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Next-generation sequencing (NGS) technologies are now established in clinical laboratories as a primary testing modality in genomic medicine. These technologies have reduced the cost of large-scale sequencing by several orders of magnitude. It is now cost-effective to analyze an individual with disease-targeted gene panels, exome sequencing, or genome sequencing to assist in the diagnosis of a wide array of clinical scenarios. While clinical validation and use of NGS in many settings is established, there are continuing challenges as technologies and the associated informatics evolve. To assist clinical laboratories with the validation of NGS methods and platforms, the ongoing monitoring of NGS testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics (ACMG) has developed the following technical standards.

Mutations in the torsinA-interacting protein 1 (TOR1AIP1) gene result in a severe muscular dystrophy with minimal literature in the pediatric population. We review a case of TOR1AIP1 gene mutation in a 16-year-old Caucasian female with a long history of muscle weakness. Extensive clinical workup was performed and MRI at time of initial presentation demonstrated no significant muscular atrophy with heterogenous STIR hyperintensity of the lower extremity muscles. MRI findings seven years later included extensive atrophy of the lower extremities, with severe progression, including the gluteal muscles, iliopsoas, rectus femoris, and obturator internus. There was also significant atrophy of the rectus abdominis and internal and external oblique muscles, and iliacus muscles. The MRI findings showed more proximal involvement of lower extremities and no atrophy of the tibialis anterior, making TOR1AIP1 the more likely genetic cause. Muscle biopsy findings supported TOR1AIP1 limb-girdle muscular dystrophy. Though rare, TOR1AIP1 gene mutation occurs in pediatric patients and MRI can aid in diagnosis and help differentiate from other types of muscular dystrophy. Genetic and pathology workup is also crucial to accurate diagnosis and possible treatment of these patients.

Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl(−)/H(+) exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and -genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH.

Contains fulltext : 202800.pdf (Publisher’s version ) (Open Access) SMARCC2 (BAF170) is one of the invariable core subunits of the ATP-dependent chromatin remodeling BAF (BRG1-associated factor) complex and plays a crucial role in embryogenesis and corticogenesis. Pathogenic variants in genes encoding other components of the BAF complex have been associated with intellectual disability syndromes. Despite its significant biological role, variants in SMARCC2 have not been directly associated with human disease previously. Using whole-exome sequencing and a web-based gene-matching program, we identified 15 individuals with variable degrees of neurodevelopmental delay and growth retardation harboring one of 13 heterozygous variants in SMARCC2, most of them novel and proven de novo. The clinical presentation overlaps with intellectual disability syndromes associated with other BAF subunits, such as Coffin-Siris and Nicolaides-Baraitser syndromes and includes prominent speech impairment, hypotonia, feeding difficulties, behavioral abnormalities, and dysmorphic features such as hypertrichosis, thick eyebrows, thin upper lip vermilion, and upturned nose. Nine out of the fifteen individuals harbor variants in the highly conserved SMARCC2 DNA-interacting domains (SANT and SWIRM) and present with a more severe phenotype. Two of these individuals present cardiac abnormalities. Transcriptomic analysis of fibroblasts from affected individuals highlights a group of differentially expressed genes with possible roles in regulation of neuronal development and function, namely H19, SCRG1, RELN, and CACNB4. Our findings suggest a novel SMARCC2-related syndrome that overlaps with neurodevelopmental disorders associated with variants in BAF-complex subunits.

The increasing quality and diminishing cost of next-generation sequencing has transformed our ability to interrogate large quantities of genetic information. This has led to a dramatic increase in the number of elective genomic tests performed. In this article, elective test denotes a test that a patient chooses to undertake without a clinical indication. The variety of elective genomic testing options is considerable. Because these offerings provide differing levels of sensitivity and specificity, it can be difficult to choose among them. A simple rubric to compare offerings is not readily available. We propose a framework designated completeness that evaluates both analytical and interpretative components of genomic tests. We then illustrate how this framework can be used to evaluate the expanding landscape of elective genomic testing.

