Hum Genet DOI 10.1007/s00439-014-1445-1
Original Investigation
Homozygous truncating PTPRF mutation causes athelia Guntram Borck · Liat de Vries · Hsin‑Jung Wu · Pola Smirin‑Yosef · Gudrun Nürnberg · Irina Lagovsky · Luis Henrique Ishida · Patrick Thierry · Dagmar Wieczorek · Peter Nürnberg · John Foley · Christian Kubisch · Lina Basel‑Vanagaite
Received: 8 January 2014 / Accepted: 9 April 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Athelia is a very rare entity that is defined by the absence of the nipple–areola complex. It can affect either sex and is mostly part of syndromes including other congenital or ectodermal anomalies, such as limb-mammary syndrome, scalp–ear–nipple syndrome, or ectodermal dysplasias. Here, we report on three children from two branches of an extended consanguineous Israeli Arab family, a girl and two boys, who presented with a spectrum of nipple anomalies ranging from unilateral hypothelia to bilateral athelia but no other consistently associated
G. Borck and L. de Vries contributed equally.
anomalies except a characteristic eyebrow shape. Using homozygosity mapping after single nucleotide polymorphism (SNP) array genotyping and candidate gene sequencing we identified a homozygous frameshift mutation in PTPRF as the likely cause of nipple anomalies in this family. PTPRF encodes a receptor-type protein phosphatase that localizes to adherens junctions and may be involved in the regulation of epithelial cell–cell contacts, peptide growth factor signaling, and the canonical Wnt pathway. Together with previous reports on female mutant Ptprf mice, which have a lactation defect, and disruption of one allele of PTPRF by a balanced translocation in a woman with amastia, our results indicate a key role for PTPRF in the development of the nipple–areola region.
C. Kubisch and L. Basel-Vanagaite jointly supervised this work. Electronic supplementary material The online version of this article (doi:10.1007/s00439-014-1445-1) contains supplementary material, which is available to authorized users. G. Borck (*) · C. Kubisch Institute of Human Genetics, University of Ulm, Ulm, Germany e-mail: guntram.borck@uni‑ulm.de
G. Nürnberg · P. Nürnberg Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany
L. de Vries (*) The Jesse Z And Sara Lea Shafer Institute For Endocrinology And Diabetes, National Center for Childhood Diabetes, Schneider Children’s Medical Center of Israel, Petah Tikva, Israel e-mail: [email protected]
L. H. Ishida Department of Plastic Surgery, University of São Paulo School of Medicine, São Paulo, Brazil
L. de Vries · P. Smirin‑Yosef · I. Lagovsky · L. Basel‑Vanagaite Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel H.-J. Wu · J. Foley Medical Sciences, Indiana University School of Medicine, Bloomington, IN, USA P. Smirin‑Yosef · I. Lagovsky · L. Basel‑Vanagaite Felsenstein Medical Research Center, Rabin Medical Center, Petah Tikva, Israel
P. Thierry Centre Hospitalier Intercommunal de la Haute-Saône, Vesoul, France D. Wieczorek Institut für Humangenetik, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Germany P. Nürnberg Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
13
Hum Genet
Introduction
Methods
Athelia, or congenital absence of nipples, is a rare condition that is most frequently associated with additional congenital or ectodermal anomalies (Ishida et al. 2005). Several terms have been used to classify abnormal breast development including athelia (presence of the mammary gland with absence of the nipples), amazia (absence of the mammary gland but presence of the nipples), and amastia (absence of both the mammary gland and the nipples). Athelia or hypothelia has been reported as an isolated sporadic or familial finding as well as in association with various genetic syndromes. It occurs in TP63-associated limb-mammary syndrome (MIM 603543) and acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome (MIM 103285), TBX3-associated ulnar-mammary syndrome (MIM 181450), KCTD1-associated scalp–ear–nipple syndrome (MIM 181270), FIG4-associated Yunis–Varon syndrome (MIM 216340), WNT10A-associated Schöpf– Schulz–Passarge syndrome (MIM 224750), ectodermal dysplasias, and other syndromes. Although these genes appear to be required for normal morphogenesis of the breasts and nipples, the spatiotemporal expression patterns and molecular networks underlying mammary and nipple development are not fully understood. While several families with isolated autosomal-dominant athelia/amastia have been described (Nelson and Cooper 1982; Fraser 1956; Wilson et al. 1972), autosomal-recessive athelia is rare. Only one family with possible autosomal-recessive inheritance of nonsyndromic athelia/amastia has been reported to our knowledge (Kowlessar and Orti 1968). Here, we describe two interrelated consanguineous families in which three individuals had a previously unidentified autosomal-recessive condition characterized by hypothelia or athelia and minor dysmorphic features with an unusual shape of the eyebrows. We have identified a homozygous mutation in PTPRF as likely causative of this syndromic form of hypothelia/athelia.
