An Expanded Role for HLA Genes: HLA-B Encodes a microRNA that Regulates IgA and Other Immune Response Transcripts.

We describe a novel functional role for the HLA-B locus mediated by its intron-encoded microRNA (miRNA), miR-6891-5p. We show that in vitro inhibition of miR-6891-5p impacts the expression of nearly 200 transcripts within the B-lymphoblastoid cell line (B-LCL) COX, affecting a large number of metabolic pathways, including various immune response networks. The top affected transcripts following miR-6891-5p inhibition are those encoding the heavy chain of IgA. We identified a conserved miR-6891-5p target site on the 3'UTR of both immunoglobulin heavy chain alpha 1 and 2 (IGHA1 and IGHA2) transcripts and demonstrated that this miRNA modulates the expression of IGHA1 and IGHA2. B-LCLs from IgA-deficient patients expressed significantly elevated levels of miR-6891-5p when compared with unaffected family members. Upon inhibition of miR-6891-5p, IgA mRNA expression levels were increased, and IgA secretion was restored in the B-LCL of an IgA-deficient patient. These findings indicate that miR-6891-5p regulates IGHA1 and IGHA2 gene expression at the posttranscriptional level and suggest that increase in miR-6891-5p levels may contribute to the etiology of selective IgA deficiency.

Mung bean nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.

Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.

Mosaic maternal uniparental disomy of chromosome 15 in Prader-Willi syndrome: utility of genome-wide SNP array.

Prader-Willi syndrome is caused by the loss of paternal gene expression on 15q11.2-q13.2, and one of the mechanisms resulting in Prader-Willi syndrome phenotype is maternal uniparental disomy of chromosome 15. Various mechanisms including trisomy rescue, monosomy rescue, and post fertilization errors can lead to uniparental disomy, and its mechanism can be inferred from the pattern of uniparental hetero and isodisomy. Detection of a mosaic cell line provides a unique opportunity to understand the mechanism of uniparental disomy; however, mosaic uniparental disomy is a rare finding in patients with Prader-Willi syndrome. We report on two infants with Prader-Willi syndrome caused by mosaic maternal uniparental disomy 15. Patient 1 has mosaic uniparental isodisomy of the entire chromosome 15, and Patient 2 has mosaic uniparental mixed iso/heterodisomy 15. Genome-wide single-nucleotide polymorphism array was able to demonstrate the presence of chromosomally normal cell line in the Patient 1 and trisomic cell line in Patient 2, and provide the evidence that post-fertilization error and trisomy rescue as a mechanism of uniparental disomy in each case, respectively. Given its ability of detecting small percent mosaicism as well as its capability of identifying the loss of heterozygosity of chromosomal regions, genome-wide single-nucleotide polymorphism array should be utilized as an adjunct to the standard methylation analysis in the evaluation of Prader-Willi syndrome.

Succinate dehydrogenase kidney cancer: an aggressive example of the Warburg effect in cancer.

PURPOSE: Recently, a new renal cell cancer syndrome has been linked to germline mutation of multiple subunits (SDHB/C/D) of the Krebs cycle enzyme, succinate dehydrogenase. We report our experience with the diagnosis, evaluation and treatment of this novel form of hereditary kidney cancer. MATERIALS AND METHODS: Patients with suspected hereditary kidney cancer were enrolled on a National Cancer Institute institutional review board approved protocol to study inherited forms of kidney cancer. Individuals from families with germline SDHB, SDHC and SDHD mutations, and kidney cancer underwent comprehensive clinical and genetic evaluation. RESULTS: A total of 14 patients from 12 SDHB mutation families were evaluated. Patients presented with renal cell cancer at an early age (33 years, range 15 to 62), metastatic kidney cancer developed in 4 and some families had no manifestation other than kidney tumors. An additional family with 6 individuals found to have clear cell renal cell cancer that presented at a young average age (47 years, range 40 to 53) was identified with a germline SDHC mutation (R133X) Metastatic disease developed in 2 of these family members. A patient with a history of carotid body paragangliomas and an aggressive form of kidney cancer was evaluated from a family with a germline SDHD mutation. CONCLUSIONS: SDH mutation associated renal cell carcinoma can be an aggressive type of kidney cancer, especially in younger individuals. Although detection and management of early tumors is most often associated with a good outcome, based on our initial experience with these patients and our long-term experience with hereditary leiomyomatosis and renal cell carcinoma, we recommend careful surveillance of patients at risk for SDH mutation associated renal cell carcinoma and wide surgical excision of renal tumors.