While the diagnostic success of genomic sequencing expands, the complexity of this testing should not be overlooked. Numerous laboratory processes are required to support the identification, interpretation, and reporting of clinically significant variants. This study aimed to examine the workflow and reporting procedures among US laboratories to highlight shared practices and identify areas in need of standardization. Surveys and follow-up interviews were conducted with laboratories offering exome and/or genome sequencing to support a research program or for routine clinical services. The 73-item survey elicited multiple choice and free-text responses that were later clarified with phone interviews. Twenty-one laboratories participated. Practices highly concordant across all groups included consent documentation, multiperson case review, and enabling patient opt-out of incidental or secondary findings analysis. Noted divergence included use of phenotypic data to inform case analysis and interpretation and reporting of case-specific quality metrics and methods. Few laboratory policies detailed procedures for data reanalysis, data sharing, or patient access to data. This study provides an overview of practices and policies of experienced exome and genome sequencing laboratories. The results enable broader consideration of which practices are becoming standard approaches, where divergence remains, and areas of development in best practice guidelines that may be helpful.Genet Med advance online publication 03 Novemeber 2016.

Diagnosis at the edges of our knowledge calls upon clinicians to be data driven, cross-disciplinary, and collaborative in unprecedented ways. Exact disease recognition, an element of the concept of precision in medicine, requires new infrastructure that spans geography, institutional boundaries, and the divide between clinical care and research. The National Institutes of Health (NIH) Common Fund supports the Undiagnosed Diseases Network (UDN) as an exemplar of this model of precise diagnosis. Its goals are to forge a strategy to accelerate the diagnosis of rare or previously unrecognized diseases, to improve recommendations for clinical management, and to advance research, especially into disease mechanisms. The network will achieve these objectives by evaluating patients with undiagnosed diseases, fostering a breadth of expert collaborations, determining best practices for translating the strategy into medical centers nationwide, and sharing findings, data, specimens, and approaches with the scientific and medical communities. Building the UDN has already brought insights to human and medical geneticists. The initial focus has been on data sharing, establishing common protocols for institutional review boards and data sharing, creating protocols for referring and evaluating patients, and providing DNA sequencing, metabolomic analysis, and functional studies in model organisms. By extending this precision diagnostic model nationally, we strive to meld clinical and research objectives, improve patient outcomes, and contribute to medical science.

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 ( NACC1 ) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10 −14 ). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1 .

Early B cell factor 3 (EBF3) is a member of the highly evolutionarily conserved Collier/Olf/EBF (COE) family of transcription factors. Prior studies on invertebrate and vertebrate animals have shown that EBF3 homologs are essential for survival and that loss-of-function mutations are associated with a range of nervous system developmental defects, including perturbation of neuronal development and migration. Interestingly, aristaless-related homeobox (ARX), a homeobox-containing transcription factor critical for the regulation of nervous system development, transcriptionally represses EBF3 expression. However, human neurodevelopmental disorders related to EBF3 have not been reported. Here, we describe three individuals who are affected by global developmental delay, intellectual disability, and expressive speech disorder and carry de novo variants in EBF3. Associated features seen in these individuals include congenital hypotonia, structural CNS malformations, ataxia, and genitourinary abnormalities. The de novo variants affect a single conserved residue in a zinc finger motif crucial for DNA binding and are deleterious in a fly model. Our findings indicate that mutations in EBF3 cause a genetic neurodevelopmental syndrome and suggest that loss of EBF3 function might mediate a subset of neurologic phenotypes shared by ARX-related disorders, including intellectual disability, abnormal genitalia, and structural CNS malformations.

Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ∼10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.

