Novel homozygous nonsense mutations in the luteinizing hormone

ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY

Novel homozygous nonsense mutations in the luteinizing hormone receptor (LHCGR) gene associated with 46,XY primary amenorrhea Imen Ben Hadj Hmida, Ph.D.,a,b Soumaya Mougou-Zerelli, M.D., Ph.D.,a Anis Hadded, M.D.,c Sarra Dimassi, M.D., Ph.D.,a Molka Kammoun, M.D., Ph.D.,a Joelle Bignon-Topalovic, B.Sc.,b Mohamed Bibi, M.D.,d Ali Saad, M.D., Ph.D.,a Anu Bashamboo, Ph.D.,b and Ken McElreavey, Ph.D.b a Laboratory of Human Cytogenetics, Molecular Genetics and Reproductive Biology, Farhat Hached University Hospital, Sousse, Tunisia; b Human Developmental Genetics, Institut Pasteur, Paris, France; c Department of Gynecology and Obstetrics, Farhat Hached University Hospital, Sousse, Tunisia; and d Department of Gynecology and Obstetrics, Fattouma Bourguiba Teaching Hospital, Monastir, Tunisia

Objective: To determine the genetic cause of 46,XY primary amenorrhea in three 46,XY girls. Design: Whole exome sequencing. Setting: University cytogenetics center. Patient(s): Three patients with unexplained 46,XY primary amenorrhea were included in the study. Intervention(s): Potentially pathogenic variants were confirmed by Sanger sequencing, and familial segregation was determined where parents' DNA was available. Main Outcome Measure(s): Exome sequencing was performed in the three patients, and the data were analyzed for potentially pathogenic mutations. The functional consequences of mutations were predicted. Result(s): Three novel homozygous nonsense mutations in the luteinizing hormone receptor (LHCGR) gene were identified:c.1573 C/T, p.Gln525Ter, c.1435 C/T p.Arg479Ter, and c.508 C/T, p.Gln170Ter. Conclusion(s): Inactivating mutations of the LHCGR gene may be a more common cause of 46,XY primary amenorrhea than previously considered. (Fertil SterilÒ 2016;106:225–9. Ó2016 by American Society for Reproductive Medicine.) Key Words: Exome sequencing, disorders of sex development (DSD), primary amenorrhea, LHCGR mutation Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/hmidai-lhcgr-46xy-primary-amenorrhea/

L

H has an essential role in both male and female sex differentiation, pubertal development, and reproduction (1). The human luteinizing hormone receptor gene (LHCGR) gene consists of 12 exons and spans >80 kb on chromosome 2p21. The exon 11 encodes the transmembrane

domain, which transduces the signal of hormone binding to the large extracellular domain (encoded by exons 2–10) (2). LHCGR expression is predominantly gonadal, with high expression in testicular Leydig cells and ovarian thecal as well as differentiated granulosa cells (3). In male fetal devel-

Received August 31, 2015; revised February 24, 2016; accepted March 3, 2016; published online March 22, 2016. I.B.H.H. has nothing to disclose. S.M.-Z. has nothing to disclose. A.H. has nothing to disclose. S.D. has nothing to disclose. M.K. has nothing to disclose. J.B.-T. has nothing to disclose. M.B. has nothing to disclose. A.S. has nothing to disclose. A.B. has nothing to disclose. K.M. has nothing to disclose. Supported by a research grant from the EuroDSD in the European Community's Seventh Framework Programme FP7/2007–2013 under grant agreement no. 201444 (to A.B. and K.M.). This work was also supported by a grant from the Tunisian Ministry of Higher Education and Research. Reprint requests: Dr. Ken McElreavey, Ph.D., Human Developmental Genetics, Institut Pasteur, 25 Rue du Dr Roux FR, Paris Cedex 15 75724, France (E-mail: [email protected]). Fertility and Sterility® Vol. 106, No. 1, July 2016 0015-0282/$36.00 Copyright ©2016 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2016.03.008 VOL. 106 NO. 1 / JULY 2016

opment, LHCGR in Leydig cells responds to hCG, which is secreted by the placenta, and induces an increased androgen synthesis. Later in fetal development, androgen synthesis from Leydig cells is maintained by LH production from the anterior pituitary (3). LHCGR is essential for male development since fetal androgen synthesis mediates the differentiation of the external genitalia into the male phenotype. Consistent with its function, heterozygous activating mutations in LHCGR are associated with gonadotropin-independent precocious puberty and Leydig cell hyperplasia (1, 3). In contrast, homozygous or compound heterozygous inactivating 225


TABLE 1 Phenotypes of the three unrelated cases of 46,XY primary amenorrhea. Variable Ancestry Karyotype Assigned sex Age, height, weight Phenotype

Case I Tunisian 46,XY Female 19 y, 107 kg, 176 cm Development of breasts at 12 y. Stage of puberty: P2S2A2. Normal female external genitalia. Primary amenorrhea. Absence of upper vagina and ultrasound. Gonads not observed by ultrasound.

Case II

Case III

Tunisian 46,XY Female 29 y, 59 kg, 175 cm Normal female external genitalia. Primary amenorrhea. Absence of upper vagina and uterus by MRI. Gonads not observed by MRI. Absence of secondary sexual characteristics.

Tunisian 46,XY Female 22 y, 74 kg, 176 cm Normal female external genitalia. Primary amenorrhea. Absence of upper vagina and uterus by ultrasound. Gonads observed by ultrasound with appearance of testicular feminization. Normal secondary sexual characteristics.

Ben Hadj Hmida. LHCGR and 46,XY primary amenorrhea. Fertil Steril 2016.

or loss-of-function mutations in 46,XY individuals result in resistance to hormone stimulation. The first reports of LHCGR-inactivating mutations were described in men with Leydig cell hypoplasia (LCH) leading to pseudohermaphroditism (4). This is due to the inability of the fetal testes to respond to hCG, resulting in a lack of androgens to masculinize the fetal internal and external genitalia and either female external genitalia or a spectrum of genital malformations (3). LCH has two major forms. Type I is a very rare severe form of 46,XY disorder of sex development (DSD) with complete disruption of receptor function resulting in a completely female phenotype. The more common type II form of LCH is associated with 46,XY male pseudohermaphroditism with micropenis and/or hypospadias as the cardinal features (3, 5, 6). The varying degree of Leydig cell hyperplasia comes from the fact that for the most part the mutations in LHCGR do not cause a complete loss of function as the receptor may and retain some degree of responsiveness to hCG and LH (3, 5, 6). The degree of masculinization correlates well with the responsiveness of the mutated receptor to hormone stimulation. In the present study, we report three cases of 46,XY females who presented with primary amenorrhea. Using an exome sequencing approach, we identified three independent and novel homozygous nonsense mutations in the LHCGR gene.