A hemoglobin variant associated with neonatal cyanosis and anemia.

Globin-gene mutations are a rare but important cause of cyanosis. We identified a missense mutation in the fetal Gγ-globin gene (HBG2) in a father and daughter with transient neonatal cyanosis and anemia. This new mutation modifies the ligand-binding pocket of fetal hemoglobin by means of two mechanisms. First, the relatively large side chain of methionine decreases both the affinity of oxygen for binding to the mutant hemoglobin subunit and the rate at which it does so. Second, the mutant methionine is converted to aspartic acid post-translationally, probably through oxidative mechanisms. The presence of this polar amino acid in the heme pocket is predicted to enhance hemoglobin denaturation, causing anemia.

Identification of intragenic deletions and duplication in the FLCN gene in Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé syndrome (BHDS), caused by germline mutations in the folliculin (FLCN) gene, predisposes individuals to develop fibrofolliculomas, pulmonary cysts, spontaneous pneumothoraces, and kidney cancer. The FLCN mutation detection rate by bidirectional DNA sequencing in the National Cancer Institute BHDS cohort was 88%. To determine if germline FLCN intragenic deletions/duplications were responsible for BHDS in families lacking FLCN sequence alterations, 23 individuals from 15 unrelated families with clinically confirmed BHDS but no sequence variations were analyzed by real-time quantitative PCR (RQ-PCR) using primers for all 14 exons. Multiplex ligation-dependent probe amplification (MLPA) assay and array-based comparative genomic hybridization (aCGH) were utilized to confirm and fine map the rearrangements. Long-range PCR followed by DNA sequencing was used to define the breakpoints. We identified six unique intragenic deletions in nine patients from six different BHDS families including four involving exon 1, one that spanned exons 2-5, and one that encompassed exons 7-14 of FLCN. Four of the six deletion breakpoints were mapped, revealing deletions ranging from 5688 to 9189 bp. In addition, one 1341 bp duplication, which included exons 10 and 11, was identified and mapped. This report confirms that large intragenic FLCN deletions can cause BHDS and documents the first large intragenic FLCN duplication in a BHDS patient. Additionally, we identified a deletion "hot spot" in the 5'-noncoding-exon 1 region that contains the putative FLCN promoter based on a luciferase reporter assay. RQ-PCR, MLPA and aCGH may be used for clinical molecular diagnosis of BHDS in patients who are FLCN mutation-negative by DNA sequencing.