The study was approved by the Ethics Committee of the University of Ulm. Prior to genetic analysis, informed consent was obtained from the patients’ parents. Genomic DNA was extracted from leukocytes from peripheral venous blood in EDTA by standard procedures. Single nucleotide polymorphism (SNP) genotyping was performed using Affymetrix Human Mapping NspI 250K arrays (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions. To map the candidate locus, we performed a genome-wide homozygosity mapping analysis as described previously (Borck et al. 2011). For mutation screening of candidate genes located in the linkage interval, we designed intronic primers to PCR amplify coding exons and the respective exon–intron boundaries using genomic DNA of an affected individual. Primer pairs for amplification of the coding exons and approximately 50 base pairs (bp) of flanking intronic sequences are available on request. PCR products were sequenced on an ABI 3730 DNA Analyzer with BigDye chemistry v3.1 (Applied Biosystems). Sequence traces were assembled, aligned, and analyzed with the Seqman software (DNASTAR Lasergene). PTPRF mutation nomenclature is based on transcript NM_002840.3 (31 coding exons) in which the translation start codon is located in exon 3. An additional coding exon, present for example in transcript AK127007.1, was also sequenced. Cosegregation of the mutation with athelia/hypothelia in the family was tested by sequencing the PCR product amplified from genomic DNA of all participating family members. Cell sorting for nipple connective tissue fibroblasts from the female keratin 14 promoter-driven parathyroid hormone-related protein (K14-PTHrP) mouse and ventral dermal fibroblasts from female wild-type mouse was performed using CD140 antibodies (eBioscience) and methodology developed by H.-J. Wu and J. Foley (manuscript in preparation). RNA isolation from isolated cells and snapfrozen tissues, as well as quantitative reverse transcriptase PCR (qRT-PCR) was performed as described (Cho et al. 2004; Nickerson et al. 2012).
P. Nürnberg Cologne Excellence Cluster on Cellular Stress Responses in Aging‑Associated Diseases (CECAD), University of Cologne, Cologne, Germany
Results
L. Basel‑Vanagaite Rabin Medical Center, Raphael Recanati Genetics Institute, Beilinson Campus, Petah Tikva, Israel
Patient 1
L. Basel‑Vanagaite Pediatric Genetics, Schneider Children’s Medical Center of Israel, Petah Tikva, Israel
13
Clinical reports
A 2-year 8-month-old girl (individual V:3, Fig. 2a) was referred to the genetics clinic for evaluation of a chest abnormality (Fig. 1). The mother, aged 25 years, and the father, aged 30 years, are first cousins of Israeli Arab origin
Hum Genet
Fig. 1 Clinical features of three children with hypothelia–athelia. a–c Patient 1, d and e patient 2, f–i patient 3. Note characteristic eyebrow shape (a, d, g), nipple anomalies (b, e, h), minor anomalies of the ears (c, i), and bilateral single transverse palmar creases (f)
(Fig. 2a). Routine fetal ultrasound examination did not reveal any abnormalities in V:3. Birth was by vaginal delivery at 40 weeks and birth weight was 3,300 g (25–50th centile). On physical examination at 2 years 8 months, her height was 85 cm (3rd centile), weight was 13 kg (10th centile), and occipitofrontal circumference was 49 cm (50th centile). She had arched eyebrows with an inverted V-shape of the lateral part of the eyebrows, small earlobes, flat philtrum, broad nasal tip, mildly anteverted nares and absent left areola and nipple (Fig. 1a–c). Neither a breast bud nor a nipple was visualized on ultrasound examination of the left side of the chest. The right breast bud and nipple were of normal size and morphology. The pectoralis muscles were present and normally developed and there were no limb malformations. Genital examination was normal and she had normal motor and cognitive development. Deciduous teeth