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2.  Nasal Embryonic LHRH Factor (NELF) Mutations in Patients with Normosmic Hypogonadotropic Hypogonadism and Kallmann Syndrome 
Fertility and sterility  2011;95(5):1613-20.e1-7.
Study 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
Patients
168 IHH/KS patients along with unrelated controls were studied for NELF mutations.
Intervention
NELF coding regions/splice junctions were subjected to PCR-based DNA sequencing, Eleven additional IHH/KS genes were sequenced in three patients with NELF mutations.
Main Outcome Measure
Mutations were confirmed by SIFT, RT-PCR, and western blot analysis.
Results
Three novel NELF mutations absent in 372-ethnically matched controls were identified in 3/168(1.8%) IHH/KS patients. One IHH patient had compound heterozygous NELF mutations (c.629-21C>G and c.629-23G>C); 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.
Conclusions
Our findings suggest that NELF is associated with normosmic IHH and KS, either singly or in combination with a mutation in another gene.
doi:10.1016/j.fertnstert.2011.01.010
PMCID: PMC3888818  PMID: 21300340
Nasal embryonic LHRH factor; hypogonadotropic hypogonadism; Kallmann syndrome; gonadotropin-releasing hormone (GnRH); GnRH neuron migration
4.  The prevalence of digenic mutations in patients with normosmic hypogonadotropic hypogonadism and Kallmann syndrome 
Fertility and sterility  2011;96(6):1424-1430.e6.
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.
doi:10.1016/j.fertnstert.2011.09.046
PMCID: PMC3573697  PMID: 22035731
Digenic mutations; idiopathic hypogonadotropic hypogonadism; Kallmann syndrome
6.  A Mosaic Activating Mutation in AKT1 Associated with the Proteus Syndrome 
The New England journal of medicine  2011;365(7):611-619.
BACKGROUND
The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state.
METHODS
We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots.
RESULTS
Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation.
CONCLUSIONS
The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K–AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.)
doi:10.1056/NEJMoa1104017
PMCID: PMC3170413  PMID: 21793738
7.  Birth of a healthy infant following preimplantation PKHD1 haplotyping for autosomal recessive polycystic kidney disease using multiple displacement amplification 
Purpose
To develop a reliable preimplantation genetic diagnosis protocol for couples who both carry a mutant PKHD1 gene wishing to conceive children unaffected with autosomal recessive polycystic kidney disease (ARPKD).
Methods
Development of a unique protocol for preimplantation genetic testing using whole genome amplification of single blastomeres by multiple displacement amplification (MDA), and haplotype analysis with novel short tandem repeat (STR) markers from the PKHD1 gene and flanking sequences, and a case report of successful utilization of the protocol followed by successful IVF resulting in the birth of an infant unaffected with ARPKD.
Results
We have developed 20 polymorphic STR markers suitable for linkage analysis of ARPKD. These linked STR markers have enabled unambiguous identification of the PKHD1 haplotypes of embryos produced by at-risk couples.
Conclusions
We have developed a reliable protocol for preimplantation genetic diagnosis of ARPKD using single-cell MDA products for PKHD1 haplotyping.
doi:10.1007/s10815-010-9432-5
PMCID: PMC2922704  PMID: 20490649
Preimplantation genetic diagnosis; Autosomal recessive polycystic kidney disease; Multiple displacement amplification; PKHD1 haplotype analysis; Linkage analysis with linked STR markers
8.  Zoom‐in comparative genomic hybridisation arrays for the characterisation of variable breakpoint contiguous gene syndromes 
Journal of Medical Genetics  2007;44(1):e59.
Contiguous gene syndromes cause disorders via haploinsufficiency for adjacent genes. Some contiguous gene syndromes (CGS) have stereotypical breakpoints, but others have variable breakpoints. In CGS that have variable breakpoints, the extent of the deletions may be correlated with severity. The Greig cephalopolysyndactyly contiguous gene syndrome (GCPS‐CGS) is a multiple malformation syndrome caused by haploinsufficiency of GLI3 and adjacent genes. In addition, non‐CGS GCPS can be caused by deletions or duplications in GLI3. Although fluorescence in situ hybridisation (FISH) can identify large deletion mutations in patients with GCPS or GCPS‐CGS, it is not practical for identification of small intragenic deletions or insertions, and it is difficult to accurately characterise the extent of the large deletions using this technique. We have designed a custom comparative genomic hybridisation (CGH) array that allows identification of deletions and duplications at kilobase resolution in the vicinity of GLI3. The array averages one probe every 730 bp for a total of about 14 000 probes over 10 Mb. We have analysed 16 individuals with known or suspected deletions or duplications. In 15 of 16 individuals (14 deletions and 1 duplication), the array confirmed the prior results. In the remaining patient, the normal CGH array result was correct, and the prior assessment was a false positive quantitative polymerase chain reaction result. We conclude that high‐density CGH array analysis is more sensitive than FISH analysis for detecting deletions and provides clinically useful results on the extent of the deletion. We suggest that high‐density CGH array analysis should replace FISH analysis for assessment of deletions and duplications in patients with contiguous gene syndromes caused by variable deletions.