A rapid, noninvasive immunoassay for frataxin: utility in assessment of Friedreich ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by reduced amounts of the mitochondrial protein frataxin. Frataxin levels in research studies are typically measured via Western blot analysis from patient fibroblasts, lymphocytes, or muscle biopsies; none of these is ideal for rapid detection in large scale clinical studies. Recently, a rapid, noninvasive lateral flow immunoassay was developed to accurately measure picogram levels of frataxin protein and shown to distinguish lymphoblastoid cells from FRDA carriers, patients and controls. We expanded the immunoassay to measure frataxin directly in buccal cells and whole blood from a large cohort of controls, known carriers and patients typical of a clinical trial population. The assay in buccal cells shared a similar degree of variability with previous studies conducted in lymphoblastoid cells (~10% coefficient of variation in controls). Significant differences in frataxin protein quantity were seen between the mean group values of controls, carriers, and patient buccal cells (100, 50.2, and 20.9% of control, respectively) and in protein extracted from whole blood (100, 75.3, and 32.2%, respectively), although there was some overlap between the groups. In addition, frataxin levels were inversely related to GAA repeat length and correlated directly with age of onset. Subjects with one expanded GAA repeat and an identified frataxin point mutation also carried frataxin levels in the disease range. Some patients displaying an FRDA phenotype but carrying only a single identifiable mutation had frataxin levels in the FRDA patient range. One patient from this group has a novel deletion that included exons 2 and 3 of the FXN gene based on multiplex ligation-dependent probe amplification (MLPA) analysis of the FXN gene. The lateral flow immunoassay may be a useful means to noninvasively assess frataxin levels repetitively with minimal discomfort in FRDA patients in specific situations such as clinical trials, and as a complementary diagnostic tool to aid in identification and characterization of atypical patients.

Paraganglioma, neuroblastoma, and a SDHB mutation: Resolution of a 30-year-old mystery.

Familial paraganglioma/pheochromocytoma (PGL/PCC) is genetically heterogenous with mutations in three of the four subunits of the heterotetrameric mitochondrial complex II enzyme succinate dehydrogenase (SDH) being causally responsible for the majority of cases. In addition to PGL/PCC an array of non-paraganglial tumors have been described in affected individuals. We present a 30-year follow-up on the family of a deceased patient who synchronously developed malignant neuroblastoma (NBL), PCC, and renal cell carcinoma (RCC). Other family members with late onset disease have come to our attention, and molecular study revealed a mutation in the SDHB gene. Despite the embryologic relationship, NBL has been seen in only two previous patients with familial PGL/PCC, both with deletions of the SDHB gene. Review of the literature suggests the lack of a reported association between NBL and familial PGL/PCC may be an ascertainment bias. We further suggest that study of the SDH genes in NBL survivors who develop secondary solid tumors, particularly RCC, may correct this bias, and provide for more effective and comprehensive tumor screening in this patient population.

Interpretation of association signals and identification of causal variants from genome-wide association studies.

GWAS have been successful in identifying disease susceptibility loci, but it remains a challenge to pinpoint the causal variants in subsequent fine-mapping studies. A conventional fine-mapping effort starts by sequencing dozens of randomly selected samples at susceptibility loci to discover candidate variants, which are then placed on custom arrays or used in imputation algorithms to find the causal variants. We propose that one or several rare or low-frequency causal variants can hitchhike the same common tag SNP, so causal variants may not be easily unveiled by conventional efforts. Here, we first demonstrate that the true effect size and proportion of variance explained by a collection of rare causal variants can be underestimated by a common tag SNP, thereby accounting for some of the "missing heritability" in GWAS. We then describe a case-selection approach based on phasing long-range haplotypes and sequencing cases predicted to harbor causal variants. We compare this approach with conventional strategies on a simulated data set, and we demonstrate its advantages when multiple causal variants are present. We also evaluate this approach in a GWAS on hearing loss, where the most common causal variant has a minor allele frequency (MAF) of 1.3% in the general population and 8.2% in 329 cases. With our case-selection approach, it is present in 88% of the 32 selected cases (MAF = 66%), so sequencing a subset of these cases can readily reveal the causal allele. Our results suggest that thinking beyond common variants is essential in interpreting GWAS signals and identifying causal variants.

A novel von Hippel-Lindau point mutation presents as apparently sporadic pheochromocytoma.

Von Hippel Lindau disease is a common cause of apparently sporadic pheochromocytomas. Herein, we describe a 20-year-old man with an apparently sporadic pheochromocytoma associated with a novel, relatively conservative germline Gly104Val VHL gene mutation, which is localized within exon 1 of the VHL gene corresponding to the beta -domain of the VHL protein (pVHL). The nearly asymptomatic patient's father also carries the same mutation. Similar to other mutations localized in the same codon, the Gly104Val VHL mutation seems to have an attenuated disease phenotype.