had a normal shape. The anterior and posterior segments of the eyes were evaluated using a slit lamp and found to be normal. The parents had normal eyebrows and the paternal nipples and maternal breasts and nipples appeared normal. Patient 2 This 20-month-old boy (individual V:4, Fig. 2a) is the brother of patient 1. He was born at term with hypoplasia of the left nipple. Birth weight was 3,100 g. He had arched eyebrows with an inverted V-shape of the lateral part of the eyebrows, flat philtrum, broad nasal tip, anteverted nares, hypoplastic left areola and hypoplastic left inverted nipple
(Fig. 1d, e). Genital examination was normal including normal penile size. At the age of 1 year 8 months, his height was 83 cm (near 50th centile), weight 12.5 (near 50th centile) and occipitofrontal circumference between 50 and 75th centile. Patient 3 This 13-year-old boy is the first cousin of patients 1 and 2. He was born to consanguineous parents (Fig. 2a) and has bilateral athelia. On physical examination at the age of 13 years (Fig. 1f–i), his height was 154 cm (25th centile) and weight 47.5 kg (50th centile); thus, his body mass index was 20 kg/m2 (50–75th centile). His eyebrow shape is similar to that of his affected cousins. He also has a slightly pointed upper ear helix. He has bilateral single transverse palmar creases. He had previously undergone orchidopexy for bilateral undescended testis and meatotomy for meatal stenosis. Pubertal development occurred spontaneously and was appropriate for his age. Physical examination revealed a normally appearing penis with a length of 4.6 cm (mean ± SD for his age is 6.4 ± 1.1 cm; mean −2 SD is 3.7 cm). Both testes were palpated in the normally developed scrotal sac (testes 8 ml each). Kidneys were of normal size and structure as shown by renal ultrasound. In addition, he had small permanent mandibular incisors and widely spaced teeth. Nails were normal and sweating was reported to be normal. An endocrinological evaluation revealed normal fT4 and TSH; and basal levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that were
13
Hum Genet
normal for his Tanner stage. Routine biochemical analyses showed normal values for nonfasting glucose, calcium and phosphorus, cholesterol and triglycerides. None of the three patients had obesity, hypertension, or acanthosis nigricans. Molecular analyses We performed a genome-wide linkage analysis with 250K SNP arrays followed by homozygosity mapping to identify the chromosomal region that harbors the mutation underlying this autosomal-recessive disorder. After genotyping of samples from two affected individuals (V:1 and V:3) and V:3 and V:4’s unaffected parents (IV:9 and IV:10; Fig. 2a) and using a reduced marker panel of ~20,000 SNPs we identified linkage to a genomic region on chromosome 1p34.2–p32.3 with a maximum LOD score of 3.31. This LOD score was the maximum expected from simulation analysis, and the region on chromosome 1p was the only region within the genome with a LOD score that exceeded 3.0 (Fig. 2b). Considering all the SNPs from the 250K SNP array, the region of shared homozygosity was determined to be flanked by SNP markers rs633297 and rs17115767 after haplotype reconstruction, defining a critical region of 15.89 Mb. This region contained >150 genes, none of which has previously been found to be mutated in syndromes featuring athelia. Based on their known or presumed function in sexual or mammary gland development, we selected three candidate genes from the linkage region. Sequencing of the Doublesex- and Mab-3-Related Transcription factor genes DMRTA2 and DMRTB1 did not reveal any mutations. In exon 12 of PTPRF (protein tyrosine phosphatase, receptor type, F; MIM 179590), we identified a homozygous 2 bp deletion that removed one of four consecutive TG dinucleotides (c.1847_1848delTG; Fig. 2c). This mutation was predicted to induce a translational frameshift