IRB00003835), and consent to genetic testing was obtained from adult probands or from the parents when the patient was under 18 years.

Conventional Cytogenetic Analysis Conventional cytogenetic analysis of R-banded chromosomes at a resolution of 450–550 bands was performed using the peripheral blood lymphocytes of the patients and their parents.

Fluorescence In Situ Hybridization Fluorescence in situ hybridization using a commercial SRY probe (SRY; Vysis) labeled in orange and an X centromere probe (X CEP; Vysis) labeled in green was performed according to the manufacturer's instructions (Vysis). Briefly, 12 mL of probe mix was added to metaphase slides and denatured for 5 minutes at 72 C. After overnight hybridization at 37 C, slides were washed for 5 minutes in 2 SSC/NP40 (Vysis) at 72 C. Finally, the slides were mounted with 15 mL of 4,6diamidino-2-phenyllindole (Vysis) and analyzed using a fluorescence in situ hybridization station (CytoVision, Applied Imaging).

MATERIALS AND METHODS Subjects The three patients studied are of the same origin in Sidi Bouzid region of Tunisia. They consulted for primary amenorrhea associated with DSD. The clinical details of each patient are described in Table 1, and the hormonal profile in Table 2. Case I was born to nonconsanguineous parents, whereas cases II and III were born to a first cousin consanguineous marriage. There was no history of DSD in any of the three families. The pedigrees of the three families are shown in Supplemental Figure 1 and the magnetic resonance imaging (MRI) of case II is shown in Figure 1. This study was approved by the local French ethical committee (2014/18NICB, registration no. 226

TABLE 2 Hormonal profiles for the three cases presenting with 46,XY primary amenorrhea. Hormone LH (mIUI/mL) FSH (mIUI/mL) PRL (ng/mL) E2 (pg/mL)

Adult range, XY male

Case I

Case II

Case III

1.1–7 1.7–12 1.5–19 0–50

40.8 28.1 8.1 –

20 22 7.3 15

31 59 – 10


VOL. 106 NO. 1 / JULY 2016

Fertility and Sterility®

FIGURE 1

(A) MRI of the pelvis, sagittal section, T2-weighted, of case II. 1 ¼ bladder; 2 ¼ pubic symphysis; 3 ¼ vagina; 4 ¼ rectum; 5 ¼ uterus; L5 ¼ L5 vertebral body; S ¼ sacrum; arrow ¼ absence of gonads. (B) Metaphase fluorescence in situ hybridization analysis using an a commercial SRY probe (orange) and an X centromere probe (green) was performed according to the manufacturer's instructions (Vysis). (C) Sequence chromatogram revealing a homozygous point mutation at nucleotide position c.1595 C/T/p.Gln525Ter of LHCGR in case 1 (A). Mother (B) and father (C) are heterozygous. (D) Sequence chromatogram revealing a homozygous nonsense mutation at nucleotide position c. 1457 C/T (p.Arg479Ter) of LHCGR in case 2 and (E) the c.508 C/T, (p.Gln170Ter) of LHCGR in case 3. Ben Hadj Hmida. LHCGR and 46,XY primary amenorrhea. Fertil Steril 2016.

Exome Sequencing, Alignment, and Variant Calling Clinical information and peripheral blood samples were collected from the proband and her parents after obtaining their written informed consent. Parental written informed consent was also obtained for publication. Genomic DNA from the proband and her parents was prepared from peripheral blood lymphocytes using the PAXgene system (Qiagen) following the manufacturer's instructions. Exon enrichment was performed using Agilent SureSelect Human All Exon V4. Paired-end sequencing was performed on the Illumina HiSeq2000 platform using TruSeq v3 chemistry. Read files (Fastq) were generated from the sequencing platform via the manufacturer's proprietary software. Reads were mapped using the BurrowsWheeler Aligner, and local realignment of the mapped reads around potential insertion/deletion (indel) sites was carried out with the GATK version 1.6. Duplicate reads were marked using Picard version 1.62. Additional BAM VOL. 106 NO. 1 / JULY 2016

file manipulations were performed with Samtools 0.1.18. Single nucleotide polymorphism (SNP) and indel variants were called using the GATK Unified Genotyper for each sample. SNP novelty was determined against dbSNP137. Novel variants were analyzed by a range of web-based bioinformatics tools using the EnsEMBL SNP Effect Predictor (http://www.ensembl.org/homosapiens/userdata/uploadvari ations). All variants were screened manually against the Human Gene Mutation Database Professional (Biobase) (http://www.biobase-international.com/product/hgmd). In silico analysis was performed to determine the potential pathogenicity of the variants. The biological function of genes carrying potentially pathogenic variants was examined using Entrez Gene Summary, UniProtKB Summary and Tocris Summary. Association with human disease was examined using the UniProt Disease summary, OMIM, and manual analysis of Pubmed data. Potentially pathogenic mutations were verified using classic Sanger sequencing. 227

ORIGINAL ARTICLE: REPRODUCTIVE ENDOCRINOLOGY Sanger Sequencing of LHCGR Gene The mutations identified by exome sequencing were confirmed using Sanger sequencing. The regions containing the mutations were amplified from DNA extracted using conventional techniques from peripheral blood lymphocytes of each individual. The polymerase chain reaction (PCR) primers used were F: 50 - AGACTGGCAGACAGGGAGTG - 30 and R: 50 GAGATAGGTGCCATGCAGGT - 30 (exon 11), or F: 50 -GAGCATCTGTAACACAGGCAT – 30 and R: 50 -CACCAGTGAGTGAGGAATGTG-30 (exon 6). The PCR amplification conditions were 95 C for 6 minutes followed by 37 cycles of 95 C for 20 seconds, 62 C for 20 seconds, and 72 C for 20 seconds. A final elongation step of 72 C for 5 seconds was added in each case. A total of 4 mL of each PCR fragment was then electrophoresed in a 2% agarose gel to verify the expected length of the amplified fragments. DNA sequence analysis was performed using at least 200 ng of purified DNA, 20 ng of primer, and fluorescent labeled Taq Dye Terminator reaction mix (Applied Biosystems) according to the manufacturer's instructions. DNA sequence was determined using an automated DNA sequencer.