Novel, complex interruptions of the GAA repeat in small, expanded alleles of two affected siblings with late-onset Friedreich ataxia.

Friedreich ataxia (FA) is an autosomal recessive disorder associated with expanded GAA repeats in intron 1 of the FRDA gene. Two siblings presented with a mild form of FA at >60 years of age. Both had a large expansion (>600 repeats) and a small expansion (120 repeats) by long-range PCR. Sequence analysis of the small allele revealed multiple, complex interruptions in the GAA repeat. These 2 patients presented later than predicted from their allele size alone, when compared with a large cohort of FA patients. Accounting for the interruptions in the GAA repeat, though, did not make the age of onset consistent with that noted in other patients. Three additional patients with late onset FA and small expanded alleles also exhibited interrupted GAA repeats that were not associated with inappropriately late onset. Our observations suggest that interrupted GAA repeats do not clearly impact the age of onset in FA.

Limb-girdle muscular dystrophy in the United States.

Limb-girdle muscular dystrophy (LGMD) has been linked to 15 chromosomal loci, 7 autosomal-dominant (LGMD1A to E) and 10 autosomal-recessive (LGMD2A to J). To determine the distribution of subtypes among patients in the United States, 6 medical centers evaluated patients with a referral diagnosis of LGMD. Muscle biopsies provided histopathology and immunodiagnostic testing, and their protein abnormalities along with clinical parameters directed mutation screening. The diagnosis in 23 patients was a disorder other than LGMD. Of the remaining 289 unrelated patients, 266 had muscle biopsies sufficient for complete microscopic evaluation; 121 also underwent Western blotting. From this combined evaluation, the distribution of immunophenotypes is 12% calpainopathy, 18% dysferlinopathy, 15% sarcoglycanopathy, 15% dystroglycanopathy, and 1.5% caveolinopathy. Genotypes distributed among 2 dominant and 7 recessive subtypes have been determined for 83 patients. This study of a large racially and ethnically diverse population of patients with LGMD indicates that establishing a putative subtype is possible more than half the time using available diagnostic testing. An efficient approach to genotypic diagnosis is muscle biopsy immunophenotyping followed by directed mutational analysis. The most common LGMDs in the United States are calpainopathies, dysferlinopathies, sarcoglycanopathies, and dystroglycanopathies.

A novel Indian beta-thalassemia mutation in the CACCC box of the promoter region.

This is the first report of a previously undescribed mutation in Indian subjects of the CACCC box of promoter region for beta-globin, which in combination with a common mutation produces thalassemia major in the offspring of the family.

Detection of germline deletions using real-time quantitative polymerase chain reaction in Japanese patients with von Hippel-Lindau disease.

Germline mutations of the VHL gene are responsible for VHL. Approximately 70% of VHL families display small intragenic mutations detectable by sequencing, whereas partial- or whole-gene deletions have been described in the majority of the remaining families. For such large deletions, complex genetic techniques other than sequencing might have to be used. In this study, we describe an RQ-PCR assay with TaqMan fluorescent probes to detect germline VHL deletions. The RQ-PCR primer/probe sets were designed for the three VHL coding exons as well as for the 5' promoter and 3' untranslated regions. The RQ-PCR assay for 30 normal and 10 known VHL deletion control samples demonstrated high sensitivity and specificity. We then screened 29 individuals from 19 classical VHL families (16 type 1, 2 type 2A, and one type 2B) and one PHEO family, as well as four solitary suspected cases, none displaying any sequence changes, for VHL deletions by the RQ-PCR assay. We detected germline deletions in 17 (89%) classical families including 15 type 1, one type 2A, and one type 2B. We also identified two mutation carriers and two non-carriers in our family cohort. The one PHEO family and four solitary cases did not display any deletion patterns. These findings indicated that the TaqMan-based RQ-PCR assay is a simple and potent technique for the rapid, sensitive, and specific investigation of VHL genetic diagnoses that could be used profitably before more complex large-deletion detection techniques.