To the editor Proteus syndrome (PS) is characterized by progressive, mosaic, segmental overgrowth and occurs sporadically (Biesecker 2001; 2006). The mosaic nature and sporadic occurrence with lack of familial transmission led to the hypothesis that PS is caused by a post-zygotic somatic mutation, which was confirmed with the discovery of a mosaic activating c.49G>A, p.Glu17Lys AKT1 mutation (Lindhurst et al. 2011). To date, all patients who meet the clinical diagnostic criteria for PS and have been tested have this mutation (Lindhurst and Biesecker, unpublished results). While any organ or tissue can be affected, skeletal overgrowth and dermatologic lesions are the most common manifestations of PS (Turner et al. 2004; Beachkofsky et al. 2010). Cerebriform connective tissue nevi (CCTN) are a highly specific and common lesion in patients with PS (Biesecker et al. 2001; Nguyen et al. 2004). These lesions are very firm and contain deep sulci that resemble the brain, for which the lesion is named. Histology sections of CCTN show massively expanded dermis filled with thick collagen bundles (Figure 1d-f) (McCuaig et al. 2012). Epidermal nevi (EN) can be non-syndromic or occur as part of several syndromes including PS (Happle 2010). The keratinocytic EN found in PS have a rough surface, are dark in color, usually follow the lines of Blaschko, and exhibit epidermal hyperkeratosis, papillomatosis, and acanthosis (Figure 1a-c) (Nguyen et al. 2004). EN are generally noticed in the first year of life and are stable in extent whereas CCTN grow progressively after first appearing later in the first or second year (Twede et al. 2005). Figure 1 Representative examples of an EN and CCTN. Panel a shows the rough surface of the linear EN on the infraaxillary vault in patient 101. Hematoxylin and eosin stain (b, scale bar = 100 μm; c, scale bar = 50 μm) of a skin biopsy from the ... It is unknown which cells determine the formation of these lesions. Based on the histology, we hypothesized that CCTN were generated by AKT1 p.Glu17Lys in the dermis and that EN were generated by this mutation in the epidermis. To test this hypothesis, we isolated fibroblasts and keratinocytes from lesional (CCTN or EN) and non-lesional (“normal”) skin samples and measured the level of the mutant allele in each cell type. Skin samples were collected during surgical procedures or by punch biopsies from patients with PS under an IRB-approved protocol. The epidermis was separated from the dermis by treatment with dispase and keratinocyte and fibroblast cultures were established using standard protocols (Aasen and Belmonte 2010). A single keratinocyte culture was established from each biopsy. Fibroblasts were allowed to grow out of the dermal tissue until the dish was confluent. The dermal tissue was then transferred to a new dish to allow another fibroblast culture to be established from that same tissue. The number of fibroblast cultures established from a piece of dermis ranged from one to six. DNA was isolated from cultured cells harvested between passages one and four, and the mutation level was assayed using a PCR-based restriction fragment length polymorphism (RFLP) assay as described (Lindhurst et al. 2011). This assay has a lower limit of sensitivity of 0.5%. Each DNA preparation was tested at least twice and the values were averaged. In ten of the 20 samples, DNA was also isolated from the dermal tissue used to establish the fibroblast cultures after the final transfer. Seven CCTN biopsies were obtained from the feet of three patients (Table S1). The mutant allele was not detected in CCTN keratinocyte cultures whereas the mutation level from the CCTN fibroblast cultures was 9-32%. In the dermal tissue post-culturing, the mutation level was 12-21% (Figure 2). Figure 2 Percentage of AKT1 c.49G>A, p.Glu17Lys mutation in epidermal and dermal cells and tissue. One keratinocyte culture (left panel) was established from each epidermal sample. Mutation percentages are indicated by the blue circles. Multiple fibroblast ... Nine EN samples were obtained from the hand, trunk, or neck from seven patients (Table S1). The mutation level in the keratinocyte cultures was 0-44% whereas the mutation level in the fibroblast cultures was 0-38%. The mutation level in the cultured dermal tissue was 9-25%. Interestingly, the two samples with the highest levels of mutation in the keratinocytes (101EN1 and 78EN2) had the lowest mutation percentages in dermal cells or tissue. Detection of the mutant allele in the EN keratinocytes is consistent with the identification of the AKT1 p.Glu17Lys mutation in skin scrapings of PS epidermal nevi (Wieland et al. 2013). Four biopsies from two patients were obtained from tissue that appeared normal by gross examination but was from an area that was far removed from lesional tissue (designated as unaffected) or bordered a CCTN (designated as unknown) (Table S1). No evidence of the mutant allele was present in the keratinocyte cultures. The mean mutation level in the fibroblast cultures was 6-27%. We conclude that the AKT1 p.Glu17Lys activating mutation in keratinocytes is a key determinant of EN formation. Mutations in the FGFR3, PIK3CA, and RAS genes have been identified in epidermal cells of EN not associated with PS, supporting this hypothesis (Hafner et al. 2006; 2007; 2012). Histopathologically, these keratinocytic EN are strikingly similar to those found in PS and it should be noted that PIK3CA encodes the catalytic subunit of the PI3K complex which functions upstream of AKT in the same signaling pathway. In contrast, there was no correlation of the mutation levels in fibroblasts or cultured dermal tissue with the clinical lesion type. The inability to detect an AKT1 mutation in two of the EN keratinocyte cultures could be due to a sampling artifact if the mutant cells were not uniformly distributed throughout the lesion and the sample was from an area with low-level mosaicism. It seems unlikely that the lack of mutant keratinocytes in some EN indicates that these lesions form as a result of signaling from mutant cells in the dermis, since even organoid EN, such as nevus sebaceous have been shown to result from mutant cells in the epidermis. (Groesser et al. 2012) Mutant cells were found in dermal fibroblasts of both CCTN and normal-appearing skin, suggesting that the presence of mutant cells in the dermis is necessary but not sufficient to drive the formation of CCTN. The propensity of CCTNs to develop on soles and palms in PS may reflect a greater role of AKT1 in postnatal regulation of tissue architecture at these sites than elsewhere on the skin.