and lead to a premature stop codon (p.Val616Glufs*49). It cosegregated with athelia/hypothelia in the family (Fig. 2a) and was absent from dbSNP137 and the approximately 6,400 European and African American individuals sequenced at this position by the NHLBI Exome Sequencing Project (Exome Variant Server). We next sequenced PTPRF in two additional unrelated individuals presenting with athelia. Patient 4 is a previously reported young woman from Brazil who underwent plastic surgery for congenital bilateral absence of the nipples with normal postpubertal breast development (Ishida et al. 2005), and patient 5 is a previously unreported Greek boy who presented with bilateral athelia, developmental delay and hearing impairment. We detected no PTPRF mutation in either of these two individuals, nor did we
13
Fig. 2 A truncating PTPRF mutation causes athelia. a Pedigree of the family and segregation of the PTPRF mutation. Filled symbols represent individuals with athelia or hypothelia. IV:4 and IV:5 are related but the exact degree of consanguinity is unknown. Mut = c.1847_1848delTG mutation in PTPRF (p.Val616Glufs*49). b Schematic representation of genome-wide LOD score calculations. LOD scores calculated with ALLEGRO are given along the y-axis relative to genomic position in centi Morgans (cM) on the x-axis. Note the single significant peak (LOD score 3.31), on chromosome 1 (red arrow). c PTPRF mutation. Sequence chromatograms showing part of PTPRF exon 12 in a control individual and in patient 1 (V:3) who is homozygous for the c.1847_1848delTG (p.Val616Glufs*49) mutation in PTPRF. Letters above the sequence indicate the encoded amino acids in one-letter code (color figure online)
identify a PTPRF mutation in a boy with a similar eyebrow shape (Supplementary Fig. 1), intellectual disability, and normal nipples.
Hum Genet
Fig. 3 Upregulation of Ptprf in nipple fibroblasts. a Relative mRNA levels of Ptprf in sorted fibroblasts. Transcript levels were measured by qRT-PCR analysis and relative ratios of target mRNA to Gapdh mRNA levels are shown (mean of three measures from five independent samples; bars, SD; **p
Original Investigation
Homozygous truncating PTPRF mutation causes athelia Guntram Borck · Liat de Vries · Hsin‑Jung Wu · Pola Smirin‑Yosef · Gudrun Nürnberg · Irina Lagovsky · Luis Henrique Ishida · Patrick Thierry · Dagmar Wieczorek · Peter Nürnberg · John Foley · Christian Kubisch · Lina Basel‑Vanagaite
Received: 8 January 2014 / Accepted: 9 April 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Athelia is a very rare entity that is defined by the absence of the nipple–areola complex. It can affect either sex and is mostly part of syndromes including other congenital or ectodermal anomalies, such as limb-mammary syndrome, scalp–ear–nipple syndrome, or ectodermal dysplasias. Here, we report on three children from two branches of an extended consanguineous Israeli Arab family, a girl and two boys, who presented with a spectrum of nipple anomalies ranging from unilateral hypothelia to bilateral athelia but no other consistently associated
G. Borck and L. de Vries contributed equally.
anomalies except a characteristic eyebrow shape. Using homozygosity mapping after single nucleotide polymorphism (SNP) array genotyping and candidate gene sequencing we identified a homozygous frameshift mutation in PTPRF as the likely cause of nipple anomalies in this family. PTPRF encodes a receptor-type protein phosphatase that localizes to adherens junctions and may be involved in the regulation of epithelial cell–cell contacts, peptide growth factor signaling, and the canonical Wnt pathway. Together with previous reports on female mutant Ptprf mice, which have a lactation defect, and disruption of one allele of PTPRF by a balanced translocation in a woman with amastia, our results indicate a key role for PTPRF in the development of the nipple–areola region.