Outcomes of clinical examination and genetic testing of 500 individuals with hearing loss evaluated through a genetics of hearing loss clinic.

Hearing loss (HL) occurs in approximately 2 out of every 1,000 births and is genetic in origin in approximately 50% of cases. This high incidence coupled with the increasing number of genes implicated in HL and the trend toward universal newborn screening led to the establishment of the Genetics of Hearing Loss Clinic at The Children's Hospital of Philadelphia to manage the diagnosis, genetic screening, and counseling of families with an affected child. To date 500 individuals have been evaluated from 1999 to 2004. To determine the cause of their HL and screen for syndromic forms of HL, individuals were offered a panel of tests. Depending on the type and severity of the HL, recommendations included GJB2 mutation analysis, renal and thyroid function studies, a CT scan of the temporal bones, an ophthalmology evaluation, an EKG, and, at times, additional genetic tests. Of the 500 patients evaluated 70 (14%) had a syndromic etiology for their HL. Twenty-eight different syndromic etiologies were identified. Enlarged vestibular aqueducts (EVAs) and/or Mondini malformations were seen in 18% of individuals with HL who had a CT or MRI of the temporal bones. Genetic testing of the GJB2 gene was completed for 310 of the 377 patients with bilateral sensorineural HL (82.2%). Nineteen different variants were identified in the GJB2 gene. Through GJB2 mutational analysis, clinical examination, and laboratory testing, a definitive etiologic diagnosis was established in 110/500 (22%) of patients.

Genetically characterized positive control cell lines derived from residual clinical blood samples.

BACKGROUND: Positive control materials for clinical diagnostic molecular genetic testing are in critically short supply. High-quality DNA that closely resembles DNA isolated from patient specimens can be obtained from Epstein-Barr virus (EBV)-transformed peripheral blood lymphocyte cell lines. Here we report the development of a process to (a) recover residual blood samples with clinically important mutations detected during routine medical care, (b) select samples likely to provide viable lymphocytes for EBV transformation, (c) establish stable cell lines and confirm the reported mutation(s), and (d) validate the cell lines for use as positive controls in clinical molecular genetic testing applications. METHODS: A network of 32 genetic testing laboratories was established to obtain anonymous, residual clinical samples for transformation and to validate resulting cell lines for use as positive controls. Three panel meetings with experts in molecular genetic testing were held to evaluate results and formulate a process that could function in the context of current common practices in molecular diagnostic testing. RESULTS: Thirteen laboratories submitted a total of 113 residual clinical blood samples with mutations for 14 genetic disorders. Forty-one EBV-transformed cell lines were established. Thirty-five individual point and deletion mutations were shown to be stable after 20 population doublings in culture. Thirty-three cell lines were characterized for specific mutations and validated for use as positive controls in clinical diagnostic applications. CONCLUSIONS: A process for producing and validating positive control cell lines from residual clinical blood samples has been developed. Sustainable implementation of the process could help alleviate the current shortage of positive control materials.

Early onset hereditary papillary renal carcinoma: germline missense mutations in the tyrosine kinase domain of the met proto-oncogene.