Identifying an HLA-matched sibling donor for hematopoietic stem cell transplantation (HSCT) is time-consuming and expensive, and often limited by reimbursement caps imposed by insurance providers. To improve the effectiveness and efficiency of screening for HLA-matched siblings, we developed an assay for determining HLA identity using a panel of nine informative short tandem repeat (STR) loci located throughout the HLA complex. The STR panel was assessed for accuracy in identifying HLA-matched siblings in 88 family workups comprising a total of 132 related donor and recipient typing comparisons. All sibling pairs with identical STR alleles were also HLA identical. Of the 48 pairs mismatched at one or more STR alleles, all were genotypically HLA non-identical at one or more loci. The sensitivity and specificity of STR analysis for identifying HLA-matched siblings were 91% and 100%, respectively. Three false negatives occurred due to an STR mutation or possible HLA-DPB1/DQB1 recombination. Additionally, STR genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was ambiguous, due to extensive homozygosity or shared parental haplotypes. The HLA STR assay is a reliable and rapid test that can be used to inexpensively screen potential sibling donors for HLA identity.

Purpose: We report a male child who presented at 15 months with perianal abscesses and proctitis, progressing to transmural pancolitis with colocutaneous fistulae, consistent with a Crohn disease-like illness. The age and severity of the presentation suggested an underlying immune defect; however, despite comprehensive clinical evaluation, we were unable to arrive at a definitive diagnosis, thereby restricting clinical management. Methods: We sought to identify the causative mutation(s) through exome sequencing to provide the necessary additional information required for clinical management. Results: After sequencing, we identified 16,124 variants. Subsequent analysis identified a novel, hemizygous missense mutation in the X-linked inhibitor of apoptosis gene, substituting a tyrosine for a highly conserved and functionally important cysteine. X-linked inhibitor of apoptosis was not previously associated with Crohn disease but has a central role in the proinflammatory response and bacterial sensing through the NOD signaling pathway. The mutation was confirmed by Sanger sequencing in a licensed clinical laboratory. Functional assays demonstrated an increased susceptibility to activation-induced cell death and defective responsiveness to NOD2 ligands, consistent with loss of normal X-linked inhibitor of apoptosis protein function in apoptosis and NOD2 signaling. Conclusions: Based on this medical history, genetic and functional data, the child was diagnosed as having an X-linked inhibitor of apoptosis deficiency. Based on this finding, an allogeneic hematopoietic progenitor cell transplant was performed to prevent the development of life-threatening hemophagocytic lymphohistiocytosis, in concordance with the recommended treatment for X-linked inhibitor of apoptosis deficiency. At 42 days posttransplant, the child was able to eat and drink, and there has been no recurrence of gastrointestinal disease, suggesting this mutation also drove the gastrointestinal disease. This report describes the identification of a novel cause of inflammatory bowel disease. Equally importantly, it demonstrates the power of exome sequencing to render a molecular diagnosis in an individual patient in the setting of a novel disease, after all standard diagnoses were exhausted, and illustrates how this technology can be used in a clinical setting. Genet Med 2011:13(3):255–262.