C. Kubisch and L. Basel-Vanagaite jointly supervised this work. Electronic supplementary material The online version of this article (doi:10.1007/s00439-014-1445-1) contains supplementary material, which is available to authorized users. G. Borck (*) · C. Kubisch Institute of Human Genetics, University of Ulm, Ulm, Germany e-mail: guntram.borck@uni‑ulm.de
G. Nürnberg · P. Nürnberg Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany
L. de Vries (*) The Jesse Z And Sara Lea Shafer Institute For Endocrinology And Diabetes, National Center for Childhood Diabetes, Schneider Children’s Medical Center of Israel, Petah Tikva, Israel e-mail: [email protected]
L. H. Ishida Department of Plastic Surgery, University of São Paulo School of Medicine, São Paulo, Brazil
L. de Vries · P. Smirin‑Yosef · I. Lagovsky · L. Basel‑Vanagaite Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel H.-J. Wu · J. Foley Medical Sciences, Indiana University School of Medicine, Bloomington, IN, USA P. Smirin‑Yosef · I. Lagovsky · L. Basel‑Vanagaite Felsenstein Medical Research Center, Rabin Medical Center, Petah Tikva, Israel
P. Thierry Centre Hospitalier Intercommunal de la Haute-Saône, Vesoul, France D. Wieczorek Institut für Humangenetik, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Germany P. Nürnberg Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
13
Hum Genet
Introduction
Methods
Athelia, or congenital absence of nipples, is a rare condition that is most frequently associated with additional congenital or ectodermal anomalies (Ishida et al. 2005). Several terms have been used to classify abnormal breast development including athelia (presence of the mammary gland with absence of the nipples), amazia (absence of the mammary gland but presence of the nipples), and amastia (absence of both the mammary gland and the nipples). Athelia or hypothelia has been reported as an isolated sporadic or familial finding as well as in association with various genetic syndromes. It occurs in TP63-associated limb-mammary syndrome (MIM 603543) and acro-dermato-ungual-lacrimal-tooth (ADULT) syndrome (MIM 103285), TBX3-associated ulnar-mammary syndrome (MIM 181450), KCTD1-associated scalp–ear–nipple syndrome (MIM 181270), FIG4-associated Yunis–Varon syndrome (MIM 216340), WNT10A-associated Schöpf– Schulz–Passarge syndrome (MIM 224750), ectodermal dysplasias, and other syndromes. Although these genes appear to be required for normal morphogenesis of the breasts and nipples, the spatiotemporal expression patterns and molecular networks underlying mammary and nipple development are not fully understood. While several families with isolated autosomal-dominant athelia/amastia have been described (Nelson and Cooper 1982; Fraser 1956; Wilson et al. 1972), autosomal-recessive athelia is rare. Only one family with possible autosomal-recessive inheritance of nonsyndromic athelia/amastia has been reported to our knowledge (Kowlessar and Orti 1968). Here, we describe two interrelated consanguineous families in which three individuals had a previously unidentified autosomal-recessive condition characterized by hypothelia or athelia and minor dysmorphic features with an unusual shape of the eyebrows. We have identified a homozygous mutation in PTPRF as likely causative of this syndromic form of hypothelia/athelia.
The study was approved by the Ethics Committee of the University of Ulm. Prior to genetic analysis, informed consent was obtained from the patients’ parents. Genomic DNA was extracted from leukocytes from peripheral venous blood in EDTA by standard procedures. Single nucleotide polymorphism (SNP) genotyping was performed using Affymetrix Human Mapping NspI 250K arrays (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions. To map the candidate locus, we performed a genome-wide homozygosity mapping analysis as described previously (Borck et al. 2011). For mutation screening of candidate genes located in the linkage interval, we designed intronic primers to PCR amplify coding exons and the respective exon–intron boundaries using genomic DNA of an affected individual. Primer pairs for amplification of the coding exons and approximately 50 base pairs (bp) of flanking intronic sequences are available on request. PCR products were sequenced on an ABI 3730 DNA Analyzer with BigDye chemistry v3.1 (Applied Biosystems). Sequence traces were assembled, aligned, and analyzed with the Seqman software (DNASTAR Lasergene). PTPRF mutation nomenclature is based on transcript NM_002840.3 (31 coding exons) in which the translation start codon is located in exon 3. An additional coding exon, present for example in transcript AK127007.1, was also sequenced. Cosegregation of the mutation with athelia/hypothelia in the family was tested by sequencing the PCR product amplified from genomic DNA of all participating family members. Cell sorting for nipple connective tissue fibroblasts from the female keratin 14 promoter-driven parathyroid hormone-related protein (K14-PTHrP) mouse and ventral dermal fibroblasts from female wild-type mouse was performed using CD140 antibodies (eBioscience) and methodology developed by H.-J. Wu and J. Foley (manuscript in preparation). RNA isolation from isolated cells and snapfrozen tissues, as well as quantitative reverse transcriptase PCR (qRT-PCR) was performed as described (Cho et al. 2004; Nickerson et al. 2012).