PURPOSE: Hereditary papillary renal carcinoma (HPRC) is characterized by a predisposition to multiple, bilateral papillary type 1 renal tumors caused by inherited activating missense mutations in the tyrosine kinase domain of the MET proto-oncogene. In the current study we evaluated the clinical phenotype and germline MET mutation of 3 new HPRC families. We describe the early onset clinical features of HPRC. MATERIALS AND METHODS: We identified new HPRC families of Italian (family 177), Spanish (family 223) and Cuban (family 268) descent. We evaluated their clinical features, performed MET mutation analysis by denaturing high performance liquid chromatography and DNA sequencing, and estimated age dependent penetrance and survival using Kaplan-Meier analysis. We characterized renal tumors by histology and fluorescence in situ hybridization. RESULTS: Identical germline MET c.3522G --> A mutations (V1110I) were identified in families 177 and 268 but no evidence of a founder effect was found. Affected members of family 223 carried a germline c.3906G --> C.3522G --> A MET mutation (V1238I). Age dependent penetrance but not survival was significantly earlier for the c.3522G -->A mutation than for the c.3906G --> A mutation in these HPRC families. Trisomy of chromosome 7 and papillary renal carcinoma type 1 histology were detected in papillary renal tumors. CONCLUSIONS: HPRC can occur in an early onset form. The median age for renal tumor development in these 3 HPRC families was 46 to 63 years. HPRC associated papillary renal tumors may be aggressive and metastasize, leading to mortality. Median survival age was 60 to 70 years. Families with identical germline mutations in MET do not always share a common ancestor. HPRC is characterized by germline mutations in MET and papillary type 1 renal tumor histology.

Molecular analysis of patients with synostotic frontal plagiocephaly (unilateral coronal synostosis).

Mutations in genes known to be responsible for most of the recognizable syndromes associated with bilateral coronal synostosis can be detected by molecular testing. The genetic alterations that could cause unilateral coronal synostosis are more elusive. It is recognized that FGFR and TWIST mutations can give rise to either bilateral or unilateral coronal synostosis, even in the same family. The authors undertook a prospective study of patients presenting with synostotic frontal plagiocephaly (unilateral coronal synostosis) to Children's Hospital Boston during the period from 1997 to 2000. Mutational analysis was performed on all patients and on selected parents whenever familial transmission was suspected. Intraoperative anthropometry was used in an effort to differentiate those patients in whom a mutation was detected from those in whom it was not. The anthropometric measures included bilateral sagittal orbital-globe distance, inter medial canthal distance, and nasal angulation. Macrocephaly and palpebral angulation were also considered possible determinants. There was a 2:1 female preponderance in 47 patients with synostotic frontal plagiocephaly. Mutations were found in eight of 47 patients: two patients with different single-amino-acid changes in FGFR2, three patients with FGFR3 Pro250Arg, and three patients with TWIST mutations. Another patient had craniofrontonasal syndrome for which a causative locus has been mapped to chromosome X, although molecular testing is not yet available. Two features were strongly associated with a detectable mutation in patients with synostotic frontal plagiocephaly: asymmetrical brachycephaly (retrusion of both supraorbital rims) and orbital hypertelorism. Other abnormalities in the craniofacial region and extremities were clues to a particular mutation in FGFR2, FGFR3, TWIST, or the X-linked mutation. Neither macrocephaly nor degree of nasal angulation nor relative vertical position of the lateral canthi correlated with mutational detection. An additional four patients in this study had either unilateral or bilateral coronal synostosis in an immediate relative and had anthropometric findings that predicted a mutation, and yet no genetic alteration was found. This suggests either that the authors' screening methods were not sufficiently sensitive or that perhaps there are other unknown pathogenic loci. Nevertheless, molecular testing is recommended for infants who have unilateral coronal synostosis, particularly if there are the anthropometric findings highlighted in this study or an otherwise suspicious feature in the child or a parent. Infants with either an identified or a suspected mutation usually need bilateral asymmetric advancement of the bandeau and may be more likely to require frontal revision in childhood.

Craniosynostosis with tracheal sleeve: a patient with Pfeiffer syndrome, tracheal sleeve and additional malformations in whom an FGFR2 mutation was found.

We discuss a patient with Pfeiffer syndrome who had a tracheal sleeve and an FGFR2 mutation. In the light of our findings, and previous reports of patients with craniosynostosis that also reported similar mutations, we suggest that genomic screening for FGFR2 may be useful in cases with negative FGFR2 mutation testing.