Jinghua Feng

Novel germ line DDX41 mutations define families with a lower age of MDS/AML onset and lymphoid malignancies

Recently our group and others have identified DDX41 mutations both as germ line and acquired somatic mutations in families with multiple cases of late onset myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML), suggesting that DDX41 acts as a tumor suppressor. To determine whether novel DDX41 mutations could be identified in families with additional types of hematologic malignancies, our group screened two cohorts of families with a diverse range of hematologic malignancy subtypes. Among 289 families, we identified nine (3%) with DDX41 mutations. As previously observed, MDS and AML were the most common malignancies, often of the erythroblastic subtype, and 1 family displayed early-onset follicular lymphoma. Five novel mutations were identified, including missense mutations within important functional domains and start-loss and splicing mutations predicted to result in truncated proteins. We also show that most asymptomatic mutation carriers have normal blood counts until malignancy develops. This study expands both the mutation and phenotypic spectra observed in families with germ line DDX41 mutations. With an increasing number of both inherited and acquired mutations in this gene being identified, further study of how DDX41 disruption leads to hematologic malignancies is critical.

ARMC5 Mutations Are Common in Familial Bilateral Macronodular Adrenal Hyperplasia

CONTEXT Bilateral macronodular adrenal hyperplasia (BMAH) is a rare form of adrenal Cushings syndrome. Familial cases have been reported, but at the time we conducted this study, the genetic basis of BMAH was unknown. Recently, germline variants of armadillo repeat containing 5 (ARMC5) in patients with isolated BMAH and somatic, second-hit mutations in tumor nodules, were identified. OBJECTIVE Our objective was to identify the genetic basis of familial BMAH. DESIGN We performed whole exome capture and sequencing of 2 affected individuals from each of 4 BMAH families (BMAH-01, BMAH-02, BMAH-03, and BMAH-05). Based on clinical evaluation, there were 7, 3, 3, and 4 affected individuals in these families, respectively. Sanger sequencing of ARMC5 was performed in 1 other BMAH kindred, BMAH-06. RESULTS Exome sequencing identified novel variants Chr16:g.31477540, c.2139delT, p.(Thr715Leufs*1) (BMAH-02) and Chr16:g.31473811, c.943C→T, p.(Arg315Trp) (BMAH-03) in ARMC5 (GRch37/hg19), validated by Sanger sequencing. BMAH-01 had a recently reported mutation Chr16:g.31476121, c.1777C→T, p.(Arg593Trp). Sanger sequencing of ARMC5 in BMAH-06 identified a previously reported mutation, Chr16:g. 31473688; c.799C→T, p.(Arg267*). The genetic basis of BMAH in BMAH-05 was not identified. CONCLUSIONS Our studies have detected ARMC5 mutations in 4 of 5 BMAH families tested, confirming that these mutations are a frequent cause of BMAH. Two of the 4 families had novel mutations, indicating allelic heterogeneity. Preclinical evaluation did not predict mutation status. The ARMC5-negative family had unusual prominent hyperaldosteronism. Further studies are needed to determine the penetrance of BMAH in ARMC5 mutation-positive relatives of affected patients, the practical utility of genetic screening and genotype-phenotype correlations.

A tale of two siblings: two cases of AML arising from a single pre-leukemic DNMT3A mutant clone.

A tale of two siblings: two cases of AML arising from a single pre-leukemic DNMT3A mutant clone

Delayed diagnosis leading to accelerated-phase chronic eosinophilic leukemia due to a cytogenetically cryptic, imatinib-responsive TNIP1-PDFGRB fusion gene.

Delayed diagnosis leading to accelerated-phase chronic eosinophilic leukemia due to a cytogenetically cryptic, imatinib-responsive TNIP1 – PDFGRB fusion gene

Ectrodactyly and Lethal Pulmonary Acinar Dysplasia Associated with Homozygous FGFR2 Mutations Identified by Exome Sequencing

Ectrodactyly/split hand‐foot malformation is genetically heterogeneous with more than 100 syndromic associations. Acinar dysplasia is a rare congenital lung lesion of unknown etiology, which is frequently lethal postnatally. To date, there have been no reports of combinations of these two phenotypes. Here, we present an infant from a consanguineous union with both ectrodactyly and autopsy confirmed acinar dysplasia. SNP array and whole‐exome sequencing analyses of the affected infant identified a novel homozygous Fibroblast Growth Factor Receptor 2 (FGFR2) missense mutation (p.R255Q) in the IgIII domain (D3). Expression studies of Fgfr2 in development show localization to the affected limbs and organs. Molecular modeling and genetic and functional assays support that this mutation is at least a partial loss‐of‐function mutation, and contributes to ectrodactyly and acinar dysplasia only in homozygosity, unlike previously reported heterozygous activating FGFR2 mutations that cause Crouzon, Apert, and Pfeiffer syndromes. This is the first report of mutations in a human disease with ectrodactyly with pulmonary acinar dysplasia and, as such, homozygous loss‐of‐function FGFR2 mutations represent a unique syndrome.