P. Nürnberg Cologne Excellence Cluster on Cellular Stress Responses in Aging‑Associated Diseases (CECAD), University of Cologne, Cologne, Germany
Results
L. Basel‑Vanagaite Rabin Medical Center, Raphael Recanati Genetics Institute, Beilinson Campus, Petah Tikva, Israel
Patient 1
L. Basel‑Vanagaite Pediatric Genetics, Schneider Children’s Medical Center of Israel, Petah Tikva, Israel
13
Clinical reports
A 2-year 8-month-old girl (individual V:3, Fig. 2a) was referred to the genetics clinic for evaluation of a chest abnormality (Fig. 1). The mother, aged 25 years, and the father, aged 30 years, are first cousins of Israeli Arab origin
Hum Genet
Fig. 1 Clinical features of three children with hypothelia–athelia. a–c Patient 1, d and e patient 2, f–i patient 3. Note characteristic eyebrow shape (a, d, g), nipple anomalies (b, e, h), minor anomalies of the ears (c, i), and bilateral single transverse palmar creases (f)
(Fig. 2a). Routine fetal ultrasound examination did not reveal any abnormalities in V:3. Birth was by vaginal delivery at 40 weeks and birth weight was 3,300 g (25–50th centile). On physical examination at 2 years 8 months, her height was 85 cm (3rd centile), weight was 13 kg (10th centile), and occipitofrontal circumference was 49 cm (50th centile). She had arched eyebrows with an inverted V-shape of the lateral part of the eyebrows, small earlobes, flat philtrum, broad nasal tip, mildly anteverted nares and absent left areola and nipple (Fig. 1a–c). Neither a breast bud nor a nipple was visualized on ultrasound examination of the left side of the chest. The right breast bud and nipple were of normal size and morphology. The pectoralis muscles were present and normally developed and there were no limb malformations. Genital examination was normal and she had normal motor and cognitive development. Deciduous teeth had a normal shape. The anterior and posterior segments of the eyes were evaluated using a slit lamp and found to be normal. The parents had normal eyebrows and the paternal nipples and maternal breasts and nipples appeared normal. Patient 2 This 20-month-old boy (individual V:4, Fig. 2a) is the brother of patient 1. He was born at term with hypoplasia of the left nipple. Birth weight was 3,100 g. He had arched eyebrows with an inverted V-shape of the lateral part of the eyebrows, flat philtrum, broad nasal tip, anteverted nares, hypoplastic left areola and hypoplastic left inverted nipple
(Fig. 1d, e). Genital examination was normal including normal penile size. At the age of 1 year 8 months, his height was 83 cm (near 50th centile), weight 12.5 (near 50th centile) and occipitofrontal circumference between 50 and 75th centile. Patient 3 This 13-year-old boy is the first cousin of patients 1 and 2. He was born to consanguineous parents (Fig. 2a) and has bilateral athelia. On physical examination at the age of 13 years (Fig. 1f–i), his height was 154 cm (25th centile) and weight 47.5 kg (50th centile); thus, his body mass index was 20 kg/m2 (50–75th centile). His eyebrow shape is similar to that of his affected cousins. He also has a slightly pointed upper ear helix. He has bilateral single transverse palmar creases. He had previously undergone orchidopexy for bilateral undescended testis and meatotomy for meatal stenosis. Pubertal development occurred spontaneously and was appropriate for his age. Physical examination revealed a normally appearing penis with a length of 4.6 cm (mean ± SD for his age is 6.4 ± 1.1 cm; mean −2 SD is 3.7 cm). Both testes were palpated in the normally developed scrotal sac (testes 8 ml each). Kidneys were of normal size and structure as shown by renal ultrasound. In addition, he had small permanent mandibular incisors and widely spaced teeth. Nails were normal and sweating was reported to be normal. An endocrinological evaluation revealed normal fT4 and TSH; and basal levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that were
13