doi:10.1136/jmg.2006.042473
PMCID: PMC2597909  PMID: 17098889
GLI3 ; oligonucleotide array; comparative genomic hybridization
9.  Preimplantation HLA haplotyping using tri-, tetra-, and pentanucleotide short tandem repeats for HLA matching 
Purpose
To aid couples wishing to conceive children who are HLA matched to a sibling in need of a hematopoietic progenitor cell transplant, we developed a preimplantation HLA haplotype analysis of embryos that utilizes tri-, tetra-, and pentanucleotide STR markers.
Methods
For preimplantation HLA genotyping, we use polymorphic STR markers located across the HLA and flanking regions, selecting exclusively tri-, tetra-, and pentanucleotide repeats. These markers can be resolved using either capillary electrophoresis (CE) or polyacrylamide gels.
Results
We have developed 43 reliable STR markers for preimplantation HLA matching. Selected STR markers enabled unambiguous identification of embryos whose HLA haplotypes were matched with the affected patient using polyacrylamide gel or capillary electrophoresis.
Conclusions
The use of tri-, tetra-, and pentanucleotide repeat markers and polyacrylamide gels for STR genotyping in HLA matching is a simple and cost effective approach to clinical testing.
Electronic supplementary material
The online version of this article (doi:10.1007/s10815-008-9233-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s10815-008-9233-2
PMCID: PMC2596682  PMID: 18677557
Haplotype analysis; Preimplantation genetic diagnosis; Preimplantation HLA matching; Short tandem repeats; Human leukocyte antigen
10.  The prevalence of intragenic deletions in patients with idiopathic hypogonadotropic hypogonadism and Kallmann syndrome 
Molecular Human Reproduction  2008;14(6):367-370.
Idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS) are clinically and genetically heterogeneous disorders caused by a deficiency of gonadotrophin-releasing hormone (GnRH). Mutations in three genes—KAL1, GNRHR and FGFR1—account for 15–20% of all causes of IHH/KS. Nearly all mutations are point mutations identified by traditional PCR-based DNA sequencing. The relatively new method of multiplex ligation-dependent probe amplification (MLPA) has been successful for detecting intragenic deletions in other genetic diseases. We hypothesized that MLPA would detect intragenic deletions in ∼15–20% of our cohort of IHH/KS patients. Fifty-four IHH/KS patients were studied for KAL1 deletions and 100 were studied for an autosomal panel of FGFR1, GNRH1, GNRHR, GPR54 and NELF gene deletions. Of all male and female subjects screened, 4/54 (7.4%) had KAL1 deletions. If only anosmic males were considered, 4/33 (12.1%) had KAL1 deletions. No deletions were identified in any of the autosomal genes in 100 IHH/KS patients. We believe this to be the first study to use MLPA to identify intragenic deletions in IHH/KS patients. Our results indicate ∼12% of KS males have KAL1 deletions, but intragenic deletions of the FGFR1, GNRH1, GNRHR, GPR54 and NELF genes are uncommon in IHH/KS.
doi:10.1093/molehr/gan027
PMCID: PMC2434956  PMID: 18463157
Kallmann syndrome; KAL1 gene; hypogonadotropic hypogonadism; idiopathic hypogonadotropic hypogonadism; MLPA
11.  Implementing genomic medicine in the clinic: the future is here 
Genetics in Medicine  2013;15(4):258-267.
Although the potential for genomics to contribute to clinical care has long been anticipated, the pace of defining the risks and benefits of incorporating genomic findings into medical practice has been relatively slow. Several institutions have recently begun genomic medicine programs, encountering many of the same obstacles and developing the same solutions, often independently. Recognizing that successful early experiences can inform subsequent efforts, the National Human Genome Research Institute brought together a number of these groups to describe their ongoing projects and challenges, identify common infrastructure and research needs, and outline an implementation framework for investigating and introducing similar programs elsewhere. Chief among the challenges were limited evidence and consensus on which genomic variants were medically relevant; lack of reimbursement for genomically driven interventions; and burden to patients and clinicians of assaying, reporting, intervening, and following up genomic findings. Key infrastructure needs included an openly accessible knowledge base capturing sequence variants and their phenotypic associations and a framework for defining and cataloging clinically actionable variants. Multiple institutions are actively engaged in using genomic information in clinical care. Much of this work is being done in isolation and would benefit from more structured collaboration and sharing of best practices.
Genet Med 2013:15(4):258–267
doi:10.1038/gim.2012.157
PMCID: PMC3835144  PMID: 23306799
medical genomics; practice standards

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