RESULTS Conventional cytogenetic analysis of the three patients with a female phenotype revealed a 46,XY male karyotype in all metaphases. The fluorescence in situ hybridization revealed the presence of the SRY gene on the Y chromosome (Fig. 1B). The number of paired-end reads were 25,280,296 with a mean target coverage of 69.89 (patient 1), 20,990,495 with a mean target coverage of 59.24 (patient 2), and 22,672,339 with a mean coverage of 67.62 (patient 3). The percentage of target bases with >10 coverage was 98.55%, 98.09%,

and 98.14%, respectively. The number of variants with predicted serious consequences (involving an essential splice site, a stop codon gained or lost, a complex indel, a frameshift mutation in the coding sequence, or a nonsynonymous change with predicted deleterious effect on protein function) for the three patients was 10,015, 10,407, and 10,214, respectively. Analyses of the data sets revealed several mutations that were predicted by PolyPhen2 and/ or SIFT to be deleterious for protein function. Assuming a dominant mutation was responsible for the phenotype and after filtering to remove variants with a minor allelic frequency of >0.05, the genes carrying predicted deleterious mutations in a heterozygous state in cases I, II, and III were 91, 84, and 56, respectively. Assuming a recessive or X-linked model of inheritance and after filtering to remove variants with a minor allelic frequency of >0.05, the genes carrying a serious homozygous, compound heterozygous, or hemizygous and predicted deleterious mutation were 5, 10, and 12 genes for cases I, II, and III, respectively (Supplemental Data). An analysis of these data sets revealed only one gene known to be associated with DSD, the LH receptor gene LHCGR. All three patients carried different homozygous nonsense mutations in LHCGR: case I c.1573 C/T (p.Gln525Ter), case II c.1435 C/T (p.Arg479Ter) both in exon 11, and case III (c.508 C/T, p.Gln170Ter) in exon 6. The presence of the mutation was verified in all cases by Sanger sequencing (Fig. 1C). DNA was available from the parents of patient 1 but was insufficient for exome sequencing, whereas the parents of patients 2 and 3 were not available for the study. Sanger sequencing DNA from the parents of patient 1 confirmed the presence of a heterozygous mutation (c.1595 C/T, p.Gln525Ter) in each parent. Neither the c.1573 C/T (p.Gln525Ter) mutation nor the c.508 C/T,

TABLE 3 Inactivating mutations in the LHCGR gene associated with 46,XY DSD and raised as a female. Mutation c.1635 A/C, p.Cys545Ter homozygous c.593 G/C, p.Ala593Pro homozygous c. 1847 C/A, p.Ser616Tyr homozygous

c. 1060 A/G, p. Glu354Lys homozygous c.430 G/T, p. Val144Phe homozygous c.1836 T/G, p.Tyr612Ter homozygous c. 455 T/C, p. Ile152Thr c. 537-3 C/A compound heterozygous c.1395 G/A, p.Trp465Ter homozygous

Clinical features and ancestry (reference) Two 46,XY sisters with completely female phenotype and Leydig cell hypoplasia. Ancestry unknown (10). Two 46,XY sisters presenting with complete male pseudohermaphroditism, vagina without uterus or fallopian tubes, the gonads examination found to be testes with normal Sertoli cells, but no mature Leydig cells. Patients from north/northeast Brazil (11). Three 46,XY sisters presenting with a lack of breast development and primary amenorrhea. Female external genitalia, normal clitoris, absence of posterior labial fusion, and separate urethral and vaginal openings. Gonads were palpable bilaterally in the inguinal regions. Patients from Brazil (9). Two 46,XY sisters presenting with primary amenorrhea, female external genitalia, and lack of breast development. Ancestry unknown (12). One 46,XY individual presenting a clitoral enlargement and labial synechia, a right-sided €llerian structures were not inguinal hernia, and a testis found in the inguinal region. Mu seen. Patient of Turkish origin (13). Two 46,XY sisters with completely female phenotype, who presented with primary amenorrhea and absence of breast development. Patients from the United States (14). 46,XY and 46,XX siblings: 46,XY sibling presented with female phenotype, clitoral enlargement, blind-ending vagina, and the external opening of urethra in the clitoris. € llerian structures were not observed. Siblings of Chinese origin (15). Uterus or other Mu One 46,XY female presenting with primary amenorrhea, presence of Sertoli cells in gonad with serum low T concentration. Ancestry unknown (16).


228

VOL. 106 NO. 1 / JULY 2016

Fertility and Sterility® p.Gln170Ter mutation is present in the dbSNP138 or ExAC databases (http://exac.broadinstitute.org/). However, the c.1435 C/T (p.Arg479Ter) mutation has been reported in the ExAC database in one individual in the heterozygous state with an allelic frequency of 1:6612 (MAF 0.0001512) in the Finnish population. The position of each of the mutations in the LHCGR gene is indicated in Supplemental Figure 2.