Hum Genet
normal for his Tanner stage. Routine biochemical analyses showed normal values for nonfasting glucose, calcium and phosphorus, cholesterol and triglycerides. None of the three patients had obesity, hypertension, or acanthosis nigricans. Molecular analyses We performed a genome-wide linkage analysis with 250K SNP arrays followed by homozygosity mapping to identify the chromosomal region that harbors the mutation underlying this autosomal-recessive disorder. After genotyping of samples from two affected individuals (V:1 and V:3) and V:3 and V:4’s unaffected parents (IV:9 and IV:10; Fig. 2a) and using a reduced marker panel of ~20,000 SNPs we identified linkage to a genomic region on chromosome 1p34.2–p32.3 with a maximum LOD score of 3.31. This LOD score was the maximum expected from simulation analysis, and the region on chromosome 1p was the only region within the genome with a LOD score that exceeded 3.0 (Fig. 2b). Considering all the SNPs from the 250K SNP array, the region of shared homozygosity was determined to be flanked by SNP markers rs633297 and rs17115767 after haplotype reconstruction, defining a critical region of 15.89 Mb. This region contained >150 genes, none of which has previously been found to be mutated in syndromes featuring athelia. Based on their known or presumed function in sexual or mammary gland development, we selected three candidate genes from the linkage region. Sequencing of the Doublesex- and Mab-3-Related Transcription factor genes DMRTA2 and DMRTB1 did not reveal any mutations. In exon 12 of PTPRF (protein tyrosine phosphatase, receptor type, F; MIM 179590), we identified a homozygous 2 bp deletion that removed one of four consecutive TG dinucleotides (c.1847_1848delTG; Fig. 2c). This mutation was predicted to induce a translational frameshift and lead to a premature stop codon (p.Val616Glufs*49). It cosegregated with athelia/hypothelia in the family (Fig. 2a) and was absent from dbSNP137 and the approximately 6,400 European and African American individuals sequenced at this position by the NHLBI Exome Sequencing Project (Exome Variant Server). We next sequenced PTPRF in two additional unrelated individuals presenting with athelia. Patient 4 is a previously reported young woman from Brazil who underwent plastic surgery for congenital bilateral absence of the nipples with normal postpubertal breast development (Ishida et al. 2005), and patient 5 is a previously unreported Greek boy who presented with bilateral athelia, developmental delay and hearing impairment. We detected no PTPRF mutation in either of these two individuals, nor did we
13
Fig. 2 A truncating PTPRF mutation causes athelia. a Pedigree of the family and segregation of the PTPRF mutation. Filled symbols represent individuals with athelia or hypothelia. IV:4 and IV:5 are related but the exact degree of consanguinity is unknown. Mut = c.1847_1848delTG mutation in PTPRF (p.Val616Glufs*49). b Schematic representation of genome-wide LOD score calculations. LOD scores calculated with ALLEGRO are given along the y-axis relative to genomic position in centi Morgans (cM) on the x-axis. Note the single significant peak (LOD score 3.31), on chromosome 1 (red arrow). c PTPRF mutation. Sequence chromatograms showing part of PTPRF exon 12 in a control individual and in patient 1 (V:3) who is homozygous for the c.1847_1848delTG (p.Val616Glufs*49) mutation in PTPRF. Letters above the sequence indicate the encoded amino acids in one-letter code (color figure online)
identify a PTPRF mutation in a boy with a similar eyebrow shape (Supplementary Fig. 1), intellectual disability, and normal nipples.
Hum Genet
Fig. 3 Upregulation of Ptprf in nipple fibroblasts. a Relative mRNA levels of Ptprf in sorted fibroblasts. Transcript levels were measured by qRT-PCR analysis and relative ratios of target mRNA to Gapdh mRNA levels are shown (mean of three measures from five independent samples; bars, SD; **p
