15–20 cM of DNA deleted from his maternally derived chromosome 4. Proximal chromosome 4q therefore is a candidate region for disease genes for both HIES and autism. Identification of genes disrupted or lost during the formation of the marker chromosome as well as linkage studies in kindreds with HIES or autism may help us to understand the etiology of these complex phenotypes.