DISCUSSION Mutations in the LH receptor are rare, and the phenotype ranges from completely female external genitalia, lack of breast development, low T, high LH levels, and primary amenorrhea to normal male sex differentiation with hypospadias or a micropenis. Homozygous inactivating mutations in the LH receptor can be associated with the most severe form that completely inactivates the LHCGR and thereby blocks T production. In contrast to activating LHCGR mutations, which result in gonadotropin-independent male-limited precocious puberty, no phenotype has been observed in heterozygous patients with loss-of-function mutations of the LHCGR, underlining the recessive character of these mutations (4, 6–8). Using exome sequencing, we identified three novel homozygous inactivating nonsense mutations in the LHCGR gene in three independent 46,XY girls presenting with primary amenorrhea. These mutations (c.1573 C/T, c.1435 C/T, and c.508 C/T), which have not been reported previously (Table 3), will result in the generation of a truncated protein (p.Gln525Ter, p.Arg479Ter, and p.Gln170Ter). However, the mRNA carrying these mutations is predicted to be recognized by the nonsense-mediateddecay surveillance complexes and degraded. Inactivating mutations in the LHCGR are rare, and there is an apparent genotypephenotype correlation dependent upon the degree of LHCGR inactivation. Mutations involving the LHCGR gene associated with a female phenotype are rare. Only eight homozygous or compound heterozygous mutations have been described in the LH receptor associated with female or ambiguous external genitalia where the individual has been raised as a female (Table 3). Three of the eight mutations are predicted to result in a premature termination codon. In most of these cases, the initial clinical presentation was primary amenorrhea, and six of the eight mutations were reported in affected siblings (Table 3). Where known, these individuals came from populations with a high degree of endogamy or consanguinity. The three different homozygous LHCGR nonsense mutations we describe here were found in different families from the Sidi Bouzid region of central Tunisia. The population of the Sidi Bouzid district was estimated at 122,000 in 2014 by the Tunisian National Institute of Statistics. The inhabitants of this region are considered to have descended from 11th-century invasions of Bedouins of Banu Hilal and Banu Sulaym originating from the Banu’Amir tribe in the Middle East. Consanguineous marriages are relatively common in North Africa, including Tunisia, where it is estimated that between 19% and 21% of marriages in the country are consanguineous (9). This, together with historic geographic isolation and pop-

VOL. 106 NO. 1 / JULY 2016

ulation bottlenecks, has lead to high levels of endogamy in Tunisia. To our knowledge, this is first reported homozygous mutation in the LHCGR gene from North Africa. The identification of three different homozygous nonsense mutations associated with 46,XY DSD and a completely female phenotype at one referral center suggests that the incidence of loss-of-function mutations in the LH receptor may be higher than previously considered. Further population-based studies are warranted to determine the frequency of LHCGR mutations in North African populations.

REFERENCES 1.

2. 3. 4.

5.

6. 7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

Themmen AP, Huhtaniemi IT. Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function. Endocrine Rev 2000;21:551–83. Themmen AP, Martens JW, Brunner HG. Activating and inactivating mutations in LH receptors. Mol Cell Endocrinol 1998;145:137–42. Segaloff DL. Diseases associated with mutations of the human lutropin receptor. Prog Mol Biol Transl Sci 2009;89:97–114. Latronico AC, Anasti J, Arnhold IJ, Rapaport R, Mendonca BB, Bloise W, et al. Brief report: testicular and ovarian resistance to luteinizing hormone caused by inactivating mutations of the luteinizing hormone-receptor gene. N Engl J Med 1996;334:507–12. Martens JW, Verhoef-Post M, Abelin N, Ezabella M, Toledo SP, Brunner HG, et al. A homozygous mutation in the luteinizing hormone receptor causes partial Leydig cell hypoplasia: correlation between receptor activity and phenotype. Mol Endocrinol 1998;12:775–84. Latronico AC, Arnhold IJ. Gonadotropin resistance. Endocrine Dev 2013;24: 25–32. Kossack N, Simoni M, Richter-Unruh A, Themmen AP, Gromoll J. Mutations in a novel, cryptic exon of the luteinizing hormone/chorionic gonadotropin receptor gene cause male pseudohermaphroditism. PLoS Med 2008;5:e88. Gromoll J, Schulz A, Borta H, Gudermann T, Teerds KJ, Greschniok A, et al. Homozygous mutation within the conserved Ala-Phe-Asn-Glu-Thr motif of exon 7 of the LH receptor causes male pseudohermaphroditism. Eur J Endocrinol 2002;147:597–608. Ben Halim N, Ben Alaya Bouafif N, Romdhane L, Kefi Ben Atig R, Chouchane I, Bouyacoub Y, et al. Consanguinity, endogamy, and genetic disorders in Tunisia. J Commun Genet 2013;4:273–84. Laue L, Wu SM, Kudo M, Hsueh AJ, Cutler GB Jr, Griffin JE, et al. A nonsense mutation of the human luteinizing hormone receptor gene in Leydig cell hypoplasia. Hum Mol Genet 1995;4:1429–33. Kremer H, Kraaij R, Toledo SP, Post M, Fridman JB, Hayashida CY, et al. Male pseudohermaphroditism due to a homozygous missense mutation of the luteinizing hormone receptor gene. Nat Genet 1995;9:160–4. Stavrou SS, Zhu YS, Cai LQ, Katz MD, Herrera C, Defillo-Ricart M, et al. A novel mutation of the human luteinizing hormone receptor in 46XY and 46XX sisters. J Clin Endocrinol Metab 1998;83:2091–8. Richter-Unruh A, Verhoef-Post M, Malak S, Homoki J, Hauffa BP, Themmen AP. Leydig cell hypoplasia: absent luteinizing hormone receptor cell surface expression caused by a novel homozygous mutation in the extracellular domain. J Clin Endocrinol Metab 2004;89:5161–7. Salameh W, Choucair M, Guo TB, Zahed L, Wu SM, Leung MY, et al. Leydig cell hypoplasia due to inactivation of luteinizing hormone receptor by a novel homozygous nonsense truncation mutation in the seventh transmembrane domain. Mol Cell Endocrinol 2005;229:57–64. Qiao J, Han B, Liu BL, Chen X, Ru Y, Cheng KX, et al. A splice site mutation combined with a novel missense mutation of LHCGR cause male pseudohermaphroditism. Hum Mutat 2009;30:855–65. Philibert P, Leprieur E, Zenaty D, Thibaud E, Polak M, Frances AM, et al. Steroidogenic factor-1 (SF-1) gene mutation as a frequent cause of primary amenorrhea in 46,XY female adolescents with low testosterone concentration. Reprod Biol Endocrinol 2010;8:28.

