Introme accurately predicts the impact of coding and noncoding variants on gene splicing, with clinical applications.

Predicting the impact of coding and noncoding variants on splicing is challenging, particularly in non-canonical splice sites, leading to missed diagnoses in patients. Existing splice prediction tools are complementary but knowing which to use for each splicing context remains difficult. Here, we describe Introme, which uses machine learning to integrate predictions from several splice detection tools, additional splicing rules, and gene architecture features to comprehensively evaluate the likelihood of a variant impacting splicing. Through extensive benchmarking across 21,000 splice-altering variants, Introme outperformed all tools (auPRC: 0.98) for the detection of clinically significant splice variants. Introme is available at https://github.com/CCICB/introme .

Bladder Paraganglioma Presenting as Post-Micturition Syncope.

A 22-year-old woman presented with a 12-year history of intensifying paroxysms of anxiety, palpitations and recurrent syncope following micturition. The patient was referred to endocrinology upon discovery of hypertension. An extended family history revealed metastatic phaeochromocytoma and paraganglioma in two grand-uncles. Clinical examination revealed hypertension, with a mean 24-hour ambulatory blood pressure of 150/100 mmHg. Supine plasma normetanephrines were markedly elevated with a raised 3-methoxytyramine, while plasma metanephrines were normal. Computed tomography identified a 4.4 cm mass at the right inferolateral margin of the bladder wall. Scintigraphic imaging confirmed unifocal bladder lesion uptake with no additional metastatic lesions. Following pre-operative alpha blockade, the patient underwent a partial cystectomy. Histology confirmed a paraganglioma, and SDHB staining was lost in neoplastic cells consistent with an SDHB-related paraganglioma. Plasma normetanephrine, 3-methoxytyramine and blood pressure returned to normal postoperatively. Genetic screening identified a germline heterozygous SDHB gene variant c.723C>G. Bladder paragangliomas are a rare but important differential to consider when investigating post-micturition syncope. An extended family history should be sought and suspicion for a genetic cause should be raised, especially when the condition presents at a young age. This is the first reported case describing phaeochromocytoma or paraganglioma with the SDHB gene variant c.723C>G. LEARNING POINTS: Bladder paragangliomas are a rare neuroendocrine tumour which should be considered when assessing patients with haematuria and hypertension, headache, palpitations, sweating and facial pallor with micturition.This case highlights the importance of a thorough clinical history with an extended family history and examination in the setting of micturition syncope, which can rarely occur with bladder paraganglioma.Young age at presentation, a family history of phaeochromocytoma and paraganglioma (PPGL), unusual paraganglioma location, mutifocality and aggressive disease should raise the suspicion for a genetic predisposition to PPGL.

The phenotypic spectrum associated with OTX2 mutations in humans.

Objective: The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development. Design: We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods: We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results: Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions: OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

Proteomic signatures of radioresistance: Alteration of inflammation, angiogenesis and metabolism-related factors in radioresistant oesophageal adenocarcinoma.

The clinical management of locally advanced oesophageal adenocarcinoma (OAC) involves neoadjuvant chemoradiotherapy (CRT), but as radioresistance remains a major clinical challenge, complete pathological response to CRT only occurs in 20-30% of patients. In this study we used an established isogenic cell line model of radioresistant OAC to detect proteomic signatures of radioresistance to identify novel molecular and cellular targets of radioresistance in OAC. A total of 5785 proteins were identified of which 251 were significantly modulated in OE33R cells, when compared to OE33P. Gene ontology and pathway analysis of these significantly modulated proteins demonstrated altered metabolism in radioresistant cells accompanied by an inhibition of apoptosis. In addition, inflammatory and angiogenic pathways were positively regulated in radioresistant cells compared to the radiosensitive cells. In this study, we demonstrate, for the first time, a comprehensive proteomic profile of the established isogenic cell line model of radioresistant OAC. This analysis provides insights into the molecular and cellular pathways which regulate radioresistance in OAC. Furthermore, it identifies pathway specific signatures of radioresistance that will direct studies on the development of targeted therapies and personalised approaches to radiotherapy.