Allan-Herndon-Dudley syndrome with unusual profound sensorineural hearing loss.

The Allan–Herndon–Dudley syndrome is caused by mutations in the thyroid hormone transporter, Monocarboxylate transporter 8 (MCT8). It is characterized by profound intellectual disability and abnormal thyroid function. We report on a patient with Allan–Herndon–Dudley syndrome (AHDS) with profound sensorineural hearing loss which is not usually a feature of AHDS and which may have been due to a coexisting nonsense mutation in Microphthalmia‐associated transcription factor (MITF).

Integrative genomic analysis reveals cancer-associated mutations at diagnosis of CML in patients with high risk disease.

Genomic events associated with poor outcome in chronic myeloid leukemia (CML) are poorly understood. We performed whole-exome sequencing, copy-number variation, and/or RNA sequencing for 65 patients to discover mutations at diagnosis and blast crisis (BC). Forty-six patients with chronic-phase disease with the extremes of outcome were studied at diagnosis. Cancer gene variants were detected in 15 (56%) of 27 patients with subsequent BC or poor outcome and in 3 (16%) of 19 optimal responders (P = .007). Frequently mutated genes at diagnosis were ASXL1, IKZF1, and RUNX1 The methyltransferase SETD1B was a novel recurrently mutated gene. A novel class of variant associated with the Philadelphia (Ph) translocation was detected at diagnosis in 11 (24%) of 46 patients comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were more frequent at diagnosis in patients with poor outcome: 9 (33%) of 27 vs 2 (11%) of 19 optimal responders (P = .07). Thirty-nine patients were tested at BC, and all had cancer gene variants, including ABL1 kinase domain mutations in 58%. However, ABL1 mutations cooccurred with other mutated cancer genes in 89% of cases, and these predated ABL1 mutations in 62% of evaluable patients. Gene fusions not associated with the Ph translocation occurred in 42% of patients at BC and commonly involved fusion partners that were known cancer genes (78%). Genomic analysis revealed numerous relevant variants at diagnosis in patients with poor outcome and all patients at BC. Future refined biomarker testing of specific variants will likely provide prognostic information to facilitate a risk-adapted therapeutic approach.

ARMC5 is not implicated in familial hyperaldosteronism type II (FH-II)

Germline loss-of-function mutations in the armadillo-repeat-containing 5 (ARMC5) gene are an established cause of Cushings syndrome due to bilateral macronodular adrenal hyperplasia (BMAH), 1,2 and may play a role in primary aldosteronism. 3 As familial hyperaldosteronism type II (FH-II) has a presumed genetic basis, 4 we hypothesised that germline ARMC5 mutations underlie FH-II. We interrogated whole-exome sequencing data from four FH-II families. We did not identify any pathogenic ARMC5 variants which segregated with the phenotype of primary aldosteronism.

Autosomal dominant hypocalcaemia due to a novel CASR mutation: clinical and genetic implications