229

Gene

CDS position

EGFL8 c.222C>G EPCAM c.5C>T

Amino Acid Change p.Tyr74Ter p.Ala2Val

EntrezGene_summary unknown This gene encodes a carcinoma-associated antigen and is a member of a family that includes at least two type I membrane proteins. This antigen is expressed on most normal epithelial cells and gastrointestinal carcinomas and functions as a homotypic calciumindependent cell adhesion molecule. The antigen is being used as a target for immunotherapy treatment of human carcinomas. Mutations in this gene result in congenital tufting enteropathy. (provided by RefSeq, Dec 2008)

VOL. 106 NO. 1 / JULY 2016


UniProtKB_summary unknown Function: May act as a physical homophilic interaction molecule between intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs) at the mucosal epithelium for providing immunological barrier as a first line of defense against mucosal infection. Plays a role in embryonic stem cells proliferation and differentiation. Up-regulates the expression of FABP5, MYC and cyclins A and E

UniProt_Disorders unknown SwissProt: P16422# Diarrhea 5, with tufting enteropathy, congenital (DIAR5) [MIM:613217]: An intractable diarrhea of infancy characterized by villous atrophy and absence of inflammation, with intestinal epithelial cell dysplasia manifesting as focal epithelial tufts in the duodenum and jejunum. Note¼The disease is caused by mutations affecting the gene represented in this entry & Hereditary non-polyposis colorectal cancer 8 (HNPCC8) [MIM:613244]: An autosomal dominant disease associated with marked increase in cancer susceptibility. It is characterized by a familial predisposition to early-onset colorectal carcinoma (CRC) and extra-colonic tumors of the gastrointestinal, urological and female reproductive tracts. HNPCC is reported to be the most common form of inherited colorectal cancer in the Western world. Clinically, HNPCC is often divided into two subgroups. Type I is characterized by hereditary predisposition to colorectal cancer, a young age of onset, and carcinoma observed in the proximal colon. Type II is characterized by increased risk for cancers in certain tissues such as the uterus, ovary, breast, stomach, small intestine, skin, and larynx in addition to the colon. Diagnosis of classical HNPCC is based on the Amsterdam criteria: 3 or more relatives affected by colorectal cancer, one a first degree relative of the other two; 2 or more generation affected; 1 or more colorectal cancers presenting before 50 years of age; exclusion of hereditary polyposis syndromes. The term ‘suspected HNPCC’ or ‘incomplete HNPCC’ can be used to describe families who do not or only partially fulfill the Amsterdam criteria, but in whom a genetic basis for colon cancer is strongly suspected. Note¼The disease is caused by mutations affecting the gene represented in this entry. HNPCC8 results from heterozygous deletion of 3-prime exons of EPCAM and intergenic regions directly upstream of MSH2, resulting in transcriptional read-through and epigenetic silencing of MSH2 in tissues expressing EPCAM


229.e1

SUPPLEMENTAL DATA: CASE 1

VOL. 106 NO. 1 / JULY 2016

SUPPLEMENTAL DATA: CASE 1 Continued.

Gene

CDS position

Amino Acid Change

EntrezGene_summary

LHCGR c.1573C>T p.Gln525Ter This gene encodes the receptor for both luteinizing hormone and choriogonadotropin. This receptor belongs to the G-protein coupled receptor 1 family, and its activity is mediated by G proteins which activate adenylate cyclase. Mutations in this gene result in disorders of male secondary sexual character development, including familial male precocious puberty, also known as testotoxicosis, hypogonadotropic hypogonadism, Leydig cell adenoma with precocious puberty, and male pseudohermaphtoditism with Leydig cell hypoplasia. (provided by RefSeq, Jul 2008)

MFAP5 c.442C>T

Function: Receptor for lutropinSwissProt: P22888# Familial male precocious puberty choriogonadotropic hormone. The activity (FMPP) [MIM:176410]: In FMPP the receptor is of this receptor is mediated by G proteins constitutively activated. Note¼The disease is which activate adenylate cyclase caused by mutations affecting the gene represented in this entry & Luteinizing hormone resistance (LHR) [MIM:238320]: An autosomal recessive disorder characterized by unresponsiveness to luteinizing hormone, defective sexual development in males, and defective follicular development and ovulation, amenorrhea and infertility in females. Two forms of the disorder have been defined in males. Type 1 is a severe form characterized by complete 46,XY male pseudohermaphroditism, low testosterone and high luteinizing hormone levels, total lack of responsiveness to luteinizing and chorionic gonadotropin hormones, lack of breast development, and absent development of secondary male sex characteristics. Type 2, a milder form, displays a broader range of phenotypic expression ranging from micropenis to severe hypospadias. Note¼The disease is caused by mutations affecting the gene represented in this entry Function: Component of the elastinassociated microfibrils

p.Pro148Ser This gene encodes a 25-kD microfibril-associated glycoprotein which is rich in serine and threonine residues. It lacks a hydrophobic carboxyl terminus and proline-, glutamine-, and tyrosine-rich regions, which are characteristics of a related 31-kDa microfibril-associated glycoprotein (MFAP2). The close similarity between these two proteins is confined to a central region of 60 aa where precise alignment of 7 cysteine residues occurs. The structural differences suggest that this encoded protein has some functions that are distinct from those of MFAP2. (provided by RefSeq, Jul 2008) p.Thr77Met unknown Function: Seems to have transcriptional repression activity in macrophages


UniProt_Disorders

229.e2


SBNO2 c.230C>T

UniProtKB_summary

Gene

CDS position

VOL. 106 NO. 1 / JULY 2016

RABGGT AMPD3

c.745C>G c.1066C>T

CHRDL1

c.623G>A

EPN3 KIF18B

c.1381C>T c.1639G>A

LACTBL1 c.653C>A

Amino Acid Change

EntrezGene_summary

p.Leu249Val unknown p.Arg356Trp This gene encodes a member of the AMP deaminase gene family. The encoded protein is a highly regulated enzyme that catalyzes the hydrolytic deamination of adenosine monophosphate to inosine monophosphate, a branch point in the adenylate catabolic pathway. This gene encodes the erythrocyte (E) isoforms, whereas other family members encode isoforms that predominate in muscle (M) and liver (L) cells. Mutations in this gene lead to the clinically asymptomatic, autosomal recessive condition erythrocyte AMP deaminase deficiency. Alternatively spliced transcript variants encoding different isoforms of this gene have been described. (provided by RefSeq, Jul 2008) p.Arg208His This gene encodes an antagonist of bone morphogenetic protein 4. The encoded protein may play a role in topographic retinotectal projection and in the regulation of retinal angiogenesis in response to hypoxia. Alternatively spliced transcript variants encoding different isoforms have been described. (provided by RefSeq, Jan 2009)

p.Pro461Ser p.Glu547Lys

unknown unknown

p.Ser218Ter

unknown


UniProtKB_summary unknown Function: AMP deaminase plays a critical role in energy metabolism