Differential association of CD68+ and CD163+ macrophages with macrophage enzymes, whole tumour gene expression and overall survival in advanced melanoma.

BACKGROUND: The density and phenotype of tumour-associated macrophages have been linked with prognosis in a range of solid tumours. While there is strong preclinical evidence that tumour-associated macrophages promote aspects of tumour progression, it can be challenging to infer clinical activity from surface markers and ex vivo behaviour. We investigated the association of macrophage infiltration with prognosis and functional changes in the tumour microenvironment in primary human melanoma. METHODS: Fifty-seven formalin-fixed, paraffin-embedded primary melanomas were analysed by immunohistochemical analysis of CD68, CD163, inducible nitric oxide synthase (iNOS) and arginase expression. RNA sequencing was performed on serial sections of 20 of the stained tumours to determine the influence of macrophage infiltration on gene expression. RESULTS: CD68+ cells are a functionally active subset of macrophages that are associated with increased iNOS and arginase staining and altered gene expression. In comparison, while there is a greater accumulation of CD163+ macrophages in larger tumours, these cells are comparatively inactive, with no association with the level of iNOS or arginase staining, and no effect on gene expression within the tumour. The infiltration of either subset of macrophages did not correlate to overall survival. CONCLUSIONS: Thus, melanomas contain distinct macrophage populations with diverse phenotypes, but with no observable prognostic role.

MTOR signaling orchestrates stress-induced mutagenesis, facilitating adaptive evolution in cancer.

In microorganisms, evolutionarily conserved mechanisms facilitate adaptation to harsh conditions through stress-induced mutagenesis (SIM). Analogous processes may underpin progression and therapeutic failure in human cancer. We describe SIM in multiple in vitro and in vivo models of human cancers under nongenotoxic drug selection, paradoxically enhancing adaptation at a competing intrinsic fitness cost. A genome-wide approach identified the mechanistic target of rapamycin (MTOR) as a stress-sensing rheostat mediating SIM across multiple cancer types and conditions. These observations are consistent with a two-phase model for drug resistance, in which an initially rapid expansion of genetic diversity is counterbalanced by an intrinsic fitness penalty, subsequently normalizing to complete adaptation under the new conditions. This model suggests synthetic lethal strategies to minimize resistance to anticancer therapy.

Development and validation of a targeted gene sequencing panel for application to disparate cancers.

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour's molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy.

Cost-benefit analysis of a population-based education program on the wise use of antibiotics.

OBJECTIVE: In 2005, the Do Bugs Need Drugs (DBND) program was imported to British Columbia (BC) from Alberta with the goal of reducing unnecessary antibiotic use in the community. The objective of this study was to estimate the impact of the program on antibiotic-associated costs and cost-benefit. METHODS: We used data on antibiotic prescription and costs from BC PharmaNet for the period of 1996 to 2014. We conducted interrupted time series regression to formally interpret the impact of the DBND program. RESULTS: The average monthly prescription rate fell by 14.5%, from 54.3 to 46.4 per 1000 population between 2005 and 2014. The proportionate contribution of macrolide prescription decreased from 19.2% in 2005 to 13.2% in 2014 and for quinolones decreased from 13.1% in 2005 to 12% in 2014. The proportion of prescriptions for both penicillins and tetracyclines increased by > 35.5%. Before the program, the average monthly cost of antibiotics was increasing by CAD $8.12 per 1000 population (p < 0.001). After program introduction, average monthly cost decreased by CAD $18.19 per 1000 population (p < 0.001), creating an annual savings for BC in 2014 of CAD $83.6 million. In 2014, one Canadian dollar spent on the DBND program was associated with conservative savings of CAD $76.20. CONCLUSION: Significant cost savings have been observed in association with a community antimicrobial stewardship program focused on both public and prescribers. Such programs are an effective strategy in cost-benefit terms and should therefore be considered for universal adoption in Canadian healthcare systems.