CHARGE syndrome and Kallmann syndrome (KS) are two distinct developmental disorders sharing overlapping features of impaired olfaction and hypogonadism. KS is a genetically heterogeneous disorder consisting of idiopathic hypogonadotropic hypogonadism (IHH) and anosmia, and is most commonly due to KAL1 or FGFR1 mutations. CHARGE syndrome, a multisystem autosomal-dominant disorder, is caused by CHD7 mutations. We hypothesized that CHD7 would be involved in the pathogenesis of IHH and KS (IHH/KS) without the CHARGE phenotype and that IHH/KS represents a milder allelic variant of CHARGE syndrome. Mutation screening of the 37 protein-coding exons of CHD7 was performed in 101 IHH/KS patients without a CHARGE phenotype. In an additional 96 IHH/KS patients, exons 6-10, encoding the conserved chromodomains, were sequenced. RT-PCR, SIFT, protein-structure analysis, and in situ hybridization were performed for additional supportive evidence. Seven heterozygous mutations, two splice and five missense, which were absent inor = 180 controls, were identified in three sporadic KS and four sporadic normosmic IHH patients. Three mutations affect chromodomains critical for proper CHD7 function in chromatin remodeling and transcriptional regulation, whereas the other four affect conserved residues, suggesting that they are deleterious. CHD7's role is further corroborated by specific expression in IHH/KS-relevant tissues and appropriate developmental expression. Sporadic CHD7 mutations occur in 6% of IHH/KS patients. CHD7 represents the first identified chromatin-remodeling protein with a role in human puberty and the second gene to cause both normosmic IHH and KS in humans. Our findings indicate that both normosmic IHH and KS are mild allelic variants of CHARGE syndrome and are caused by CHD7 mutations.

This article covers the rapidly advancing field of preimplantation genetic diagnosis (PGD), the molecular genetic analysis of cells taken from embryos formed through in vitro fertilization (IVF). The article focuses on current practices in patient management, relevant IVF and PGD procedures, molecular methods used in the genetic analysis, and technical difficulties that can affect test results. It discusses the growing list of indications for PGD including chromosomal disorders, monogenic disorders and human leukocyte antigen typing typing of embryos. The article also examines some of the emerging technologies being introduced into PGD.

Objective To characterize the phenotype, modes of inheritance, karyotype, and molecular basis of patients with idiopathic hypogonadotropic hypogonadism (IHH). Design Review of medical records, karyotyping, and collation of gene mutation analysis. Setting University molecular reproductive endocrinology laboratory. Patient(s) Patients with IHH. Intervention(s) Review of medical records, laboratory studies, and molecular studies. Main Outcome Measure(s) Sense of smell, severity of IHH (complete vs. incomplete), associated anomalies, karyotype, mutation analysis, and genotype/phenotype correlations were studied. Result(s) Of 315 patients with IHH, 6.3% had one or more affected relatives. Autosomal recessive inheritance was likely in most of these familial cases, but autosomal-dominant and X-linked recessive inheritance patterns were likely in some families. Complete IHH was more commonly found in males (62%), whereas incomplete IHH was more commonly observed in females (54.3%). Anosmia was present in 31.3% of males and 27.9% of females. The karyotype was normal in all 19 females tested, but was abnormal in 3 of 57 (5.3%) of males tested. Although cryptorchidism did not differ among those who were anosmic vs. normosmic, it was approximately four times more common in patients with complete IHH than incomplete IHH (15.3% vs. 3.9%). Approximately 10% of the IHH patients tested had mutations in either the GNRHR or KAL1 gene. Conclusion(s) Idiopathic hypogonadotropic hypogonadism is a heterogeneous disorder affecting fertility, in which the number of familial cases is probably underestimated. Further study of genes that regulate hypothalamic-pituitary development and function will likely reveal important information regarding the development of normal puberty in humans.