UniProt_Disorders unknown SwissProt: Q01432# Adenosine monophosphate deaminase deficiency erythrocyte type (AMPDDE) [MIM:612874]: A metabolic disorder due to lack of activity of the erythrocyte isoform of AMP deaminase. It is a clinically asymptomatic condition characterized by a 50% increase in steady-state levels of ATP in affected cells. Individuals with complete deficiency of erythrocyte AMP deaminase are healthy and have no hematologic disorders. Note¼The disease is caused by mutations affecting the gene represented in this entry

Function: Antagonizes the function of SwissProt: Q9BU40# Megalocornea 1, X-linked BMP4 by binding to it and preventing (MGC1) [MIM:309300]: An eye disorder in which its interaction with receptors. Alters the corneal diameter is bilaterally enlarged the fate commitment of neural stem (greater than 13 mm) without an increase in cells from gliogenesis to neurogenesis. intraocular pressure. It may also be referred to as Contributes to neuronal differentiation anterior megalophthalmos, since the entire of neural stem cells in the brain by anterior segment is larger than normal. Features preventing the adoption of a glial fate. of megalocornea in addition to a deep anterior May play a crucial role in dorsoventral chamber include astigmatic refractive errors, axis formation. May play a role in atrophy of the iris stroma, miosis secondary to embryonic bone formation (By decreased function of the dilator muscle, similarity). May also play an important iridodonesis, and tremulousness, subluxation, role in regulating retinal angiogenesis or dislocation of the lens. Whereas most through modulation of BMP4 actions affected individuals exhibit normal ocular function, in endothelial cells. Plays a role during complications include cataract development and anterior segment eye development glaucoma following lenticular dislocation or subluxation. Note¼The disease is caused by mutations affecting the gene represented in this entry unknown unknown Function: In complex with KIF2C, constitutes the major microtubule plus-end depolymerizing activity in mitotic cells. Its major role may be to transport KIF2C and/or MAPRE1 along microtubules unknown unknown


229.e3


VOL. 106 NO. 1 / JULY 2016


Gene

CDS position

Amino Acid Change

EntrezGene_summary

UniProtKB_summary

LHCGR

c.1435 C>T p.Arg479Ter This gene encodes the receptor for both luteinizing hormone and choriogonadotropin. This receptor belongs to the G-protein coupled receptor 1 family, and its activity is mediated by G proteins which activate adenylate cyclase. Mutations in this gene result in disorders of male secondary sexual character development, including familial male precocious puberty, also known as testotoxicosis, hypogonadotropic hypogonadism, Leydig cell adenoma with precocious puberty, and male pseudohermaphtoditism with Leydig cell hypoplasia. (provided by RefSeq, Jul 2008)

Function: Receptor for lutropinchoriogonadotropic hormone. The activity of this receptor is mediated by G proteins which activate adenylate cyclase

RHCE

c.254C>G

Function: May be part of an oligomeric complex which is likely to have a transport or channel function in the erythrocyte membrane

p.Ala85Gly


SwissProt: P22888# Familial male precocious puberty (FMPP) [MIM:176410]: In FMPP the receptor is constitutively activated. Note¼The disease is caused by mutations affecting the gene represented in this entry & Luteinizing hormone resistance (LHR) [MIM:238320]: An autosomal recessive disorder characterized by unresponsiveness to luteinizing hormone, defective sexual development in males, and defective follicular development and ovulation, amenorrhea and infertility in females. Two forms of the disorder have been defined in males. Type 1 is a severe form characterized by complete 46,XY male pseudohermaphroditism, low testosterone and high luteinizing hormone levels, total lack of responsiveness to luteinizing and chorionic gonadotropin hormones, lack of breast development, and absent development of secondary male sex characteristics. Type 2, a milder form, displays a broader range of phenotypic expression ranging from micropenis to severe hypospadias. Note¼The disease is caused by mutations affecting the gene represented in this entry

229.e4


The Rh blood group system is the second most clinically significant of the blood groups, second only to ABO. It is also the most polymorphic of the blood groups, with variations due to deletions, gene conversions, and missense mutations. The Rh blood group includes this gene which encodes both the RhC and RhE antigens on a single polypeptide and a second gene which encodes the RhD protein. The classification of Rh-positive and Rh-negative individuals is determined by the presence or absence of the highly immunogenic RhD protein on the surface of erythrocytes. A mutation in this gene results in amorph-type Rh-null disease. Alternative splicing of this gene results in four transcript variants encoding four different isoforms. (provided by RefSeq, Jul 2008)

UniProt_Disorders

Continued.

Gene

CDS position

TCEAL2

c.374C>T

TYSND1

c.338T>C

Amino Acid Change p.Ala125Val

EntrezGene_summary

UniProtKB_summary

This gene encodes a member of the transcription Function: May be involved in elongation factor A (SII)-like (TCEAL) gene family. transcriptional regulation Members of this family contain TFA domains and may function as nuclear phosphoproteins that modulate transcription in a promoter contextdependent manner. Multiple family members are located on the X chromosome. (provided by RefSeq, Jul 2008) p.Leu113Pro This gene encodes a protease that removes the Function: Peroxisomal protease N-terminal peroxisomal targeting signal (PTS2) from that mediates both the proteins produced in the cytosol, thereby facilitating removal of the leader peptide their import into the peroxisome. The encoded from proteins containing a protein is also capable of removing the C-terminal PTS2 target sequence and peroxisomal targeting signal (PTS1) from proteins in processes several PTS1the peroxisomal matrix. The full-length protein containing proteins. Catalyzes undergoes self-cleavage to produce shorter, the processing of PTS1potentially inactive, peptides. Alternative splicing proteins involved in the results in multiple transcript variants for this gene. peroxisomal beta-oxidation (provided by RefSeq, Jan 2013) of fatty acids