Pathogenic variants in PLOD3 result in a Stickler syndrome-like connective tissue disorder with vascular complications.

BACKGROUND: Pathogenic PLOD3 variants cause a connective tissue disorder (CTD) that has been described rarely. We further characterise this CTD and propose a clinical diagnostic label to improve recognition and diagnosis of PLOD3-related disease. METHODS: Reported PLOD3 phenotypes were compared with known CTDs utilising data from three further individuals from a consanguineous family with a homozygous PLOD3 c.809C>T; p.(Pro270Leu) variant. PLOD3 mRNA expression in the developing embryo was analysed for tissue-specific localisation. Mouse microarray expression data were assessed for phylogenetic gene expression similarities across CTDs with overlapping clinical features. RESULTS: Key clinical features included ocular abnormalities with risk for retinal detachment, sensorineural hearing loss, reduced palmar creases, finger contractures, prominent knees, scoliosis, low bone mineral density, recognisable craniofacial dysmorphisms, developmental delay and risk for vascular dissection. Collated clinical features showed most overlap with Stickler syndrome with variable features of Ehlers-Danlos syndrome (EDS) and epidermolysis bullosa (EB). Human lysyl hydroxylase 3/PLOD3 expression was localised to the developing cochlea, eyes, skin, forelimbs, heart and cartilage, mirroring the clinical phenotype of this disorder. CONCLUSION: These data are consistent with pathogenic variants in PLOD3 resulting in a clinically distinct Stickler-like syndrome with vascular complications and variable features of EDS and EB. Early identification of PLOD3 variants would improve monitoring for comorbidities and may avoid serious adverse ocular and vascular outcomes.

Genomic stratification and liquid biopsy in a rare adrenocortical carcinoma (ACC) case, with dual lung metastases.

Adrenocortical carcinoma is a rare malignancy with a poor prognosis and few treatment options. Molecular characterization of this cancer remains limited. We present a case of an adrenocortical carcinoma (ACC) in a 37-yr-old female, with dual lung metastases identified 1 yr following commencement of adjuvant mitotane therapy. As standard therapeutic regimens are often unsuccessful in ACC, we undertook a comprehensive genomic study into this case to identify treatment options and monitor disease progress. We performed targeted and whole-genome sequencing of germline, primary tumor, and both metastatic tumors from this patient and monitored recurrence over 2 years using liquid biopsy for ctDNA and steroid hormone measurements. Sequencing revealed the primary and metastatic tumors were hyperhaploid, with extensive loss of heterozygosity but few structural rearrangements. Loss-of-function mutations were identified in MSH2, TP53, RB1, and PTEN, resulting in tumors with mismatch repair signatures and microsatellite instability. At the cellular level, tumors were populated by mitochondria-rich oncocytes. Longitudinal ctDNA mutation and hormone profiles were unable to detect micrometastatic disease, consistent with clinical indicators of disease remission. The molecular signatures in our ACC case suggested immunotherapy in the event of disease progression; however, the patient remains free of cancer. The extensive molecular analysis presented here could be applied to other rare and/or poorly stratified cancers to identify novel or repurpose existing therapeutic options, thereby broadly improving diagnoses, treatments, and prognoses.

Whole blood human transcriptome and virome analysis of ME/CFS patients experiencing post-exertional malaise following cardiopulmonary exercise testing.

Myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) is a syndrome of unknown etiology characterized by profound fatigue exacerbated by physical activity, also known as post-exertional malaise (PEM). Previously, we did not detect evidence of immune dysregulation or virus reactivation outside of PEM periods. Here we sought to determine whether cardiopulmonary exercise stress testing of ME/CFS patients could trigger such changes. ME/CFS patients (n = 14) and matched sedentary controls (n = 11) were subjected to cardiopulmonary exercise on 2 consecutive days and followed up to 7 days post-exercise, and longitudinal whole blood samples analyzed by RNA-seq. Although ME/CFS patients showed significant worsening of symptoms following exercise versus controls, with 8 of 14 ME/CFS patients showing reduced oxygen consumption ([Formula: see text]) on day 2, transcriptome analysis yielded only 6 differentially expressed gene (DEG) candidates when comparing ME/CFS patients to controls across all time points. None of the DEGs were related to immune signaling, and no DEGs were found in ME/CFS patients before and after exercise. Virome composition (P = 0.746 by chi-square test) and number of viral reads (P = 0.098 by paired t-test) were not significantly associated with PEM. These observations do not support transcriptionally-mediated immune cell dysregulation or viral reactivation in ME/CFS patients during symptomatic PEM episodes.

Utilization of Antibiotics in Long-Term Care Facilities in British Columbia, Canada.

BACKGROUND: Antibiotic use is highly prevalent in long-term care facilities (LTCFs); a resident's annual exposure to at least 1 course of antibiotic is approximately 50% to 80%. The objective of this study was to understand the extent of antibiotic use in the population of residents in British Columbia's (BC) LTCFs from 2007 to 2014. METHODS: Antibiotic prescription data for LTCF residents was extracted from the central prescription database and linked to the physician billing plan to obtain antibiotic indication. Total defined daily dose (DDD) per 1000 residents per day was calculated. RESULTS: Our database had 381 LTCFs with an average of nearly 24,694 residents annually and 419,036 antibiotic prescriptions. Antibiotic utilization did not change dramatically between 2007 and 2014, ranging from 39.2 in 2007 to 35.2 DDD per 1000 residents per day in 2014. Although usage of most antibiotics declined, use of moxifloxacin, amoxicillin-clavulanate, doxycycline, and amoxicillin increased significantly. The indication most frequently linked to prescription was urinary tract infection (6.58 DDD per 1000 residents per day), with nitrofurantoin, ciprofloxacin, and trimethoprim/sulfamethoxazole being the most commonly prescribed agents. This was followed closely by prescriptions for respiratory infections (5.34 DDD per 1000 residents per day), with moxifloxacin being the most commonly prescribed antibiotic, primarily for upper respiratory tract infection (URTI), whereas doxycycline is used commonly for lower respiratory tract infection. Duration of antibiotic therapy in LTCF residents has decreased significantly from 9.29 days to 7.3 days per prescription in 2014. CONCLUSION: Antibiotic use in LTCFs is high relative to the general population. Our study underscores that stewardship in LTCFs should continue to focus on length of treatment, appropriate detection of urinary tract infections, and avoidance of treating URTIs with antibiotics.

Evaluation of Streck BCT and PAXgene Stabilised Blood Collection Tubes for Cell-Free Circulating DNA Studies in Plasma.

INTRODUCTION: Blood samples for studies of circulating DNA in disease are often collected in clinical settings where prompt processing of samples is not possible. In order to avoid problems associated with leukocyte lysis after prolonged blood storage, stabilised blood tubes have been developed containing preservatives that prevent cell lysis. We evaluated Streck BCT tubes and PAXgene ccfDNA tubes, as well as standard EDTA blood collection tubes, in terms of DNA yield and fragment size. METHODS: Blood was collected in EDTA, Streck BCT or PAXgene ccfDNA tubes and stored for 1 h at 4 °C, or 4 days at room temperature. DNA was extracted using the QIAamp Circulating Nucleic Acids kit, and visualised on an agarose gel or quantitated by qPCR. Ratios of a 247-base and a 115-base amplicon of the Alu repetitive element were used to infer size distribution. RESULTS: While plasma DNA in EDTA tube blood samples increased by ~10- to 20-fold after 4 days of storage at room temperature, both Streck BCT tubes and PAXgene ccfDNA tubes maintained stable plasma DNA concentrations. A slight decrease in DNA yield following 1 h of blood storage at 4 °C was observed in Streck BCT and PAXgene ccfDNA tubes relative to EDTA tubes. This decrease was reversed by increasing the proteinase digest step of the DNA extraction protocol to 60 min, as recommended by Streck tube product literature. Visualisation of the extracted DNA on an agarose gel showed that after 4 days of room temperature storage, samples collected in EDTA tubes contained abundant high-molecular weight DNA, which was partially fragmented in a ladder pattern. A slight increase in high-molecular weight DNA in samples stored for 4 days at room temperature in Streck BCT tubes was also observed, but this was not reflected in a change in large and small Alu fragment ratios as measured by qPCR. CONCLUSIONS: Tubes containing preservative to prevent cell lysis can extend the scope for blood collection in clinical settings; however, slight differences between samples collected in different tube types underscore the requirement for standardised protocols, as well as attention to sample handling.