We report a patient from a family with autosomal dominant hypocalcaemia who also had prednisolone-treated Langerhans’ cell histiocytosis and familial genetic studies identifying a novel calcium-sensing receptor (CASR) mutation. We describe an approach to define pathogenicity of a genetic variation of uncertain significance and postulate that bisphosphonates may have paradoxically increased fracture risk in our patient. In 2009, a 49-year-old woman (III:1; Fig. 1a) was referred with recurrent fractures. Past history included: (i) hypocalcaemia (age 30) – manifesting with fatigue, arthralgia and tetany; despite continuous calcitriol and calcium carbonate, she had hospitalizations with tetany and (ii) prednisolone-treated (minimum 15 mg daily) pulmonary Langerhans’ cell histiocytosis (age 44). She was a current smoker (20 pack years). Hormone replacement therapy was prescribed for menopausal symptoms since age 46. Family history revealed dominantly inherited hypocalcaemia (10 individuals spanning three generations) (Fig. 1a). Individuals generally presented in their thirties with mildly symptomatic hypocalcaemia and received calcitriol and calcium. There was no family history of minimal trauma fracture. In mid-2007, the patient fractured two ribs coughing. Lumbar spine T-score was 0 4 (+0 3 2 years earlier), and femoral neck was 0 3. Risedronate 35 mg weekly was commenced in October 2007. However, nine further minimal trauma fractures occurred prior to review. Imaging showed no lytic or mass lesions at fracture sites. Mild paraesthesiae occurred intermittently. Corrected calcium was 1 87 mmol/l [reference range (RR): 2 1–2 5 mmol/ l], phosphate 1 65 mmol/l (RR: 0 8–1 45 mmol/l), 25-hydroxy vitamin D 34 nmol/l (RR: 60–160 nmol/l), parathyroid hormone (PTH) 1 1 pmol/l (RR: 0 8–5 5 pmol/l), beta C-terminal telopeptide <70 ng/l (desirable range: <400 ng/l) and urinary calcium 9 2 mmol/24 h (RR: 2 5–7 5 mmol/24 h). Bone density was stable. Imaging showed basal ganglia, but not renal, calcification. She fractured three ribs manoeuvring during a bone scan, which showed no other pathology. She did not consent to a tetracycline-labelled iliac crest biopsy to further assess bone turnover. Cholecalciferol was added and calcium carbonate doubled to 600 mg twice daily, improving symptoms and biochemistry. Her respiratory physicians agreed to taper prednisolone. Approval for teriparatide was granted by our institutional Pharmacy Committee and risedronate ceased. Severe bony pain occurred 1 week after commencing teriparatide 20 lg daily. This resolved and recurred after cessation and reintroduction of teriparatide, respectively, which was withdrawn indefinitely. She then fractured three phalanges of her foot during a low trauma fall. Hormone replacement therapy ceased, and warfarin commenced after a pulmonary embolism. She received 5 mg zoledronic acid. One week later, she fractured a rib coughing. In 2010, she had three lower respiratory tract infections. She declined hospitalization for the third and died at home. Autopsy was not performed. Sanger sequencing of CASR performed by the Genetics Laboratories, Churchill Hospital, Oxford, UK, using germline DNA from an affected sibling (proband – genetic studies: III:2; Fig. 1a), identified a heterozygous three base pair in-frame deletion in the coding region for transmembrane domain 6 (c.2443_2445delTTC; p.Phe815del; gene sequence accession NM_000388.1). This was not in the CASR database (http://www.casrdb.mcgill.ca/), a population screen we performed (616 individuals) or the Exome Aggregation Consortium Browser (http://exac.broadinstitute.org/). Segregation analysis of the deletion and other candidate gene screening were performed. This study was approved by the Royal Adelaide Hospital Human Research Ethics Committee (Protocol: 110623); all subjects gave written consent. Whole exome capture and sequencing (Australian Genome Research Facility, Victoria) of the proband’s germline DNA was performed using Illumina TruSeq Capture protocol and HiSeq 2000 platform (Illumina, Inc, San Diego, CA, USA), respectively. This identified the CASR deletion, but no other plausible candidates. The deletion segregated with hypocalcaemia (Fig. 1a). Despite normal bone density, in 2 5 years, our patient experienced 18 fractures of the ribs and feet – not classical sites for osteoporotic fracture. The cause of this high fracture rate is likely multifactorial – with reduced mobility, smoking and longterm prednisolone contributory. However, 16 fractures occurred in the year following initiation of risedronate (Fig. 1b). Whilst bisphosphonates prevent glucocorticoid-induced bone loss and fracture, we speculate that risedronate, in the milieu of low PTH and low bone turnover, which was further reduced by prednisolone, paradoxically increased fracture risk. The patient’s beta C-terminal telopeptide was undetectable on treatment and low in the proband (132 ng/l in III:2; Fig. 1a). As approximately 10% of adults with Langerhans’ cell histiocytosis have skeletal involvement, this is a potential aetiology of fractures in our patient. However, the presentation is typically with painful swelling of the affected site(s), and imaging shows a lytic lesion or associated soft tissue mass. As our patient had no clinical or radiological evidence of skeletal involvement, we hypothesize that bony Langerhans’ cell histiocytosis was not the cause of her recurrent fractures. Over 70 CASR mutations underlying autosomal dominant hypocalcaemia have been described. We report a novel amino acid deletion. Whilst functional studies may provide further evidence of pathogenicity, the data are already compelling. The deletion is in transmembrane domain 6 where causative mutations have been described (Fig. 1c). Functional studies report amino acids in this domain are critical for maintaining an inactive configuration. The

A novel, somatic, transforming mutation in the extracellular domain of Epidermal Growth Factor Receptor identified in myeloproliferative neoplasm

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.