UniProt_Disorders


229.e5


VOL. 106 NO. 1 / JULY 2016

VOL. 106 NO. 1 / JULY 2016


Gene DEGS1

KRT80

KRT84

CDS position

Amino Acid Change

c.99A>G

p.Ile33Met

EntrezGene_summary


UniProt_Disorders

Function: Has sphingolipiddelta-4-desaturase activity. Converts D-erythrosphinganine to D-erythrosphingosine (E-sphing-4-enine)


229.e6

This gene encodes a member of the membrane fatty acid desaturase family which is responsible for inserting double bonds into specific positions in fatty acids. This protein contains three His-containing consensus motifs that are characteristic of a group of membrane fatty acid desaturases. It is predicted to be a multiple membranespanning protein localized to the endoplasmic reticulum. Overexpression of this gene inhibited biosynthesis of the EGF receptor, suggesting a possible role of a fatty acid desaturase in regulating biosynthetic processing of the EGF receptor. (provided by RefSeq, Mar 2010) c.341G>A p.Arg114His Keratins are intermediate filament proteins responsible for the structural integrity of epithelial cells and are subdivided into epithelial keratins and hair keratins. This gene's expression profile shows that it encodes a type II epithelial keratin, although structurally the encoded protein is more like a type II hair keratin. This protein is involved in cell differentiation, localizing near desmosomal plaques in earlier stages of differentiation but then dispersing throughout the cytoplasm in terminally differentiating cells. The type II keratins are clustered in a region of chromosome 12q13. Two transcript variants encoding two different fully functional isoforms have been found for this gene. (provided by RefSeq, Oct 2010) c.1688T>A p.Val563Asp The protein encoded by this gene is a member of the keratin gene family. As a type II hair keratin, it is a basic protein which heterodimerizes with type I keratins to form hair and nails. The type II hair keratins are clustered in a region of chromosome 12q13 and are grouped into two distinct subfamilies based on structure similarity. One subfamily, consisting of KRTHB1, KRTHB3, and KRTHB6, is highly related. The other lessrelated subfamily includes KRTHB2, KRTHB4, and KRTHB5. All hair keratins are expressed in the hair follicle; this hair keratin is contained primarily in the filiform tongue papilla, among other hair keratins. (provided by RefSeq, Jul 2008)

UniProtKB_summary

Continued.

Gene

CDS position

Amino Acid Change

EntrezGene_summary

LHCGR

c.508 C>T p.Gln170Ter This gene encodes the receptor for both luteinizing hormone and choriogonadotropin. This receptor belongs to the G-protein coupled receptor 1 family, and its activity is mediated by G proteins which activate adenylate cyclase. Mutations in this gene result in disorders of male secondary sexual character development, including familial male precocious puberty, also known as testotoxicosis, hypogonadotropic hypogonadism, Leydig cell adenoma with precocious puberty, and male pseudohermaphtoditism with Leydig cell hypoplasia. (provided by RefSeq, Jul 2008)

NBAS

c.6790G>C p.Asp2264His This gene encodes a protein with two leucine zipper domains, a ribosomal protein S14 signature domain and a Sec39 like domain. The protein is thought to be involved in Golgi-to-ER transport. Mutations in this gene are associated with short stature, optic nerve atrophy, and Pelger-Huet anomaly. (provided by RefSeq, Oct 2012)

SDC3

c.112G>A p.Ala38Thr

VOL. 106 NO. 1 / JULY 2016

The protein encoded by this gene belongs to the syndecan proteoglycan family. It may play a role in the organization of cell shape by affecting the actin cytoskeleton, possibly by transferring signals from the cell surface in a sugar-dependent mechanism. Allelic variants of this gene have been associated with obesity. (provided by RefSeq, Oct 2009)


UniProtKB_summary Function: Receptor for lutropinchoriogonadotropic hormone. The activity of this receptor is mediated by G proteins which activate adenylate cyclase

Function: Cell surface proteoglycan that may bear heparan sulfate (By similarity). May have a role in the organization of cell shape by affecting the actin cytoskeleton, possibly by transferring signals from the cell surface in a sugardependent mechanism

UniProt_Disorders SwissProt: P22888# Familial male precocious puberty (FMPP) [MIM:176410]: In FMPP the receptor is constitutively activated. Note¼The disease is caused by mutations affecting the gene represented in this entry & Luteinizing hormone resistance (LHR) [MIM: 238320]: An autosomal recessive disorder characterized by unresponsiveness to luteinizing hormone, defective sexual development in males, and defective follicular development and ovulation, amenorrhea and infertility in females. Two forms of the disorder have been defined in males. Type 1 is a severe form characterized by complete 46,XY male pseudohermaphroditism, low testosterone and high luteinizing hormone levels, total lack of responsiveness to luteinizing and chorionic gonadotropin hormones, lack of breast development, and absent development of secondary male sex characteristics. Type 2, a milder form, displays a broader range of phenotypic expression ranging from micropenis to severe hypospadias. Note¼ The disease is caused by mutations affecting the gene represented in this entry SwissProt: A2RRP1# Short stature, optic nerve atrophy, and Pelger-Huet anomaly (SOPH) [MIM:614800]: An autosomal recessive syndrome characterized by severe postnatal growth retardation, facial dysmorphism with senile face, small hands and feet, normal intelligence, abnormal nuclear shape in neutrophil granulocytes (Pelger-Huet anomaly), and optic atrophy with loss of visual acuity and color vision. Note¼The disease is caused by mutations affecting the gene represented in this entry


229.e7


VOL. 106 NO. 1 / JULY 2016


Gene

CDS position

Amino Acid Change

EntrezGene_summary

SMCR7 c.598G>A p.Gly200Arg This gene encodes an outer mitochondrial membrane protein that functions in the regulation of mitochondrial morphology. It can directly recruit the fission mediator dynamin-related protein 1 (Drp1) to the mitochondrial surface. The gene is located within the Smith-Magenis syndrome region on chromosome 17. Alternative splicing results in multiple transcript variants encoding different isoforms. (provided by RefSeq, Jun 2011) SYT17 TAF9B