Germline variants in familial pituitary tumour syndrome genes are common in young patients and families with additional endocrine tumours.

OBJECTIVE: Familial pituitary tumour syndromes (FPTS) account for 5% of pituitary adenomas. Multi-gene analysis via next-generation sequencing (NGS) may unveil greater prevalence and inform clinical care. We aimed to identify germline variants in selected patients with pituitary adenomas using a targeted NGS panel. DESIGN: We undertook a nationwide cross-sectional study of patients with pituitary adenomas with onset ≤40 years of age and/or other personal/family history of endocrine neoplasia. A custom NGS panel was performed on germline DNA to interrogate eight FPTS genes. Genome data were analysed via a custom bioinformatic pipeline, and validation was performed by Sanger sequencing. Multiplex ligation-dependent probe amplification (MLPA) was performed in cases with heightened suspicion for MEN1, CDKN1B and AIP mutations. The main outcomes were frequency and pathogenicity of rare variants in AIP, CDKN1B, MEN1, PRKAR1A, SDHA, SDHB, SDHC and SDHD. RESULTS: Forty-four patients with pituitary tumours, 14 of whom had a personal history of other endocrine tumours and/or a family history of pituitary or other endocrine tumours, were referred from endocrine tertiary-referral centres across Australia. Eleven patients (25%) had a rare variant across the eight FPTS genes tested: AIP (p.A299V, p.R106C, p.F269F, p.R304X, p.K156K, p.R271W), MEN1 (p.R176Q), SDHB (p.A2V, p.S8S), SDHC (p.E110Q) and SDHD (p.G12S), with two patients harbouring dual variants. Variants were classified as pathogenic or of uncertain significance in 9/44 patients (20%). No deletions/duplications were identified in MEN1, CDKN1B or AIP. CONCLUSIONS: A high yield of rare variants in genes implicated in FPTS can be found in selected patients using an NGS panel. It may also identify individuals harbouring more than one rare variant.

RNA-Seq Analysis of Gene Expression, Viral Pathogen, and B-Cell/T-Cell Receptor Signatures in Complex Chronic Disease.

Background: Chronic fatigue syndrome (CFS) remains poorly understood. Although infections are speculated to trigger the syndrome, a specific infectious agent and underlying pathophysiological mechanism remain elusive. In a previous study, we described similar clinical phenotypes in CFS patients and alternatively diagnosed chronic Lyme syndrome (ADCLS) patients­individuals diagnosed with Lyme disease by testing from private Lyme specialty laboratories but who test negative by reference 2-tiered serologic analysis. Methods: Here, we performed blinded RNA-seq analysis of whole blood collected from 25 adults diagnosed with CFS and 13 ADCLS patients, comparing these cases to 25 matched controls and 11 patients with well-controlled systemic lupus erythematosus (SLE). Samples were collected at patient enrollment and not during acute symptom flares. RNA-seq data were used to study host gene expression, B-cell/T-cell receptor profiles (BCR/TCR), and potential viral infections. Results: No differentially expressed genes (DEGs) were found to be significant when CFS or ADCLS cases were compared to controls. Forty-two DEGs were found when SLE cases were compared to controls, consistent with activation of interferon signaling pathways associated with SLE disease. BCR/TCR repertoire analysis did not show significant differences between CFS and controls or ADCLS and controls. Finally, viral sequences corresponding to anelloviruses, human pegivirus 1, herpesviruses, and papillomaviruses were detected in RNA-seq data, but proportions were similar (P = .73) across all genus-level taxonomic categories. Conclusions: Our observations do not support a theory of transcriptionally mediated immune cell dysregulation in CFS and ADCLS, at least outside of periods of acute symptom flares.