Microsatellite analysis was performed in the laboratory of Dr. W. P. Robinson. We thank I. J. Barrett, F. Bernasconi, and B. L. Lomax, of the Research Cytogenetics and Molecular Genetics Laboratories, for their technical assistance. The comments, on the manuscript, of Drs. W. P. Robinson, F. J. Dill, E. Separovic, and A. Rose are greatly appreciated. The financial assistance of the March of Dimes Research Foundation (FY96-1034) is gratefully acknowledged.

Objective To determine the prevalence of digenic mutations in patients with idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS). Design Molecular analysis of DNA in IHH/KS patients. Setting Academic medical center. Patient(s) Twenty-four IHH/KS patients with a known mutation (group 1) and 24 IHH/KS patients with no known mutation (group 2). Intervention(s) DNA from IHH/KS patients was subjected to polymerase chain reaction–based DNA sequencing of the 13 most common genes ( KAL1, GNRHR, FGFR1, KISS1R, TAC3, TACR3, FGF8, PROKR2, PROK2, CHD7, NELF, GNRH1 , and WDR11 ). Main Outcome Measure(s) The identification of mutations absent in ≥188 ethnically matched controls. Both SIFT (sorting intolerant from tolerant) and conservation among orthologs provided supportive evidence for pathologic roles. Result(s) In group 1, 6 (25%) of 24 IHH/KS patients had a heterozygous mutation in a second gene, and in group 2, 13 (54.2%) of 24 had a mutation in at least one gene, but none had digenic mutations. In group 2, 7 (29.2%) of 24 had a mutation considered sufficient to cause the phenotype. Conclusion(s) When the 13 most common IHH/KS genes are studied, the overall prevalence of digenic gene mutations in IHH/KS was 12.5%. In addition, approximately 30% of patients without a known mutation had a mutation in a single gene. With the current state of knowledge, these findings suggest that most IHH/KS patients have a monogenic etiology.

Objective To determine if mutations in NELF , a gene isolated from migratory GnRH neurons, cause normosmic idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS). Design Molecular analysis correlated with phenotype. Setting Academic medical center. Patient(s) A total of 168 IHH/KS patients as well as unrelated control subjects were studied for NELF mutations. Intervention(s) NELF coding regions/splice junctions were subjected to polymerase chain reaction (PCR)–based DNA sequencing. Eleven additional IHH/KS genes were sequenced in three patients with NELF mutations. Main Outcome Measure(s) Mutations were confirmed by sorting intolerant from tolerant, reverse-transcription (RT)-PCR, and Western blot analysis. Result(s) Three novel NELF mutations absent in 372 ethnically matched control subjects were identified in 3/168 (1.8%) IHH/KS patients. One IHH patient had compound heterozygous NELF mutations (c.629–21G>C and c.629–23C>G), and he did not have mutations in 11 other known IHH/KS genes. Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: NELF/KAL1 (c.757G>A; p.Ala253Thr of NELF and c.488_490delGTT; p.Cys163del of KAL1 ) and NELF/TACR3 (c.1160–13C>T of NELF and c.824G>A; p.Trp275X of TACR3 ). In vitro evidence of these NELF mutations included reduced protein expression and splicing defects. Conclusion(s) Our findings suggest that NELF is associated with normosmic IHH and KS, either singly or in combination with a mutation in another gene.

Objective To describe two children diagnosed with Beckwith-Wiedemann Syndrome (BWS) arising from a spontaneous conception and an assisted reproductive technology (ART) cycle from one patient with a long-standing history of subfertility. Design Case report. Setting Academic medical center. Patient(s) Two children with the morphologic features of BWS as a result of a spontaneous conception and an ART cycle from the same patient. Intervention(s) Assisted reproductive technology. Main Outcome Measure(s) Neonatal and pediatric morphologic evaluation by geneticists. Result(s) Two children with the morphologic features consistent with the criteria for the diagnosis of BWS. Conclusion(s) Patients with subfertility may be carriers for genetic disorders that can be passed to a child with or without the use of assisted reproductive technologies (ART). The use of ART may bypass natural selection mechanisms.