WSB1

UniProtKB_summary Function: Mitochondrial outer membrane protein which regulates mitochondrial fission. Promotes the recruitment and association of the fission mediator dynamin-related protein 1 (DNM1L) to the mitochondrial surface independently of the mitochondrial fission FIS1 and MFF proteins

229.e8


c.347G>A p.Arg116His unknown c.286G>T p.Ala96Ser Initiation of transcription by RNA polymerase II requires Function: Essential for cell viability. the activities of more than 70 polypeptides. The TAF9 and TAF9B are involved in protein that coordinates these activities is transcription transcriptional activation as well factor IID (TFIID), which binds to the core promoter to as repression of distinct but position the polymerase properly, serves as the scaffold overlapping sets of genes. May for assembly of the remainder of the transcription have a role in gene regulation complex, and acts as a channel for regulatory signals. associated with apoptosis. TAFs TFIID is composed of the TATA-binding protein (TBP) are components of the and a group of evolutionarily conserved proteins known transcription factor IID (TFIID) as TBP-associated factors or TAFs. TAFs may participate in complex, the TBP-free TAFII basal transcription, serve as coactivators, function in complex (TFTC), the PCAF promoter recognition or modify general transcription histone acetylase complex and factors (GTFs) to facilitate complex assembly and the STAGA transcription transcription initiation. This gene encodes a protein that is coactivator-HAT complex. TFIID similar to one of the small subunits of TFIID, TBPor TFTC are essential for the associated factor 9, and is also a subunit of TFIID. TAF9 regulation of RNA polymerase and TAF9b share some functions but also have distinct II-mediated transcription roles in the transcriptional regulatory process. (provided by RefSeq, Jul 2008) c.1185C>G p.Ilr395Met This gene encodes a member of the WD-protein subfamily. Function: Probable substrateThis protein shares a high sequence identity to mouse recognition component of a SCFand chick proteins. It contains several WD-repeats like ECS (Elongin-Cullin-SOCS-box spanning most of the protein and an SOCS box in the protein) E3 ubiquitin ligase C-terminus. Alternatively spliced transcript variants complex which mediates the encoding distinct isoforms have been found for this ubiquitination and subsequent gene. (provided by RefSeq, Jul 2008) proteasomal degradation of target proteins. Recognizes type II iodothyronine deiodinase/DIO2. Confers constitutive instability to HIPK2 through proteasomal degradation


UniProt_Disorders

Continued.

Gene

CDS position

Amino Acid Change

WWC3

EntrezGene_summary

c.1878C>G p.Asn626Lys This gene encodes a member of the WWC family of proteins, which also includes the WWC1 (KIBRA) gene product and the WWC2 gene product. The protein encoded by this gene includes a C2 domain, which is known to mediate homodimerization in the related WWC1 gene product. (provided by RefSeq, Sep 2011) ZBTB8OS c.138C>G p.His46Gln Unknown Ben Hadj Hmida. LHCGR and 46,XY primary amenorrhea. Fertil Steril 2016.

UniProtKB_summary

UniProt_Disorders


229.e9


VOL. 106 NO. 1 / JULY 2016


SUPPLEMENTAL FIGURE 1 Case 1 I II III

2

1

Case 2

2

Case 3

I

I

II

II

III

III

IV

IV V

2

Pedigrees indicating the affected 46,XY females with primary amenorrhea (solid circles). Squares represent male family members, and circles represent female family members. In each family, the female sibs were either married with children or had spontaneous puberty with regular cycles, suggesting that they are unaffected. Numbers within symbols indicate multiple siblings. Symbols with a slash represent a deceased individual. Ben Hadj Hmida. LHCGR and 46,XY primary amenorrhea. Fertil Steril 2016.

VOL. 106 NO. 1 / JULY 2016

229.e10


SUPPLEMENTAL FIGURE 2 1-MKQRFSALQLLKLLLLLQPPLPRALREALCPEPCNCVPDGALRCPGPTAGLTRLSLAYLP -60

61-VKVIPSQAFRGLNEVIKIEISQIDSLERIEANAFDNLLNLSEILIQNTKNLRYIEPGAFI -120 c.508 C>T, p.Gln170Ter 121-NLPRLKYLSICNTGIRKFPDVTKVFSSESNFILEICDNLHITTIPGNAF QGMNNESVTLK-180

181-LYGNGFEEVQSHAFNGTTLTSLELKENVHLEKMHNGAFRGATGPKTLDISSTKLQALPSY -240

241-GLESIQRLIATSSYSLKKLPSRETFVNLLEATLTYPSHCCAFRNLPTKEQNFSHSISENF-300

301-SKQCESTVRKVNNKTLYSSMLAESELSGWDYEYGFCLPKTPRCAPEPDAFNPCEDIMG YD-360

361-FLRVLIWLINILAIMGNMTVLFVLLTSRYKLTVPRFLMCNLSFADFCMGLYLLLIASVDS-420 c.1435 C>T p.Arg479Ter 421-QTKGQYYNHAIDWQTGSGCSTAGFFTVFASELSVYTLTVITLERWHTITYAIHLDQKLRL-480 c.1573C>T, p.Gln525Ter 481-RHAILIMLGGWLFSSLIAMLPLVGVSNYMKVSICFPMDVETTLSQVYILTILILNVVAFF-540

541-IICACYIKIYFAVRNPELMATNKDTKIAKKMAILIFTDFTCMAPISFFAISAAFK VPLIT-600

601-VTNSKVLLVLFYPINSCANPFLYAIFTKTFQRDFFLLLSKFGCCKRRAELYRRKDFSAYT-660

661-SNCKNGFTGSNKPSQSTLKLSTLHCQGTALLDKTRYTEC -699 The LHCGR protein sequence. The position of the three homozygous nonsense mutations is indicated by the red arrow. For each mutation, the truncated transcripts are predicted to be eliminated by nonsense mediated decay. The transmembrane domains are highlighted in red, and the genomic exon boundaries are indicated by a black arrow. Ben Hadj Hmida. LHCGR and 46,XY primary amenorrhea. Fertil Steril 2016.

229.e11

VOL. 106 NO. 1 / JULY 2016