Metagenomic Investigation of Plasma in Individuals with ME/CFS Highlights the Importance of Technical Controls to Elucidate Contamination and Batch Effects.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease causing indefinite fatigue. ME/CFS has long been hypothesised to have an infectious cause; however, no specific infectious agent has been identified. We used metagenomics to analyse the RNA from plasma samples from 25 individuals with ME/CFS and compare their microbial content to technical controls as well as three control groups: individuals with alternatively diagnosed chronic Lyme syndrome (N = 13), systemic lupus erythematosus (N = 11), and healthy controls (N = 25). We found that the majority of sequencing reads were removed during host subtraction, thus there was very low microbial RNA content in the plasma. The effects of sample batching and contamination during sample processing proved to outweigh the effects of study group on microbial RNA content, as the few differences in bacterial or viral RNA abundance we did observe between study groups were most likely caused by contamination and batch effects. Our results highlight the importance of including negative controls in all metagenomic analyses, since there was considerable overlap between bacterial content identified in study samples and control samples. For example, Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodes were found in both study samples and plasma-free negative controls. Many of the taxonomic groups we saw in our plasma-free negative control samples have previously been associated with diseases, including ME/CFS, demonstrating how incorrect conclusions may arise if controls are not used and batch effects not accounted for.

Partial Loss of Function of the GHRH Receptor Leads to Mild Growth Hormone Deficiency.

OBJECTIVE: Recessive mutations in GHRHR are associated with severe isolated growth hormone deficiency (IGHD), with a final height in untreated patients of 130 cm ± 10 cm (-7.2 ± 1.6 SDS; males) and 114 ± 0.7 cm (-8.3 ± 0.1 SDS; females). DESIGN: We hypothesized that a consanguineous Pakistani family with IGHD in three siblings (two males, one female) would have mutations in GH1 or GHRHR. RESULTS: Two novel homozygous missense variants [c.11G>A (p.R4Q), c.236C>T (p.P79L)] at conserved residues were identified in all three siblings. Both were absent from control databases, aside from pR4Q appearing once in heterozygous form in the Exome Aggregation Consortium Browser. The brothers were diagnosed with GH deficiency at 9.8 and 6.0 years (height SDS: -2.24 and -1.23, respectively), with a peak GH of 2.9 µg/liter with low IGF-1/IGF binding protein 3. Their sister presented at 16 years with classic GH deficiency (peak GH <0.1 µg/liter, IGF-1 <3.3 mmol/liter) and attained an untreated near-adult height of 144 cm (-3.0 SDS); the tallest untreated patient with GHRHR mutations reported. An unrelated Pakistani female IGHD patient was also compound homozygous. All patients had a small anterior pituitary on magnetic resonance imaging. Functional analysis revealed a 50% reduction in maximal cAMP response to stimulation with GHRH by the p.R4Q/p.P79L double mutant receptor, with a 100-fold increase in EC50. CONCLUSION: We report the first coexistence of two novel compound homozygous GHRHR variants in two unrelated pedigrees associated with a partial loss of function. Surprisingly, the patients have a relatively mild IGHD phenotype. Analysis revealed that the pP79L mutation is associated with the compromise in function, with the residual partial activity explaining the mild phenotype.